Joven Et Al-2013-Journal of Comparative Neurology

Expression Patterns of Pax6 and Pax7 in the AdultBrain of a Urodele Amphibian, Pleurodeles waltl

Alberto Joven, Ruth Morona, Agustın Gonz�alez, and Nerea Moreno*

Department of Cell Biology, Faculty of Biology, University Complutense, 28040 Madrid, Spain

ABSTRACTExpression patterns of Pax6, Pax7, and, to a lesser

extent, Pax3 genes were analyzed by a combination of

immunohistochemical techniques in the central nervous

system of adult specimens of the urodele amphibian

Pleurodeles waltl. Only Pax6 was found in the telen-

cephalon, specifically the olfactory bulbs, striatum, sep-

tum, and lateral and central parts of the amygdala. In

the diencephalon, Pax6 and Pax7 were distinct in the

alar and basal parts, respectively, of prosomere 3. The

distribution of Pax6, Pax7, and Pax3 cells correlated

with the three pretectal domains. Pax7 specifically la-

beled cells in the dorsal mesencephalon, mainly in the

optic tectum, and Pax6 cells were the only cells found

in the tegmentum. Large populations of Pax7 cells

occupied the rostral rhombencephalon, along with lower

numbers of Pax6 and Pax3 cells. Pax6 was found in

most granule cells of the cerebellum. Pax6 cells also

formed a column of scattered neurons in the reticular

formation and were found in the octavolateral area. The

rhombencephalic ventricular zone of the alar plate

expressed Pax7. Dorsal Pax7 cells and ventral Pax6

cells were found along the spinal cord. Our results

show that the expression of Pax6 and Pax7 is widely

maintained in the brains of adult urodeles, in contrast

to the situation in other tetrapods. This discrepancy

could be due to the generally pedomorphic features of

urodele brains. Although the precise role of these tran-

scription factors in adult brains remains to be deter-

mined, our findings support the idea that they may also

function in adult urodeles. J. Comp. Neurol. 521:2088–

2124, 2013.

VC 2012 Wiley Periodicals, Inc.

INDEXING TERMS: Pax genes; immunohistochemistry; segmental organization; telencephalon; diencephalon; brain

evolution

The central nervous system (CNS) of urodele amphib-

ians (newts and salamanders) has long been studied by

comparative neuroanatomists (Herrick, 1927, 1948; Kicli-

ter and Ebbesson, 1976; Fritzsch and Himstedt, 1980;

Northcutt and Kicliter, 1980), and its simplified organiza-

tion of neurons and fiber systems was considered by

some researchers to be common to all tetrapods. Newer

data generated by modern techniques, however, allowed

identification of the neuroanatomical features that char-

acterize most brain regions in amniotes, although their

homologs could not be identified in amphibians. This was

particularly true for urodeles, where only limited cell

migration from the ventricular lining exists in adults, and

most neurons are crowded into a dense periventricular

cell layer, and nuclei and distinct structures could be rec-

ognized only as local condensations (see ten Donkelaar,

1998). Thus, many features of the brain of urodeles seem

to be less differentiated than in tetrapod outgroups,

including cartilaginous and bony fishes, and some charac-

ters in urodele brains are even more simple than those in

agnathan brains (Northcutt, 1984, 1987; Roth et al.,

1992, 1993). This situation was explained as a case of

secondary simplification of the brain, implying that uro-

dele brains are more primitive than their phylogenetic

position as tetrapods would indicate (Northcutt, 1987;

Roth et al., 1992, 1993). Our knowledge of the organiza-

tion of urodele brains increased with the use of specific

tract-tracing and immunohistochemical techniques,

which allowed identification of distinct neuronal popula-

tions, revealing that the organization of the nonsegre-

gated periventricular cell layer is much more complex

Additional Supporting Information may be found in the online version ofthis article.

Grant sponsor: Spanish Ministry of Science and Technology; Grantnumbers: BFU2009-12315 and BFU2012-31687.

*CORRESPONDENCE TO: Nerea Moreno, PhD, Departamento deBiologıa Celular, Facultad de Biologıa, Universidad Complutense, 28040Madrid, Spain. E-mail: [email protected]

VC 2012 Wiley Periodicals, Inc.

Received September 12, 2012; Revised November 21, 2012; AcceptedNovember 27, 2012

DOI 10.1002/cne.23276

Published online December 10, 2012 in Wiley Online Library(wileyonlinelibrary.com)

2088 The Journal of Comparative Neurology | Research in Systems Neuroscience 521:2088–2124 (2013)

RESEARCH ARTICLE

than previously thought, and that most features of the

neurochemical systems in the brains of urodeles are

shared with amniotes (Wicht and Himstedt, 1988; Bar-

roso et al., 1993; Gonz�alez et al., 1993; Krug et al., 1993;

Marın et al., 1997a–d; Westhoff and Roth, 2002; Laberge

et al., 2006; Morona and Gonz�alez, 2008, 2009).

Recently, localization of genetic markers in the CNS,

particularly transcription factors, has proved useful for

studying characteristics of brain organization that are con-

served across species. Most of these markers have been

used to interpret brain specification and regionalization

during development but, in addition, a number of these

transcription factors display regionally restricted expres-

sion domains in adult brains, with their borders corre-

sponding precisely with morphological landmarks. The

expression patterns of conserved markers in vertebrates

are best interpreted according to the current neuromeric

models of the brain (Gilland and Baker, 1993; Marın and

Puelles, 1995; Puelles et al., 1996; Fritzsch, 1998; Cam-

bronero and Puelles, 2000; Diaz et al., 2000; Puelles and

Rubenstein, 2003; Straka et al., 2006). Thus, in spite of

the apparently simple organization of the forebrain in uro-

deles, most features of amniote brains can be identified by

analyzing the combined patterns of distribution of specific

transcription factors expressed in adult amniotes (Moreno

and Gonz�alez, 2007; Bardet et al., 2008).

Pax genes encode a family of highly conserved tran-

scription factors characterized by the presence of a

paired domain that confers sequence-specific binding to

DNA; in addition, Pax transcription factors may also have

an octapeptide motif and part or all of a homeobox DNA-

binding domain (Balczarek et al., 1997; Chi and Epstein,

2003; Vorobyov and Horst, 2006; Lang et al., 2007; Wang

et al., 2010). Among the Pax genes, Pax7 has the paired

domain, the octapeptide motif, and the homeobox do-

main, whereas Pax6 lacks the octapeptide motif. Both

Pax6 and Pax7 are expressed in regionally restricted pat-

terns in the developing brain and are involved in neuronal

proliferation, brain regionalization, cell differentiation,

and neuronal survival (Wehr and Gruss, 1996; Lang et al.,

Abbreviations

Acc Nucleus accumbensal Anterior lobe of the hypophysisAOB Accessory olfactory bulbAv Anteroventral tegmental nucleusBH Basal hypothalamusBST Bed nucleus of the stria terminalisCb CerebellumCeA Central amygdalaDB Diagonal band of BrocaDCN Dorsal column nucleusdh Dorsal hornDi DiencephalonDN Dorsal nucleus of the octavolateral areaDP Dorsal palliumepi Epiphysisepl External plexiform layerGc Central grayGCL Granular cell layergl Glomerular layerHb Habenulahc Habenular commissurehht Hypothalamo-hypophyseal tractHis Sulcus of HisHyp Hypothalamusigl Internal granular layeril Intermediate lobe of the hypophysisIN Intermediate nucleus of the octavolateral areaINL Inner nuclear layerIp Interpeduncular nucleusIPL Inner plexiform layerIpn Interpeduncular neuropilIs Isthmic nucleusIV Trochlear nucleusIVv Fourth ventricleiz Intermediate zoneLA Lateral amygdalaLC Laterocaudal mesencephalic nucleusLc Locus coeruleusLDT Laterodorsal tegmental nucleusLL Lateral line nucleusLP Lateral palliumMa Mammillary nucleusMeA Medial amygdalaMes Mesencephalonml Mitral layerMOB Main olfactory bulbMP Medial pallium

NCPm Nucleus of the commissura posterior, magnocellular partnl Neural lobe of the hypophysisnhy NeurohypophysisNPv Nucleus of the periventricular organnV Trigeminal nerveOB Olfactory bulbONL Outer nuclear layerOPL Outer plexiform layerOT Optic tectump1-3b Basal part of prosomeres 1-3p1-3 Prosomeres 1-3P PalliumPA Pallidumpar Paraphysispc Posterior commissurePd Posterodorsal tegmental nucleusPdi Posterodorsal tegmental nucleus, isthmic partPO Preoptic areaPOC Preoptocommissural areaPv Posteroventral tegmental nucleusPvi Posteroventral tegmental nucleus, isthmic partPThE Prethalamic eminencer0 Isthmus (rhombomere 0)r1-8 Rhombomeres 1-8Ra Raphe nucleiRh RhombencephalonRi Inferior reticular nucleusrinf Infundibular recessRm Medial reticular nucleusRs Superior reticular nucleussc Spinal cordsco Subcommissural organSd Septum dorsalisSe SeptumSl Septum lateralisSN Substantia nigraSPV Supraopto paraventricular areaStr StriatumTel Telencephalontgd Dorsal tegmentumTs Torus semicircularisTub Tuberal areaVm Motor trigeminal nucleusVP Ventral palliumVTA Ventral tegmental areaVZ Ventral zone of the octavolateral area

Pax6 and Pax7 in the brain of adult urodeles

The Journal of Comparative Neurology | Research in Systems Neuroscience 2089

2007; Thomson et al., 2007; Osumi et al., 2008; Wang

et al., 2008). Interestingly, Pax6 and Pax7 are also

expressed in adult brains in restricted and well-localized

cell groups and regions (Walther and Gruss, 1991; Stoy-

kova and Gruss, 1994; Kawakami et al., 1997; Shin et al.,

2003; Thompson and Ziman, 2011; Duan et al., 2012),

suggesting their involvement in the maintenance of dis-

tinct neuronal identity (Ninkovic et al., 2010), in physio-

logical functions in mature neurons (Stoykova and Gruss,

1994; Shin et al., 2003), and as key regulators of a cell’s

measured response to a dynamic environment (Blake

et al., 2008). In terms of neuroanatomical distribution in

various adult mammals, Pax6 is expressed in retinal cells,

telencephalon, diencephalon, ventral mesencephalon,

cerebellum, and pons/medulla (Stoykova and Gruss,

1994; Kohwi et al., 2005; Maekawa et al., 2005; Nacher

et al., 2005; Stanescu et al., 2007), whereas Pax7 is

expressed in the superior colliculus and in specific nuclei

of the pons/medulla and thalamus (Stoykova and Gruss,

1994; Shin et al., 2003, Thomas et al., 2007; Thompson

et al., 2007, 2008).

Fragmentary data from amphibians, particularly the anu-

ran Xenopus laevis, show a spatiotemporal sequence of

Pax6 and Pax7 expression comparable to that in amniotes

(Bachy et al., 2002; Moreno et al., 2008a; Morona et al.,

2011). The present study reports our further research on

brain organization in urodele amphibians; specifically, we

analyzed the distribution of Pax6- and Pax7-immunoreac-

tive cells (Pax6 and Pax7 cells, respectively) in the brain of

Pleurodeles waltl, and placed our results in the context of

the neuromeric paradigm. In addition, the localization of

Pax3, which is a paralogous gene to Pax7 but with very re-

stricted expression in adults (Stoykova and Gruss, 1994;

Maczkowiak et al., 2010), was also investigated. We chose

P. waltl because this urodele species has been used in neu-

roanatomical studies with modern immunohistochemical

techniques (see Marın et al., 1997b; ten Donkelaar, 1998;

Moreno and Gonz�alez, 2007; Morona and Gonz�alez, 2008,

2009; Joven et al., 2012).

The immunohistochemical techniques employed in the

present study allow high-resolution analysis of expressing

cells (Hitchcock et al., 1996; Wullimann and Rink, 2001;

Gonz�alez and Northcutt, 2009; Ferreiro-Galve et al.,

2012). To precisely identify the cell groups expressing

Pax6 and/or Pax7, we used double- and triple-labeling

techniques to simultaneously reveal several neuronal

markers, which in turn served as landmarks for brain

regions, as previously reported (Gonz�alez and Smeets,

1991, 1992; Naujoks-Manteuffel et al., 1994; Gonz�alez

et al., 1996; Marın et al., 1997b; Morona and Gonz�alez,

2008, 2009). These markers included the c-amino butyric

acid (GABA), calbindin D-28k (CB), calretinin (CR), choline

acetyltransferase (ChAT), nitric oxide synthase (NOS),

serotonin (5-HT), tyrosine hydroxylase (TH), and the tran-

scription factors Nkx2.1 and Nkx2.2. Our results show

that the expression of Pax6 and Pax7 is widely main-

tained in adult P. waltl brains, and the patterns of distribu-

tion are largely comparable to those reported for other

vertebrates, in particular mammals.

MATERIALS AND METHODS

Animals and tissue processingAdult specimens of the urodele amphibian Pleurodeles

waltl (n ¼ 14) were obtained from laboratory stock in the

Department of Cell Biology, University Complutense, Ma-

drid. The seven males and seven females showed active

aquatic behavior and were kept in aquariums that repli-

cated their natural conditions, with a 12/12-hour light/

dark cycle. The original research reported here was per-

formed according to the regulations and laws of the Euro-

pean Union (86/609/EEC) and Spain (Royal Decree 1201/

2005) for the care and handling of animals in research.

The animals were anesthetized by immersion in a 0.4

mg/ml solution of tricaine methanesulfonate (MS222,

Sigma Chemical Co., St. Louis, MO) and perfused trans-

cardially with 0.9% NaCl, followed by the fixative MEMFA

(0.1M MOPS [4-morpholinopropanesulphonic acid], 2 mM

EGTA [ethylene glycol tetraacetic acid], 1 mM MgSO4,

3.7% formaldehyde). The brain, eyes, and spinal cord

were dissected out and postfixed �24 hours in the same

fixative solution at 4�C. Subsequently, they were

immersed in a solution of 30% sucrose in 0.1 M phos-

phate buffer (PB; pH 7.4) for 4–6 hours at 4�C until they

sank. For sectioning on a freezing microtome (Thermo

Scientific, Pittsburgh, PA; Microm HM 450) the tissue was

embedded in a solution of 20% gelatin with 30% sucrose

in PB, and stored overnight in formaldehyde diluted 1:10

in 30% sucrose in PB at 4�C. For sectioning on a cryostat

(Leica CM1850), tissue was preembedded at 37�C over-

night in 3.5% gelatin with 30% sucrose in PB, and subse-

quently embedded in 7.5% gelatin with 30% sucrose in PB

and quickly frozen with dry ice. In both cases, brains were

sectioned at 25–40 lm in the transverse, sagittal, or hori-

zontal planes and collected in PB or on SuperFrost slides

(Thermo Scientific Menzel Gl€aser Superfrost plus) in four

series of adjacent sections. In some cases the slides

were treated with citrate buffer (10 mM sodium citrate

dehydrate, Sigma Chemical), pH 6, for antigen retrieval

for a period of 25 minutes at 70�C, followed by 25

minutes at room temperature, and then rinsed again with

PB just before the immunohistochemical procedure.

ImmunohistochemistryImmunohistofluorescence procedures were conducted

for different primary antibodies, all of which were diluted

Joven et al.

2090 The Journal of Comparative Neurology |Research in Systems Neuroscience

in 5–10% normal goat serum in PB with 0.1% Triton X-100

(Sigma) and 2% bovine serum albumin (BSA, Sigma). Dif-

ferent protocols were carried out on free-floating sec-

tions, with incubation in the primary antibodies for 72

hours at 4�C, or for 16–24 hours at room temperature in

the antigen retrieval pretreated slides. The dilution of

each primary antibody used is detailed in Table 1.

Single-staining protocols for the detection of Pax6,

Pax7, and Pax3 were carried out on the free-floating sec-

tions or on the antigen retrieval pretreated slides as fol-

lows: 1) Incubation for 72 hours at 4�C (free-floating sec-

tions) or 16–24 hours at room temperature (slides) in the

dilution of each primary serum (see Table 1) in PB with

0.1% Triton X-100. 2) According to the species in which

the primary antibody was raised, the second incubations

were conducted with the appropriately labeled secondary

antibody diluted 1:500 for 90 minutes at room tempera-

ture: Alexa 594-conjugated goat anti-rabbit (red fluores-

cence; Molecular Probes, Eugene, OR; catalog reference:

A11037), Alexa 488-conjugated goat anti-mouse (green

fluorescence; Molecular Probes; catalog reference:

A21042).

For brightfield immunohistochemistry, free-floating

sections were rinsed twice in PB, treated with 1% H2O2 in

PB for 20 minutes to reduce endogenous peroxidase ac-

tivity, rinsed again three times in PB, incubated in the pri-

mary antibody dilution (mouse anti-Pax6 or mouse anti-

Pax7) with 0.025% Triton in PB, revealed with biotinylated

horse anti-mouse (1:100; Vector, Burlingame, CA; catalog

reference: BA-2000), rinsed three times in PB, and visual-

ized by the ABC-DAB kit method (Vector, SK4100).

To study the relative distribution of two proteins in the

same sections, the two-step protocol for immunohisto-

fluorescence was used, with cocktails of pairs of primary

TABLE 1.

List of Primary Antibodies

Name Immunogen Commercial supplier MW (kDa) Dilution

CB E. coli-produced recombinant ratcalbindin D-28k

Monoclonal mouse anti-calbindin D-28k;Swant, Bellinzona, Switzerland; Cat.No. 300

28 1:500

CB E. coli-produced recombinant ratcalbindin D-28k

Polyclonal rabbit anti-calbindin D-28k;Swant, Bellinzona, Switzerland; Cat.No. CB-38a

28 1:500

ChAT Human placental cholineacetyltransferase

Polyclonal goat anti-ChAT; Chemicon,Temecula, CA; Cat. No. AB144P

68 1:100

CR E. coli-produced recombinant humancalretinin

Polyclonal rabbit anti-calretinin; Swant,Bellinzona, Switzerland; Cat. No. 7699/4

29 1:1,000

GABA c-Aminobutyric acid (GABA) conjugatedto BSA

Polyclonal rabbit anti-c-aminobutyric acid;Sigma, St. Louis, MO; Cat. No. A2052

0.0103 1:3,000

Nkx2.1 Amino acids 110–122 from theamino terminus

Polyclonal rabbit anti-TTF; BiopatImmunotechnologies, Caserta, Italy;Cat. No. PA 0100

42-37 1:500

Nkx2.2 E. coli-derived recombinant chickNKX2.2NK2 transcription factorrelated

Monoclonal mouse anti-Nkx2.2; DevelopmentalStudies Hybridoma Bank, Iowa City, IA;Cat. No. 74.5A5

30 1:500

NOS Recombinant rat NOS Polyclonal sheep-anti-NOS K205 antibody;Dr. P.C. Emson, Babraham Institute

155 1:20,000

PAX3 E. coli-derived recombinant quailPAX3. aa 298-481 of theC-terminal region of the quail Pax3

Monoclonal mouse anti-Pax3; DevelopmentalStudies Hybridoma Bank, Iowa City, IA;Cat. No. PAX3

42 1:250

PAX6 E. coli-derived recombinant chick PAX6.aa 1–223 of the chick Pax6

Monoclonal mouse anti-Pax6; DevelopmentalStudies Hybridoma Bank, Iowa City, IA; Cat.No. PAX6

46 1:250

PAX6 Peptide sequence: QVPGSEPDMS-QYWPRLQ of the C-terminus of themouse PAX6 protein

Polyclonal rabbit anti-Pax6; Covance, CA;Cat. No. PBR-278

46 1:300

PAX7 E. coli-derived recombinant chick PAX7.aa 352-523 of the chick Pax7

Monoclonal mouse anti-Pax7; DevelopmentalStudies Hybridoma Bank, Iowa City, IA;Cat. No. PAX7

55 1:500

TH Protein purified from ratpheochromocytoma

Polyclonal rabbit anti-TH; ChemiconInternational, Temecula, CA;Cat. No. AB152

62 1:1,000

TH TH purified from rat PC12 cells Monoclonal mouse anti-TH; ImmunoStar,Hudson, WI; Cat.ue No. 22941

62 1:1,000

5-HT Serotonin conjugated to BSA withparaformaldehyde

Polyclonal rabbit anti-5-HT; Incstar,Stillwater, MN; Cat. No: 20080

0.176 1:1,000



antibodies (always developed in different species), at the

same dilutions and conditions specified in Table 1.

According to the species in which the primary antibody

was raised, the second incubations were conducted with

the appropriate fluorescent-labeled secondary antibody

cocktails diluted in PB for 90 minutes at room tempera-

ture: Alexa 594-conjugated goat anti-rabbit (1:500), Alexa

488-conjugated goat anti-mouse (1:500), Alexa 594-con-

jugated donkey anti-goat (1:500; Molecular Probes; cata-

log reference: A11058), Alexa 594-conjugated chicken

anti-rabbit (1:500; Molecular Probes; catalog reference:

A21442), fluorescein-conjugated rabbit anti-sheep

(1:500; Vector; catalog reference: FI-6000), or biotinyl-

ated horse anti-mouse (1:100; Vector; catalog reference:

BA-2000), the latter followed by 90 minutes incubation at

room temperature with AMCA-conjugated streptavidin

complex (1:500; Vector; catalog reference: SA-5008).

Except for those cases in which AMCA-conjugated strep-

tavidin was used, sections were stained with the nuclear

marker H€oechst (Sigma-Aldrich, St. Louis, MO; H€oechst

33258) to facilitate interpretation of the results. In all

cases, after being rinsed the sections were mounted on

glass slides and coverslipped with Vectashield mounting

medium (Vector Laboratories; catalog number: H1000).

Western blot analysisTwo animals were anesthetized in MS222 and the

brains were quickly removed and mechanically homoge-

nized in an equal volume of cold buffer (5 mM EDTA, 20

mM Tris, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Nonidet

P40; Roche, Mannheim, Germany) supplemented with

protease and phosphatase inhibitors (50 lg/ml phenyl-

methyl-sulfonyl fluoride, 10 lg/ml aprotinin, 25 lg/ml

leupeptin, and 100 nM orthovanadate; all from Sigma).

Samples of the supernatants, each containing 50 lg of

protein, were applied in each lane of a 12% polyacryl-

amide gel (#161-0801, Bio-Rad, Hercules, CA) and sepa-

rated by sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) with a Mini-Protean system

(Bio-Rad). Samples of a rat brain and molecular weight

standards (Precision Plus Protein Dual color Standards,

Bio-Rad) were run in other lanes. The separated samples

in the gel were transferred to a nitrocellulose membrane

(Bio-Rad). Nonspecific binding sites were blocked by incu-

bation overnight in Tris-HCl buffer (TBS) containing 0.1%

Tween-20 (TBST) and 5% nonfat milk, at 4�C. The blots

were then incubated for 24 hours at 4�C in primary anti-

body dilution. After rinsing in TBS, the blots were incu-

bated in horseradish peroxidase-coupled secondary goat

anti-mouse or goat anti-rabbit antisera (Jackson Immu-

noResearch, West Grove, PA; diluted 1:15,000) for 2

hours at room temperature. Immunoreactive bands were

detected using an enhanced chemiluminescence system

(Super Signal West Pico Chemiluminescent Substrate,

Pierce, Thermo Scientific, Rockford, IL). Photographs

were taken after applying an autoradiographic film to the

membrane, in darkness for 1–4 minutes.

Controls and specificity of the antibodiesGeneral controls for the immunohistochemical reaction

included: 1) western blot (see the previous section); 2)

staining some selected sections with preimmune mouse,

rabbit, or goat serum instead of the primary antibody; 3)

controls in which either the primary and/or the second-

ary antibody was omitted. In all these negative controls

the immunostaining was eliminated. In addition, all the

antibodies used have been tested, under identical condi-

tions, in tissues devoid of antigen (rat brain slices at lev-

els revealing no expression), as negative control, and in

tissues positive for the antigen (rat brain slices at levels

expressing the antigen). In all cases the controls were

satisfactory. The specificity of the antibodies used has

been assessed by the commercial companies (Table 1),

and, in addition, immunoblotting was conducted (see

above). The western blots of brain extract of P. waltl

showed that all antibodies used labeled a single band,

which corresponded well (with minor variations) to the

bands labeled in the rat lanes (Fig. 1). In the cases of

Pax6, CB, and TH, monoclonal and polyclonal antibodies

were used (Table 1) with fully comparable results in the

pattern of immunostaining.

The specificity of the antibodies against CB and CR

was assessed by the commercial supplier (Swant, Bellin-

zona, Switzerland; see Table 1). Particularly in anurans

(Xenopus laevis and Rana perezi) and urodeles (P. waltl

and Ambystoma tigrinum), the specificity was reported in

a previous study (Morona and Gonz�alez, 2008), in which

the same pattern of staining was observed, and the west-

ern blot analysis also revealed a single band at the same

appropriate molecular weight as that of the major product

detected in rat brain extract (about 29 kDa; see Morona

and Gonz�alez, 2008).

The ChAT antiserum used was raised against human

placental ChAT, and its specificity was analyzed by immu-

noblot (and western blot) performed in rat, guinea pig,

and rabbit in which a band in the range of 68–70 kDa was

always observed (see manufacturer’s data sheet). In addi-

tion, western blot analysis of protein extracts from brains

of dogfish, sturgeon, trout, and diverse amphibians,

including P. waltl, showed the presence of similar bands

of 68–72 kDa (Anad�on et al., 2000; Morona and Gonz�alez,

2009). The band observed in the western blot from Pleu-

rodeles (see Morona and Gonz�alez, 2009) corresponds to

that of the rat brain extract at the expected molecular

weight in relation to the published nucleotide sequence

for rat ChAT (NCBI accession number XM_001061520).

Joven et al.


Furthermore, the staining with this antibody colocalizes

with the mRNA distribution of the same enzyme by in situ

hybridization histochemistry (Oh et al., 1992).

The GABA antiserum was developed in rabbit using

GABA-BSA as the immunogen. The antibody was isolated

by immunospecific methods of purification and antigen-

specific affinity isolation, with the removal of essentially

all rabbit serum proteins, including immunoglobulins, that

did not specifically bind to GABA. Rabbit anti-GABA shows

positive binding with GABA in a dot blot assay, and nega-

tive binding with BSA (Sigma data sheet). In amphibians

the pattern of staining obtained with this antibody was

similar to that obtained with in situ hybridization for

GAD67 gene, which encodes an isoform of glutamic acid

decarboxylase, an enzyme involved in the synthesis of

GABA (compare results in Brox et al., 2003, and Moreno

and Gonz�alez, 2007). The pattern of staining observed

with this antibody was identical to that described for P.

waltl in previous immunohistochemical studies (Naujoks-

Manteuffel et al., 1994).

Thyroid transcription factor 1 (known as TTF-1 or

Nkx2.1) is a homeodomain containing transcription factor

expressed in restricted regions of the brain (Lazzaro

et al., 1991). It was recently characterized by western

blot in brain tissue from the turtle Pseudemys scripta and

the anuran Xenopus laevis with comparable results

Figure 1. Identification by western blots of protein bands recognized in Pleurodeles waltl for the rabbit anti-Nkx2.1 antiserum (a), mouse

anti-Nkx2.2 antibody (b), goat anti-NOS antiserum (c), mouse anti-Pax3 antibody (d), mouse anti-Pax6 antibody (e), rabbit anti-Pax6 antise-

rum (f), mouse anti-Pax7 antibody (g), mouse anti-TH antibody (h), and rabbit anti-TH antiserum (i). The bands seen in each of the lanes

corresponding to the urodele brain extracts are compared with the corresponding band stained for rat brain extracts. The expected molec-

ular weight is indicated for each transcription factor or enzyme, and the molecular weight standard is represented on the right of each

photograph.



(Moreno et al., 2010, 2012a). Western blot analysis with

the anti-Nkx2.1 serum also detected a single band for P.

waltl at the same molecular weight as that of the major

product detected in rat brain extract (about 42 kDa; Fig.

1a), corresponding to the TTF-1 protein according to the

nucleotide sequence for rat Nkx2.1 (NCBI accession

numbers NM_013093, XM_001079296, and

XM_216720). Finally, staining with this antiserum in all

species studied colocalizes with the mRNA distribution of

the same protein using in situ hybridization (Lazzaro

et al., 1991; Marın et al., 2000; Bachy and R�etaux, 2006;

Garcıa-L�opez et al., 2008).

The anti-Nkx2.2 monoclonal antibody was developed by

Dr. T.M. Jessell (Columbia University, New York, NY). The

DNA region of NK2 transcription factor in chicks was

cloned by polymerase chain reaction (PCR) into the E. coli

expression vector. Recombinant protein was expressed

and purified. The monoclonal antibody was generated by

immunization of mice with the recombinant protein. It has

been tested in mice, rats, chicks, and humans (see Devel-

opmental Studies Hybridoma Bank, Iowa City, IA, data-

sheet). The specificity of the Nkx2.2 antibody has been con-

firmed by an absence of labeling in Nkx2.2�/� mice (Cai

et al., 2010). Western blot analysis with the anti-Nkx2.2

antibody detected a single band at the same molecular

weight as that of the major product detected in rat brain

extract (Fig. 1b). This same antibody has also been tested

by western blot with chicken brain extract: two bands of 43

kDa and 28 kDa were obtained, and two isoforms were sug-

gested (Ferr�an et al., 2009). The band observed in P. waltl

corresponds well to the band of 28 kDa observed in chicks

and the turtle Pseudemys scripta (Moreno et al., 2012b).

The polyclonal NOS antiserum was raised in sheep

against recombinant rat NOS, and its specificity has been

previously described (Herbison et al., 1996). Antibody spec-

ificity against neuronal NOS was assessed by liquid phase

adsorption experiments (Simonian and Herbison, 1996). In

addition, the specificity of the antiserum for detection of

NOS in P. waltl and many other species was found to reveal

a staining pattern identical to that previously reported to

match the NADPH-diaphorase histochemical reaction

(Gonz�alez et al., 1996; Smeets et al., 1997; Moreno et al.,

2002). This antiserum recognizes a single band of about

155 kDa, similar to the band labeled in the lane of the rat

brain extract (Fig. 1c). This coincides with the expected mo-

lecular weight of the neuronal NOS (NCBI accession num-

ber NM_052799 XM_346438) in rats.

The anti-Pax3 antibody was developed by Dr. C.P.

Ordahl (Developmental Studies Hybridoma Bank). The

DNA region corresponding to amino acids 298–481 of

the C-terminal region of quail Pax3 was cloned by PCR

into the E. coli expression vector. In western blots of

chicken brain tissue, the Pax3 antibody detects a single

band at the expected molecular weight, and the spatio-

temporal distribution obtained by immunohistochemistry

coincides with the mRNA expression pattern (Williams

and Ordahl, 1994; Ferr�an et al., 2009). In the western

blot performed for the present analysis, the bands

obtained in the P. waltl lanes corresponded with those for

rat brain extract (Fig. 1d).

The mouse anti-Pax6 antibody (Kawakami et al., 1997)

was developed by Dr. A. Kawakami (Division of Biological

Science, University of Tokyo, Japan). The DNA region cor-

responding to amino acids 1–223 of chick Pax6 was

cloned by PCR into the E. coli expression vector.

Recombinant protein was expressed and purified. The

monoclonal antibody was generated by immunization of

mice with the recombinant protein. It has been tested in

turtles, chickens, mice, and rats (see Developmental

Studies Hybridoma Bank data sheet; Moreno et al.,

2010). In Pleurodeles, the western blot analysis with the

Pax6 antibody detected a single band at the same molec-

ular weight as the major product detected in rat brain

extract (about 46 kDa; Fig. 1e), and it also coincided with

the band observed in Pseudemys (Moreno et al., 2010)

brain extracts. In Pleurodeles we had better staining

results (i.e., more intensity and less background) when

mouse anti-Pax6 (DSHB) was used than with the antibody

raised in rabbit by Covance (Denver, PA; see below). The

Pax6 antibody detects two major and two minor products

in western blots of chicken brain, suggesting the exis-

tence of Pax6 isoforms (Kawakami et al., 1997) and the

results of the immunoreaction produced essentially the

same topographic localization as the mRNA expression

pattern in the brain (Ferr�an et al., 2009). Comparatively,

in Pleurodeles we observed better staining results (i.e.,

more intensity and less background) when mouse anti-

Pax6 (DSHB) was used than with the antibody raised in

rabbit by Covance (see below).

The Pax6 serum was generated against a sequence

that is highly conserved (see Table 1). The antibody was

subsequently purified on a Protein A column and is useful

in studying brain, neuronal, and olfactory development in

eukaryotes (see Covance data sheet). The western blot

for the brain extract of P. waltl shows a band that corre-

sponded well to that in the rat lane, coincides with the

calculated molecular weight (Fig. 1f), and is similar to

that obtained with the monoclonal anti-Pax6 (Fig. 1e).

However, it should be noted that slight differences were

observed in the bands labeled in the western blots with

the polyclonal and monoclonal antibodies (compare Fig.

1e,f). This could be due to the fact that each antibody rec-

ognizes different domains within the Pax6 protein (see Ta-

ble 1). The results of the immunoreaction produced the

same topographic localization of labeled cells as the

monoclonal antibody.

Joven et al.


The anti-Pax7 antibody (Kawakami et al., 1997) was

developed by Dr. A. Kawakami (Division of Biological Sci-

ence, Nagoya University Graduate School of Science,

Nagoya, Japan). The DNA region corresponding to amino

acids 352–523 of chick Pax7 was cloned by PCR into the

E. coli expression vector. Recombinant protein was

expressed and purified. The monoclonal antibody was

generated by immunization of mice with the recombinant

protein. It has been tested in chicken, mouse, zebrafish,

rat, human, Xenopus, turtle, and axolotl (see Developmen-

tal Studies Hybridoma Bank data sheet; Morona et al.,

2011, 2012b). In western blots of chicken brain tissue

the Pax7 antibody detects three bands and the spatio-

temporal distribution pattern obtained by immunohisto-

chemistry corresponded with the mRNA expression pat-

terns (Ferr�an et al., 2009). In Pleurodeles, the western

blot analysis with the Pax7 antibody detected a single

band at the same molecular weight as the major product

detected in rat brain extract (about 55 kDa; see Fig. 1g)

and also coincides with the band observed in Xenopus

laevis (Morona et al., 2011) and Pseudemys scripta (Mor-

eno et al., 2012b) brain extracts.

The serotonin antiserum was developed in rabbit using

serotonin coupled to BSA with paraformaldehyde as im-

munogen. It was quality control-tested using standard im-

munohistochemical methods by the supplier, who

reported no detectable crossreactivity with tryptamine, 5-

methoxytryptamine, L-tryptophan, 5-hydroxytryptophan,

dopamine, norepinephrine, or adrenaline (see data

sheet). The antiserum demonstrates strongly positive

labeling of rat hypothalamus, raphe nuclei, and spinal

cord using indirect immunofluorescent and biotin/avidin-

horseradish peroxidase (HRP) techniques, and it has

been tested by the supplier in a wide range of inverte-

brate and vertebrate species, including urodeles (see

data sheet). Additionally, the specificity of this antibody

was tested by western blot or preadsortion assays in

lampreys (Villar-Cervi~no et al., 2006; Barreiro-Iglesias,

et al., 2008, 2009), sharks (Carrera et al., 2008), turtles

(Trujillo-Cen�oz et al., 2007), mice (Fortune and Lurie,

2009), and macaques (Hsu and Price, 2009) and the

results in this species have been satisfactory and

included in the JCN Antibody Database.

The specificity of the monoclonal mouse anti-TH anti-

body was corroborated by western blot analysis in rats,

mice, ferrets, cats, and Aplysia (see specification data

sheet; ImmunoStar, Hudson, WI), the turtle Pseudemys

scripta (Moreno et al., 2010), two lungfishes (L�opez et al.,

2012), and diverse amphibians, including P. waltl (Morona

and Gonz�alez, 2009), in which it selectively labels a single

band at �62 kDa. The western blot performed with brain

extracts of P. waltl revealed a comparable single band at

the same molecular weight for the monoclonal and poly-

clonal anti-TH antibodies used (about 62 kDa; Fig. 1h,i),

and the pattern of staining on the sections was identical.

ImagingThe sections were analyzed with an Olympus BX51

microscope equipped for fluorescence with appropriate fil-

ter combinations. Selected sections were photographed

using a digital camera (Olympus DP70). To confirm actual

colocalization of two markers in the same neurons, the

double-labeled samples were studied with a Leica spectral

confocal laser scanning microscope (TCS-SP2). The argon

488-nm and helium/neon lasers were used, respectively,

to excite the green or red fluorophores. Image series were

acquired in steps of 0.8 or 1 lm along the z-axis, and col-

lapsed images were obtained from an average of 10–12

optical sections. Photomicrographs were adjusted for con-

trast and brightness with Adobe PhotoShop CS4 (Adobe

Systems, San Jose, CA) and were mounted on plates using

Canvas 11 (ACS Systems International, Santa Clara, CA).

RESULTS

The antibodies used in the present study resulted in

distinct labeling patterns throughout the brain of Pleuro-

deles waltl that were consistent for all individuals exam-

ined. The distribution of Pax6 and Pax7 cells was largely

segregated, but in some regions extensive codistribution

was noted. In general, Pax6 cells were located in telence-

phalic, diencephalic, and brainstem regions, whereas

Pax7 cells were restricted to the brainstem and part of

the diencephalon. In addition, Pax3 cells were located

only in the caudal diencephalon and upper rhombence-

phalon. With the exceptions noted in the description of

each area, labeling was observed in cells of the mantle

zone, and when labeling was detected in cells of the ven-

tricular zone these cells were always weakly immunoreac-

tive. The combined localization of Pax6 and Pax7 cells

and the cells containing TH, ChAT, NOS, GABA, CB, CR,

Nkx2.1, and Nkx2.2 was a useful tool for identifying the

precise signature of diverse cell groups not distinguish-

able on the basis of cytoarchitecture. Actually, a particu-

lar feature of the CNS of urodeles is the limited cell migra-

tion from the periventricular layer, even in adults, which

probably represents a case of secondary simplification

(see ten Donkelaar, 1998). Thus, in Nissl-stained material

distinct nuclei and boundaries of main brain regions are

not recognizable (Fig. 2a,c). Therefore, the distinct immu-

noreactivity found for Pax6 and Pax7 in specific cell

groups has helped to characterize brain subdivisions

where anatomical landmarks are not evident (Fig. 2b,d),

as well as helping to specify the possible relationships of

Pax-expressing cells with some of the neuronal systems

characterized chemically in urodele brains. In those



double-labeled sections where actual colocalization of

the two markers in the same cells could not be estab-

lished with the normal fluorescence microscopy, the sec-

tions were analyzed under confocal microscopy.

The patterns of labeling for Pax6 and Pax7 are

described below from rostral to caudal. The drawings in

Figures 3 and 4 correspond to a series of transverse and

sagittal sections and are intended to aid in the descrip-

tion. The reduced distribution of Pax3 cells is also shown

in the drawings. The data are further presented as photo-

graphs of series of conventional transverse sections, sin-

gle-labeled for Pax6 or Pax7 (Figs. 5, 6). In addition, some

horizontal single-labeled sections are shown to clarify the

actual location and boundaries of the labeled cell groups

(Fig. 7). We will comment on the codistribution/colocali-

zation of Pax6 and Pax7, and between these and the

other markers used (Figs. 8–10), describing the precise

location and identification of the cells that contain these

transcription factors.

The results were analyzed primarily within the context

of recently proposed subdivisions of the telencephalon

(Moreno and Gonz�alez, 2007) and the neuromeric organi-

zation of the brain, following current models validated for

many vertebrates, including amphibians (prosencephalon:

Puelles and Rubenstein, 1993, 2003; midbrain: Dıaz

et al., 2000; rhombencephalon: Gilland and Baker, 1993;

Marın and Puelles, 1995; Cambronero and Puelles, 2000;

Aroca and Puelles, 2005; Straka et al., 2006). A similar

segmental organization of adult urodele brains was

recently supported by the distribution of calcium-binding

proteins and other neurochemical markers (Morona and

Gonz�alez, 2008, 2009).

ProsencephalonThe early prosencephalon (primary prosencephalon)

divides into the secondary prosencephalon (telencepha-

lon and hypothalamus) and the diencephalon. The

Figure 2. Photomicrographs of a transverse (a,b) and a sagittal (c,d) section showing Nissl staining (a,c) and specific Pax6 (b) and Pax7

(d) immunolabeling. Within the densely crowded periventricular cell layer observed in the Nissl-stained sections, labeling for either Pax6

(in this case revealed with the polyclonal antiserum) or Pax7 allows the recognition of particular cell groups otherwise indistinct, as shown

in the prethalamus (p3) for Pax6 (b) and the pretectum (p1), tectum, and rhombomere 1 (r1) for Pax7 (d). For abbreviations, see list. Scale

bars ¼ 200 lm.

Joven et al.


Figure 3. Diagrams (a–y) of transverse sections through the brain of Pleurodeles waltl (levels indicated in the upper scheme of a lateral

view of the brain) showing the distribution of immunoreactive cells for Pax6, Pax7, and Pax3. For abbreviations, see list. Scale bar ¼ 500

lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]



diencephalon is subdivided into three prosomeres (p1–

p3) identified by morphological constrictions and the

expression domains of regulatory genes (Puelles and

Rubenstein, 1993, 2003). The pretectum forms in p1, the

thalamus in p2, and the prethalamus in p3.

TelencephalonThe large telencephalic hemispheres and the evagi-

nated olfactory bulbs formed at their rostral tip con-

tained abundant Pax6 cells (Figs. 3a–c, 4a,b, 5a, 7a–d).

Numerous Pax6-expressing bulbar neurons were la-

beled in the internal granule cell layer and, to a lesser

extent, around the glomeruli in both the main and

accessory olfactory bulbs. Only weakly labeled cells

were detected in the ventricular zone. Double-labeled

sections revealed that most Pax6 cells contained CR,

and virtually all the catecholaminergic cells located in

the bulbs (TH-positive) represented a subpopulation of

the Pax6 cells, located primarily in the external part of

the granular cell layer (Fig. 8a). Furthermore, the scat-

tered CB-positive neurons found in the bulbs were also

Pax6 cells (Fig. 8b).

Figure 4. Diagrams (a–d) of sagittal sections through the brain of Pleurodeles waltl (levels indicated in the upper scheme of a dorsal view

of the brain) showing the distribution of immunoreactive cells for Pax6, Pax7, and Pax3. For abbreviations, see list. Scale bar ¼ 500 lm.

[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Joven et al.


In the telencephalic hemispheres most Pax6 cells were

found in subpallial regions, distributed in the septum, ba-

sal ganglia, and amygdaloid complex. At rostral levels, a

large population of Pax6 cells occupied the ventral part of

the hemispheres, in the region of nucleus accumbens

(Figs. 3c,d, 4a,b, 5a,b, 7a). The position of nucleus

Figure 5. Photomicrographs of single-stained transverse sections (using the polyclonal antiserum) showing the localization of Pax6 cells in

the telencephalon (a–f, from rostral to caudal), the diencephalon (g–j, from rostral to caudal), the mesencephalic tegmentum (k), the cen-

tral gray in rhombomere 1 (l), and in the inner nuclear and ganglion cell layers of the retina (m). Arrowheads in i (a higher magnification

of the framed area in h) point to superficial labeled cells in the habenula. For abbreviations, see list. Scale bars ¼ 200 lm in a–h,j–l; 100

lm in i; 50 lm in m.



accumbens within the basal ganglia is not well defined in

urodeles and, unlike in anurans, TH staining does not dis-

tinguish a particular dense neuropil that could determine

its boundaries. It has been proposed that nucleus accum-

bens is located in a position slightly lateral and ventral in

the rostral aspect of the hemisphere (Gonz�alez and

Smeets, 1991; Marın et al., 1998a,b). Double-labeling for

TH and Pax6 revealed a conspicuous group of Pax6 cells

in close apposition to the TH-labeled neuropil in the ros-

tral ventromedial aspect of the hemisphere (Fig. 8d), and

these cells extended medioventrally, accompanying an

NOS-positive cell population that occupied distinct septal

regions more caudally (Fig. 8c). Thus, in rostral levels of

the septum, Pax6 cells were detected in the dorsal

Figure 6. Photomicrographs of single-stained transverse sections showing the localization of Pax7 cells in the diencephalon (a–c, from

rostral to caudal), the mesencephalon and isthmic (r0) region (d–f, from rostral to caudal), the large rhombomere 1 (r1) and cerebellum

(g,h), and the ventricular zone of the rhombencephalic ventral zone of the alar plate at mid and caudal levels (i,j, respectively). Arrowhead

in c points to scattered cells that extend from p3 into the mammillary region. Arrowheads in h point to scattered labeled cells in the cere-

bellum and in i and j to the extent of the ventricular expression. For abbreviations, see list. Scale bars ¼ 200 lm.

Joven et al.


Figure 7. Photomicrographs of horizontal (a–o) and transverse (p) sections single-stained for either Pax6 or Pax7 (indicated on each pho-

tomicrograph); in all horizontal sections rostral is to the left. The monoclonal (a–h) and polyclonal (m,p) anti-Pax6 were used. a–d: Sections

through the telencephalon (from ventral to dorsal) showing the distribution of Pax6 cells in the olfactory bulbs and the telencephalic hemi-

spheres. e–h: Sections through the diencephalon (from ventral to dorsal) showing the abundant distribution of Pax6 cells in the prethala-

mus (p3) and pretectum (p1), with only few cells in the dorsal habenula (p2). i,j: Sections through the dorsal pretectum (p1) and optic

tectum showing the distribution of Pax7 cells. Section i is adjacent to section g. The higher magnification of photograph j illustrates the

localization of the Pax7 cells in the deep cell layer at caudal tectal levels. k: Section through the hypophysis showing the Pax7 cells in the

intermediate lobe. l: Section through the basal part of p3, the mammillary region, and the r1 Pax7 cell populations. m,n: Adjacent sections

showing the distinct Pax6 and Pax7 staining in the cerebellum and dorsal part of r1. o: Higher magnification of the dorsal Pax7 cell popu-

lation in r1. p: Transverse section showing the right side of the spinal cord and Pax6 cells at intermediate levels of the ventral horn. For

abbreviations, see list. Scale bars ¼ 200 lm in a–e,g,i,k–n; 100 lm in f,h,j; 50 lm in o,p.



septum, and more caudally this cell population increased

and extended to the diagonal band of Broca (Figs. 3e,f,

4c,d, 5b–d, 7b). In addition, Pax6 cells were found in the

lateral septum where some expression was noted in the

ventricular zone (Fig. 5b). Double-labeling experiments

demonstrated that the septal Pax6 cells were codistrib-

uted with NOS-positive neurons, but actual colocalization

in the same neurons was not found.

The most abundant Pax6 cell population in the telen-

cephalon was localized in the ventrolateral hemisphere

(Figs. 3d,e, 4a,b, 5b,c, 7a–c). These conspicuously la-

beled cells formed a subpopulation of the thick periven-

tricular cell layer of the striatum and were located medial

to the TH-positive fibers that occupied the striatal neuro-

pil in the double-labeled sections (Fig. 8e). Most striatal

Pax6 cells were found in the deep portion of the cell layer

and included weakly labeled cells in the ventricular zone,

primarily at rostral levels (Figs. 5b,c, 8d–f). Of note, dou-

ble labeling for TH and, most important, NOS revealed

that the Pax6 cells extended dorsally into a region charac-

terized by low TH innervation and the presence of NOS-

positive cells and fibers (Fig. 8e,f). This region was identi-

fied as the urodele counterpart of the anuran lateral amyg-

dala, in the ventral pallium (see below; Moreno and

Gonz�alez, 2007). Finally, with regard to the basal ganglia in

urodeles, the presence of pallidal regions has been demon-

strated by the expression of the gene Nkx2.1 (Gonz�alez

et al., 2002; Moreno and Gonz�alez, 2007), and double

labeling for Nkx2.1 and Pax6 demonstrated that these sub-

stances do not codistribute. In addition to the pallidum,

the bed nucleus of the stria terminalis, which expresses

Nkx2.1 in amphibians (Moreno et al., 2012a), and the

region of the basal cholinergic system (revealed with ChAT

immunohistochemistry) also lacked Pax6 cells (Fig. 8g,h).

Within the telencephalon of urodeles, the amygdala

pars medialis and pars lateralis (see Northcutt and Kicli-

ter, 1980) have been recently reinterpreted in P. waltl on

the basis of their neurochemical features and their con-

nections with the olfactory and vomeronasal systems, the

hypothalamus, and the brainstem. Counterparts of the

lateral, medial, and central components of the amygdala

in anurans have also been proposed (Moreno and

Gonz�alez, 2007).

As noted, the lateral amygdala is here considered a

part of the ventral pallium that contains abundant NOS-

positive cells and fibers and, as shown in anurans, is the

only pallial component of the amygdaloid complex (see

Moreno and Gonz�alez, 2006, 2007). The lateral amygdala

in Pleurodeles contained abundant Pax6 cells that were a

dorsal continuation of the striatal cells (Figs. 3d–h, 8f,g),

which extended caudally above the other components of

the amygdaloid complex, up to the caudal tip of the telen-

cephalic hemisphere (Figs. 3f–i, 8g).

The central amygdala of urodeles has been proposed

to reside in the caudal striatal region (within the limits of

the former pars lateralis of the amygdala) and to be char-

acterized by its striatal nature, connections, and large

NOS-positive neurons (Marın et al., 1998a; Moreno and

Gonz�alez, 2007). Pax6 cells were numerous in the central

amygdala (Figs. 3f,g, 4b, 5d,e, 8g,h), and combined immu-

nohistochemistry for Pax6 with Nkx2.1 and ChAT defined

the boundaries of the striatal regions (striatum proper

and central amygdala) (Fig. 8g,h).

The third component of the amygdala, the medial

amygdala, which was defined by its relationship with the

vomeronasal fibers arising in the accessory bulb and by

its subpallial nature (Moreno and Gonz�alez, 2008), was

virtually devoid of Pax6 cells (Figs. 3h,i, 5e,f, 8g). In addi-

tion, the preoptic area, in the proximity of the medial

amygdala, is currently considered a subpallial component

of the telencephalon (see Medina, 2008; Moreno and

Gonz�alez, 2011) that expresses Nkx2.1 in both its com-

missural and main parts (Fig. 8g), and it did not show Pax-

labeled cells (Figs. 3g–i, 4b–d).

HypothalamusAccording to the prosomeric model, the hypothala-

mus is the part of the secondary prosencephalon that is

topologically ventral to the telencephalic preoptic area

and rostral to the diencephalon. Two alar regions

(supraoptoparaventricular and suprachiasmatic) and

two basal regions (tuberal and mammillary) are gener-

ally recognized anatomically (reviewed in Medina, 2008;

Moreno and Gonz�alez, 2011). Pax6 expression was not

detected in any hypothalamic region, and only a few

Pax7 cells were seen scattered in the ventrocaudal

region of the basal mammillary region (Figs. 3o, 4b).

These cells were identified by double labeling with 5-

HT, which labeled the cerebrospinal contacting cells of

the nucleus of the periventricular organ (Fig. 8i,i’), and

TH, which labeled abundant catecholaminergic neurons

in this mammillary region (Fig. 8j). These scattered cells

appeared to be continuous with labeled cells in the ba-

sal part of the rostral diencephalon (p3) and could cor-

respond to migrated neurons into the mammillary

region (see below). Of note, a conspicuous Pax7 cell

population was observed in the hypophysis (Figs. 3p,

4b–d, 6f, 9g,s). These cells belonged to the intermedi-

ate lobe, as their location matched that of the terminal

field formed by TH fibers (Fig. 9h). In addition, some

weakly labeled Pax6 cells were often noted in the ante-

rior hypophyseal lobe.

DiencephalonThe distinct distribution of different Pax-labeled cells in

the diencephalon allowed easy identification of the three

Joven et al.


prosomeres (p1–p3) that constitute the diencephalon,

clarifying their boundaries with the hypothalamus (ros-

trally) and the mesencephalon (caudally). Because of flex-

ure in the longitudinal axis of the brain, these three proso-

meres in Pleurodeles are bent such that in conventional

transverse sections they are cut obliquely or almost

Figure 8



horizontally, whereas in horizontal sections they are cut

transversely (see Figs. 3, 4).

The prethalamic eminence is currently considered to

occur dorsally within the rostral prosomere 3 (p3). The

rest of the alar region of p3 comprises the prethalamus

(formerly called the ventral thalamus). The most striking

population of Pax6 cells in Pleurodeles was observed in

the alar region of p3 (Figs. 3j–m, 4b,c, 5g,h, 7e,f). A few

scattered cells in the ventral part of the prethalamic emi-

nence showed Pax6 expression, and also the ventricular

zone of this dorsal territory was weakly labeled (Fig. 7e).

The rostral part of the prethalamus contained a conspicu-

ous population of Pax6 cells that were intensely immuno-

reactive. The boundary between these cells in the alar

region of p3 and the topologically rostral suprachiasmatic

hypothalamic region was clearly defined by the combina-

tion of Pax6 and Nkx2.2 immunolabeling (Fig. 8o). The

Pax6 cells were closely packed together, and ventricular

expression was difficult to discern, although moderate

expression was apparent in some cases (Fig. 5g,h). The

Pax6 cells extended caudally as a band close to the ven-

tricle. Near the boundary with the caudally located p2, a

group of cells was seen to detach from the ventricle (Figs.

3j–m, 4c, 5g,h, 8l,r). Notably, at caudal levels of p3, close

to p2, the Pax6 cells located more peripherally showed

the most intense Pax6 reactivity (Figs. 5h, 7f). Towards

the basal part of p3, the Pax6 cells separated increasingly

from the ventricle, and some were even seen to

extend into the basal region adjacent to the hypothala-

mus (Fig. 5j).

Some Pax7 cells also formed a population within p3

(Figs. 3m, 4b–d, 6b,c), primarily ventral to the Pax6 cell.

Actually, the most dorsal Pax7 cells were likely located in

the ventral alar portion of p3, but most of the labeled cells

were seen in the basal part of this prosomere. Thus, in

conventional horizontal sections, which through p3 are

practically transverse sections, the Pax6 cells appear

clearly in the alar part of the segment. Close to the basal

border, there is an area free of Pax6 but containing

Nkx2.2 cells (Fig. 8l,p). Pax7 cells occupy the basal part

of this area, without actual overlapping with the Nkx2.2

cells (compare photomicrographs in Fig. 8k,l). The dorsal

part of the basal territory occupied by Pax7 cells formed

a boundary with the adjacent dorsal part of the basal

hypothalamus, as revealed by double labeling for Pax7

and 5-HT (Fig. 8m). In addition, the ventralmost extent of

the Pax7 cells was confirmed in the basal region of p3 by

double labeling some cells for Pax7 and Nkx2.1 (Fig. 8n);

the location of Pax7 cells in the region of the posterior tu-

bercle (basal p3) was also confirmed by double labeling

for Pax7 and TH, which labeled large catecholaminergic

neurons in this region (Fig. 8q).

The large diencephalic p2, which contains the habenula

in its dorsal part and the thalamus (formerly termed dor-

sal thalamus) in its alar portion, was practically devoid of

Pax-labeled cells. Only scattered Pax6 and Pax7 cells

were seen to border the dorsal part of the habenula and

reach the habenular commissure (Figs. 3j, 5h,i, 7h). Most

of these cells were double-labeled for Pax6 and Pax7.

Double labeling with CB and ChAT revealed the absence

of Pax cells in the thalamus, served to identify Pax cells in

the dorsal habenula, which is strongly positive for CB and

ChAT in Pleurodeles, and revealed the presence of the

Pax cells among the fibers that cross in the habenular

commissure (Fig. 8r,s). It should be noted that the epiphy-

sis also labeled for Pax6 (Figs. 3j,k, 4c,d, 8r) at these

Figure 8. Photomicrographs of double-labeled transverse (a–k,m–s) and horizontal (l) sections through the forebrain. The detected mole-

cules are indicated on each photomicrograph. a,b: Pax6 labeling of granule cells in the olfactory bulbs, showing that those most closely

located to the glomeruli are catecholaminergic cells (TH-immunoreactive) and that some of them contain CB; arrowheads point to some

double-labeled cells. c–h: Localization of the Pax6 cells in the telencephalic hemisphere, from rostral to caudal levels, showing their distri-

bution relative to that of the NOS-positive cells (c,f), the terminal fields formed by TH-positive fibers in the region of the nucleus accum-

bens and the striatum (d,e), and in relation to the Nkx2.1 cells in the ventromedial region of the bed nucleus of the stria terminalis (g)

and the cholinergic cells of the basal telencephalon (h). i,j: The most rostrally located cells expressing Pax7 are scattered in the mammil-

lary region, identified in relation to the nucleus of the periventricular organ labeled for 5-HT around the lateral recess of the infundibular

ventricle (i and the higher magnification in i’), and around TH-labeled cells (j). k: Pax6 and Pax7 cells occupy the pretectum (p1) defining

the three main domains, whereas in p3 both groups of labeled cells are segregated. l: Horizontal section at the level marked in k, showing

the dorsorostral labeling of p3 with Pax6, and the ventrocaudal labeling for Pax7. m–o: The basal Pax7 cell population of p3 forms a

boundary with the caudal hypothalamus, as seen with double-labeling for 5-HT (m), and Nkx2.1 (n), whereas the most densely labeled

Pax6 cell group in the alar p3 borders the suprachiasmatic nucleus, labeled for Nkx2.2 (o). p: The boundary between p3 and p2 is high-

lighted by double-labeling for Nkx2.2, and the gap free of Pax6 cells in p3 is seen filled with Nkx2.2 cells (asterisks in l,p). q: Pax7 cells in

the basal p3 are located lateral to the TH cells in this segment. r: Pax6 labeling with CB highlights the boundary between the thalamus

and the prethalamus and shows Pax6 cells in the epiphysis. s: the ChAT-positive cells of the dorsal habenula are traversed by fibers of

the habenular commissure where Pax6 cells are scattered. The monoclonal (a,b,h,r,s) or polyclonal (c–g,l–p) anti-Pax6 antibody was used

according to the requirements of the combinations. A magenta-green version of this figure is provided as Supporting Figure 1. For abbrevi-

ations, see list. Scale bars ¼ 100 lm in a,b,l,n,o,p,s; 200 lm in c–h,k,m,q,r; 50 lm in i,j.

Joven et al.


Figure 9



levels, whereas the large paraphysis that extends ros-

trally between the two telencephalic hemispheres

showed intense Pax7 immunoreactivity (Figs. 3f–i, 9i).

Within the caudal diencephalic prosomere p1, abun-

dant codistribution of cells labeled for Pax6, Pax7, and

Pax3 facilitated identification of the three main pretectal

(alar p1) domains: commissural, juxtacommissural, and

precommissural (Figs. 3k–n, 4b–d, 5j, 6a–c, 7g–I, 8k,m,

9a–f). Moderate immunoreactivity for the three Pax

genes was found in the roof plate, particularly in the sub-

commissural organ where Pax7 labeling was the most

intense (Figs. 6a,b, 7g,h, 9a–e). In the commissural do-

main, the three Pax factors were widely codistributed

(Fig. 9a,e). In adult specimens the commissural domain

occupied most of the dorsal alar plate, and different ra-

dial strata were observable. Pax6 cells were distributed

in the periventricular and outer intermediate strata,

whereas Pax7 cells were more abundant in the deep in-

termediate stratum and part of the outer intermediate

stratum (Fig. 9a–e). The juxtacommissural domain

showed more or less homogeneous distribution of Pax6

and Pax7 cells (Fig. 9d–f). The precommissural domain

of the pretectum was practically devoid of Pax-labeled

cells (Fig. 9e,f), although weakly labeled Pax3 cells were

present from ventricular to deep intermediate strata,

but absent in the most outer stratum (Fig. 9c). The cor-

rect localization of the different Pax-labeled cell popula-

tions in the pretectum was assessed by immunohisto-

chemistry for other markers of this region, such as CB,

CR, GABA, and TH. In particular, a relatively large popu-

lation of double Pax7/GABA cells was found in the juxta-

commissural domain (Fig. 9b). The TH commissural pre-

tectal neurons expressed Pax7 (Fig. 9d). Finally, in the

basal part of p1 only a band of Pax6 cells was detected

in the caudal portion and continued caudally in the mes-

encephalic tegmentum (Fig. 9f).

MesencephalonThe mesencephalon is considered here as a single seg-

ment that can be divided into longitudinal bands (dorsal,

lateral, basal, and medial) and cellular strata. Rostrally,

the pretectomesencephalic boundary passes through the

posterior commissure, whereas the transverse isthmome-

sencephalic boundary passes behind the oculomotor nu-

cleus. Within the alar mesencephalon, Pax7 cells clearly

occupied the dorsal part corresponding to the optic tec-

tum, with only a few cells extending into the torus semi-

circularis. In turn, Pax6 cells were located in a concise

band along the dorsal tegmentum of the mesencephalon

(Figs. 3m–r, 4b–d, 5k, 6d–g).

Within the optic tectum of Pleurodeles, a thick

periventricular cell layer is covered by a superficial

fiber zone formed by axons and dendrites. Many

Pax7 cells are distributed along the tectum and tend

to be more numerous in the deeper zone of the peri-

ventricular cell layer; moderate immunolabeling was

also present in the ventricular zone (Figs. 6d–g, 7i,j).

Only a few Pax3 cells were noted in the dorsal part

of the tectum at rostral levels (Figs. 3m,n, 4d). Dou-

ble labeling for Pax7 and CR served to localize the

tectal boundaries, but colocalization of both markers

in the same tectal neurons was not observed (Fig.

9k). In contrast, double labeling for Pax7 and NOS

(Fig. 9l) or GABA (Fig. 9m) revealed abundant coloc-

alization in Pax7 tectal cells.

Pax6 cells in the mesencephalon were observed in the

dorsal portion of the tegmentum in a longitudinal band

that appeared to be a caudal continuation of the Pax6

cells in the basal part of p1. They were clearly separated

from the cholinergic cells of the oculomotor nucleus and

the dopaminergic cells of the ventral tegmental area

located in the ventral (medial) band of the mesencephalic

tegmentum (Fig. 9n,o). These Pax6 cells were located

Figure 9. Photomicrographs of double-labeled transverse sections (only d is sagittal and h is horizontal) through the brain showing the

detected molecules that are indicated on each photomicrograph. a–f: In the pretectal part of p1 Pax7, Pax6 and Pax3 are distributed in the

commissural and juxtacommissural domains, where they are codistributed with cells expressing GABA (b) and TH (d,f). g,h: Pax7 cells are

located in the interpeduncular nucleus at the level of the superior raphe nucleus (g) and in the intermediate lobe of the hypophysis where

the cells intermingle with a profuse TH fiber-labeling (h is a horizontal section). i: Pax7 immunoreactivity in the paraphysis. j: separate labeling

for Pax7 in the dorsal mesencephalon and Pax6 in the tegmentum. k–m: Pax7 in the optic tectal cells colocalizes, in part, with NOS (l) and

GABA (m), arrowheads indicate some double-labeled cells. n–q: the band of Pax6 cells in the mesencephalic tegmentum is located dorsal to

the cholinergic (n), catecholaminergic (o), and CB-positive (p) cells but ventral to the tegmental cells labeled for Nkx2.2 (q). r,r’: Pax7 cells in

the interpeduncular nucleus are mainly separated from the GABA-positive cells, although the confocal analysis reveals some double-labeled

cells (arrowhead in the higher magnification in r’). s: Simultaneous localization of Pax7 cells in the interpeduncular nucleus above the interpe-

duncular neuropil in r1. t: The rostral extent of the Pax7 cells in the interpeduncular reaches the isthmic tegmentum (r0), medial to the troch-

lear nucleus. u,v: A large population of Pax7 cells is located dorsally in the rostral r1, at the level of the rostral raphe nucleus (u) that

extends ventromedially in the caudal part of this segment into the central gray, where Pax6 cells are located (v). The monoclonal (n–p) or pol-

yclonal (a,c,d,f,j,q,v) anti-Pax6 antibody was used according to the requirements of the combinations. A magenta-green version of this figure

is provided as Supporting Figure 2. For abbreviations, see list. Scale bars ¼ 100 lm in a–i,l,r–u; 200 lm in k,j,v; 50 lm in r’.

Joven et al.


close to the CB-positive cells in the basal mesencephalic

band (Fig. 9p) and were observed ventrally adjacent to

the alar–basal border along the mesencephalon, marked

by a longitudinal band of Nkx2.2-positive cells (Fig. 9q).

The caudal tip of this Pax6 cell band ended abruptly at

the border with the isthmic tegmentum (Fig. 10a).

Figure 10. Photomicrographs of double-labeled sagittal (a,b), transverse (c–h,j–o), and horizontal (i) sections showing the molecules indi-

cated on each photomicrograph. a: Distinct localization of Pax7 and Pax6 cells in r1 and the caudal mesencephalon. b,c: A small group of

Pax3 cells is located close to the ventricle at the level of the superior reticular nucleus. d–i: The large population of Pax7 cells in r1 is ros-

trally medial to the isthmic nucleus (d) and continues caudally in the superior reticular nucleus, medial to the laterodorsal tegmental nucleus

(e,f,i) and in the central gray (e,g,h). j: Pax6 cells are seen in the lateral margin of the cerebellum and the central gray, at the level of the

locus coeruleus. k: The Pax7 cells in the central gray are distinct from the GABA cells located in this region. l: Double-labeled cells for Pax6

and calbindin are abundant in the ventral zone of the rostral octavolateral area and in the central gray (yellow cells). m: Pax7 nuclear staining

does not correspond to motor neurons of the facial nucleus. n: In the rostral spinal cord, scattered Pax6 cells are located in the ventral horn,

and Pax7 cells are grouped medially in the dorsal horn. o: The cell nuclei labeled for Pax6 in the ventral horn do not correspond to somato-

motor neurons. The monoclonal (j,l,m,o) or polyclonal (a,b,g,n) anti-Pax6 antibody was used according to the requirements of the combina-

tions. A magenta-green version of this figure is provided as Supporting Figure 3. For abbreviations, see list. Scale bars ¼ 200 lm in a,e,g–j;

100 lm in b,d,f,k,l,n; 50 lm in c,m,o.



IsthmusWe considered this structure to be a single segment,

currently named rhombomere 0 (r0). The isthmus is not

well defined in urodeles, but the distribution of calcium-

binding proteins and other territorial markers indicates

that in Pleurodeles it is a curved neuromere with irregular

boundaries, being thinner medially and dorsally, and grad-

ually thickening ventrally and laterally (Morona and

Gonz�alez, 2009). Its main cell groups are the dorsal isth-

mic nucleus and the ventral trochlear nucleus. Within the

isthmus, only a few Pax7-labeled cells were identified as

rostral continuations of more caudally located groups in

r1. Thus, scattered Pax7 cells enter medial to the cholin-

ergic isthmic nucleus into the caudal alar part of r0 (Fig.

10a,d). In turn, in the ventral tegmentum, close to the

midline, a rostral group of Pax7 cells of the interpeduncu-

lar nucleus was localized medial to the trochlear nucleus

(Fig. 9t).

RhombencephalonWe consider the caudal brainstem to be formed by the

transverse subdivisions of rhombomeres 1–7 (r1–r7) and

the long r8, which is not clearly defined and probably rep-

resents more than one segment (Cambronero and

Puelles, 2000). We evaluated the pattern of Pax immuno-

reactivity under this paradigm, because in urodeles the

segmentation can be inferred by examining the organiza-

tion of the segmentally patterned motor nuclei (Marın

et al., 1997b; Straka et al., 2006; Morona and Gonz�alez,

2009). Double labeling for ChAT was commonly con-

ducted to analyze the precise location of structures la-

beled for the Pax transcription factors. The most numer-

ous Pax-labeled cells were found in r1, a quite large and

complex territory. The extent of this rhombomere in Pleu-

rodeles can be assessed because it contains well-defined

cell groups whose anatomical names have been con-

firmed by neurochemical and hodological studies. Primar-

ily cholinergic, catecholaminergic, GABAergic, nitrergic,

and serotonergic cell groups thus helped to identify the

correct location of the Pax-labeled cells in this rhombo-

mere (Figs. 9r–u, 10e,f,h–l).

Pax7 cells were the most abundant in r1 (Figs. 3p–s,

4b–d, 6g,h, 7l,n,o). In the ventral (basal) part of this seg-

ment, the Pax7 cells in the interpeduncular nucleus

formed a column whose rostral tip was localized in r0

(see above; Figs. 9t, 10a). These cells were separated

from the ventricle and were located above the large neu-

ropil formed by CB- and ChAT-labeled fibers arising from

the habenula (Fig. 9s). At rostral levels of r1, the Pax7

cells of the interpeduncular nucleus were intermingled

with the rostral serotonergic cells of the raphe column,

but no colocalization was found (Fig. 9u). Also close to

the interpeduncular cells, GABAergic cells were seen to

extend primarily in the superior reticular nucleus (Fig. 9r).

In this case, however, analysis under confocal microscopy

revealed a few double-labeled cells (Fig. 9r’). Also in the

r1 basal plate, Pax7 and Pax6 cells were present in the

griseum centrale, up to the border of r2 marked by the

rostral neurons of the trigeminal motor nucleus. The Pax7

cells in this position were located more rostrally than the

Pax6 cells, and in the caudal r1 the Pax7 cells occupied

more lateral positions, leaving the medial part of the gri-

seum centrale for the Pax6 cells (Figs. 5l, 9v, 10g). The

Pax6 and Pax7 cells of the griseum centrale formed popu-

lations that were mostly separate from the GABA-positive

cells, which were located ventromedial to the Pax cells

(Fig. 10k). Notably, in these basal regions of r1 no Pax

labeling was found in the ventricular zone.

Pax7 cells were conspicuous in the dorsal part of r1

(Figs. 6g,h, 7l,n,o, 9r,u,v, 10a,d). In the rostral part of r1,

scattered cells were seen in the superior reticular nucleus

from ventral regions close to the interpeduncular nucleus

and the griseum centrale, to the most dorsal regions,

where a large group of closely packed Pax7 cells was

located mediocaudal to the isthmic nucleus and close to

the ventricle, just rostral and ventral to the cerebellum

(Figs. 6g,h, 7l,n,o, 9r,u,v, 10a,d). This large cell population

constituted the border of the isthmus, but some cells

located more dorsally could extend within r0 (Figs. 7o,

10a). At caudal levels within r1, these Pax7 cells contin-

ued to be numerous in the superior reticular nucleus, just

medial to the cholinergic and nitrergic cells of the latero-

dorsal tegmental nucleus (Fig. 10e,f) and, even more cau-

dally in r1, medial to the noradrenergic (TH-positive) cells

of the locus coeruleus (Fig. 10h,i). Of note, beginning at

these levels of r1, extending into the cerebellum, and con-

tinuing caudally in the hindbrain up to the obex, weak to

moderate Pax7 expression was found in the ventricular

zone of the rhombencephalic alar plate, although the dor-

salmost part of the ventricle lacked this labeling (Figs.

3s–x, 6i,j, 10i).

A small group of Pax3 cells was detected within a re-

stricted zone of the reticular formation of r1 (Figs. 3r,s,

4b, 10b,c). These cells were found close to the ventricle

in the dorsal part of the superior reticular nucleus, as

observed in sections double-labeled for CB, for example

(Fig. 10c).

The cerebellum is an outgrowth of the rostral hindbrain

and is poorly developed in urodeles. It comprises two lat-

eral auriculae and a smaller medial corpus cerebelli. Most

granule cells were Pax6-positive and occupied the auricu-

lae and the caudal part of the vertically oriented cerebel-

lar plate (corpus cerebelli; Figs. 3r,s, 4c,d, 7m, 10b,j).

Comparatively few Pax7 cells were also scattered in the

granule cell layer (Fig. 6h); as previously noted, weak

Joven et al.


labeling for Pax7 was also found in the caudal ventricular

zone of the cerebellum.

Pax-labeled cells were scarce in the rhombencephalon

caudal to r1. Pax7 cells were practically restricted to the

aforementioned ventricular zone of the ventromedial part

of the alar plate, with only a few cells separated from the

ventricle. In turn, Pax6 cells were found dorsoventrally in

the dorsal and ventral zones of the octavolateral area

and, in the ventral zone, many of the cells were double-la-

beled for CB (Fig. 10l). In addition, Pax6 cells were scat-

tered in the reticular formation, some of them among the

branchiomotor neurons, primarily of the trigeminal and fa-

cial nuclei, but double labeling for ChAT revealed that the

Pax6 cells were not motoneurons (Fig. 10m). In addition,

weakly labeled Pax6 cells were detected in the dorsal

rhombencephalic alar plate, mainly in regions of the dor-

sal nucleus of the octavolateral area, as a continuation of

the cells observed at the lateral aspect of the cerebellum

(Fig. 3n–t). At caudal levels of the rhombencephalon,

Pax6 cells were also weakly labeled in the region of the

nucleus of the solitary tract and the dorsal column nu-

cleus (Figs. 3w,x, 4c,d).

Spinal cordBoth Pax6 and Pax7 cells were found in the segments

of the spinal cord that were analyzed (cervical, thoracic,

and lumbar). Caudal to the obex, where the central canal

of the spinal cord is formed, Pax7 ventricular expression

was low to nonexistent, but a population of labeled cells

was located medially in the dorsal gray (Figs. 3y, 4d,

10n). In contrast, Pax6 spinal cells were found in the in-

termediate ventral zone of the ventral horn (Figs. 3y, 4d,

7p, 10n) in a constant pattern throughout the spinal cord.

Double labeling for ChAT demonstrated that Pax6 cells in

the spinal cord represent a column of scattered cells

above the somatomotor neurons (Fig. 10o).

RetinaThe retina was analyzed in two specimens in the pres-

ent study, because of the extensive literature concerning

the presence of Pax6 in vertebrate retinae and its critical

importance during development. We corroborated that

important cell populations of Pax6 cells were located in

the deep inner nuclear layer (close to the inner plexiform

layer) and in the ganglion cell layer in the retina in Pleuro-

deles (Fig. 5m). Considering their position, these Pax6

cells may correspond to amacrine cells and ganglion

cells, but this was not further investigated in our study.

DISCUSSION

In the present study we examined expression patterns

of Pax6, Pax7, and, to a lesser extent, Pax3 genes in the

central nervous system of adult P. waltl. Using sensitive

immunohistochemical methods to detect the transcrip-

tion factors encoded by these Pax genes, we found that

they are distributed in unique patterns that mark specific

regions of the brain in urodeles. This allowed us to ana-

lyze the regionalization of particular areas and nuclei of

the brain that could be identified by their Pax expression

patterns. In topographically defined nuclei, primarily in

the diencephalon and brainstem (Neary and Northcutt,

1983; Puelles et al., 1996; Morona and Gonz�alez, 2008,

2009) expression patterns often correlated with pro-

posed, histologically defined boundaries. Importantly, in

the telencephalon of adult Pleurodeles, Pax expression is

particularly abundant in subpallial regions; due to the

combinations of labeling used, and other regional

markers, these regions could be analyzed in relation to

the basal ganglia, amygdaloid complex, and septal subdi-

visions (Moreno and Gonz�alez, 2007).

The prospect of identifying particular cell groups is of

special importance in the brains of urodele amphibians,

as cell migrations from the ventricular lining are limited,

and distinct regions and nuclei are usually hidden within a

thick periventricular cell layer (see ten Donkelaar, 1998).

In this group of vertebrates, distinct structures can be

distinguished only by immunohistochemical and hodo-

logic techniques, which serve to suggest homologies with

other vertebrates. Therefore, analysis of gene expression

patterns that are retained in adult brains and conserved

across species has become a useful tool for comparative

purposes (Moreno et al., 2004, 2005, 2010, 2012b). For

comparisons across species, the currently adopted para-

digm of brain segmentation based on spatially restricted

gene expression patterns has proven extremely useful

(Gilland and Baker, 1993; Marın and Puelles, 1995;

Puelles et al., 1996; Fritzsch, 1998; Cambronero and

Puelles, 2000; Diaz et al., 2000; Puelles and Rubenstein,

2003; Straka et al., 2006). As expected, urodele brains fit

perfectly in this paradigm, and Pax-expressing cell groups

provide independent confirmation of many boundaries

and help to characterize subdivisions and regions that

cannot be distinguished cytoarchitectonically.

Pax expression in the adult brainPax transcription factors are involved in several devel-

opmental processes in metazoans; for example, they act

as tissue-specific transcriptional regulators that play key

roles in organogenesis (cell fate and patterning), cell pro-

liferation, and disease (Chalepakis et al., 1993; Noll,

1993; Stuart et al., 1994; Wehr and Gruss, 1996; Balczar-

eck et al., 1997; Mansouri et al., 1999; Chi and Epstein,

2002; Haubst et al., 2004; Lang et al., 2007; Buckingham

and Relaix, 2007; Blake et al., 2008; Wang et al., 2008).

Moreover, they also appear to be involved in maintaining



pluripotency, not only during development but also

throughout adulthood, in subsets of tissue-specific cell

populations characterized as stem/progenitor cells, as

well as subpopulations of mature nerve cells within re-

stricted regions, with the ability to respond to environ-

mental signals (Chi and Epstein, 2002; Maekawa, 2005;

Thomas et al., 2007; Thompson et al., 2007, 2008; Blake

et al., 2008; Fedstova et al., 2008; Osumi et al., 2008).

Pax expression has been reported in distinct cell

masses in diverse regions of the CNS in some representa-

tives of all major vertebrate classes. Although most of the

data are restricted to specific regions during develop-

ment, the expression patterns of each described subpo-

pulation are highly comparable across vertebrate species

(Haubst et al., 1994, Stoykova and Gruss, 1994; Stuart

et al., 1994; Kawakami et al., 1997; Murakami et al.,

2001; Derobert, 2002; Pritz and Ruan, 2009; Duan et al.,

2012). In the case of amphibians, a previous study of Xen-

opus, based on in situ hybridization techniques, found

that Pax6 expression continued in the brain of juvenile

and reproductive adults, in regions of the forebrain

including the dorsal portion of the septum and the pretha-

lamus (Moreno et al., 2008a). The situation in adult Pleu-

rodeles contrasts with that in anurans, and this discrep-

ancy could be due to technical differences (in situ

hybridization versus immunohistochemistry) or to true dif-

ferences reflecting ‘‘embryonic-like’’ properties of urodele

brains, given the secondary simplification or pedomorphic

features (Nothcutt, 1987; Roth et al., 1993).

Comparative regional analysis of Pax6 andPax7 expression in the CNS acrossvertebrates

In the following sections the main features of the distri-

bution of Pax expression in the brain of P. waltl will be dis-

cussed from rostral to caudal levels in relation to data

available for other species. We will generally limit the dis-

cussion to Pax distribution in adult brains, and where

data are not available for adults we offer insights gained

from studies where Pax expression has been reported

only for certain developmental stages.

TelencephalonFollowing topographical criteria in urodele amphibians,

and according to previous authors (Northcutt and Kicliter,

1980; Moreno and Gonz�alez 2007; Morona and Gonz�alez,

2008; Joven et al., 2012), the main subdivisions of the tel-

encephalon include the main and accessory olfactory

bulbs, pallial (ventral, lateral, dorsal, and medial pallia)

and subpallial (striatum, pallidum, and preoptic region)

territories, and compartments with a dual pallial-subpal-

lial origin (septum and amygdaloid complex; see Moreno

and Gonz�alez, 2007a,b). In cell subpopulations of these

telencephalic regions Pax6 is expressed in adult Pleuro-

deles in a pattern similar to that in mammals, where Pax6

is the only Pax-gene to be expressed in the most rostral

forebrain areas in the adult CNS (Stoykowa and Gruss,

1994).

Olfactory bulbsIn urodeles the well-developed olfactory bulbs are in the

rostral tip of the telencephalic hemispheres and show the

same layered organization and neuronal populations as in

amniotes (ten Donkelar, 1998; Laberge, 2008). Pax6 cells

are present in the main and accessory olfactory bulbs of

Pleurodeles, mainly within the granule cell layer. Unlike in

mammals (Stoykova and Gruss, 1994; Duan et al., 2012),

no differences in their distribution in the two bulbs were

noted. The presence of Pax6 in the olfactory bulbs is a con-

served feature in vertebrates and has been reported from

lampreys through mammals, including humans (Stoykova

and Gruss, 1994; Hauptmann and Gerster, 2000; Puelles

et al., 2000; Franco et al., 2001; Murakami et al., 2001;

Derobert et al., 2002; Hauptmann, 2002; Moreno et al.,

2008a; Verdiev et al., 2009; Moreno et al., 2010, 2012a;

Duan et al., 2012; Ferreiro-Galve et al., 2012). Most data

obtained in mice demonstrate that Pax6 is essential for

the formation of the olfactory placode, olfactory bulb, and

olfactory cortex (Nomura et al., 2007). Furthermore, it is

required for the differentiation of granule and periglomeru-

lar cells in the postnatal and adult olfactory bulb (Dello-

vade et al., 1998; Hack et al., 2005; Kohwi et al., 2005).

Specifically, Pax6 cooperates with Dlx2 in adult neuro-

blasts to specify the dopaminergic identity of periglomeru-

lar neurons (Hack et al., 2005; Kohwi et al., 2005; Brill

et al., 2008). These dopaminergic neurons continue to

express Pax6 throughout life, regulating their survival by in-

hibiting programmed cell death (Ninkovic et al., 2010). In

addition, Pax6 is expressed in interneurons that also

express calcium-binding proteins, and it is required for the

differentiation and/or maintenance of specific subtypes of

interneurons in adults (Haba et al., 2009). Interestingly, in

Pleurodeles virtually all TH-immunoreactive cells (dopami-

nergic cells; Gonz�alez and Smeets, 1991) and abundant

cells containing calbindin are positive for Pax6, as reported

in zebrafish (Wullimann and Rink, 2002) and mice (Hack

et al., 2005; Kohwi et al., 2005; Verga~no-Vera et al., 2006;

Baltan�as et al., 2009; Chevigny et al., 2012), suggesting

that the implication of Pax6 in dopaminergic and calbindin-

containing cell differentiation and/or maintenance is a

conserved feature in vertebrates.

PalliumThe selective regional expression of Pax6 in the telen-

cephalic ventricular zone indicates that it as a general

Joven et al.


marker for the pallium during development (Puelles et al.,

2000), and multiple functions have been demonstrated

for Pax6 during the formation of the cortex (Yun et al.,

2001; Bachy et al., 2002; Bishop et al., 2002; Jimenez

et al., 2002; Muzio et al., 2002; Schuurmans and Guille-

mot, 2002; Assimacopoulos et al., 2003; Talamillo et al.,

2003; Carney et al., 2006; O’Leary et al., 2007; Quinn

et al., 2007; Tamai et al., 2007; Gopal and Golden, 2008;

Pi~non et al., 2008; Tuoc et al., 2009; Ceci et al., 2010;

Cocas et al., 2011; Georgala et al., 2011). Although no

Pax6 expression was revealed by in situ hybridization in

adult murine cerebral cortex (Stoykova and Gruss, 1994),

neurons expressing Pax6 in the subventricular zone of

adult mice were reported in a recent immunohistochemi-

cal study (Duan et al., 2012). Also in the pallium of young

turtles (Pseudemys scripta), immunohistochemical techni-

ques demonstrated Pax6 expression in the ventricular

and subventricular zones of the pallium (Moreno et al.,

2010). In the pallium of anamniotes, various conditions

have been observed. In zebrafish (Wullimann and Rink,

2001), anurans (Moreno et al., 2008), and urodeles (pres-

ent results) the pallium seems to lose Pax6 expression af-

ter development, with the exception of the most ventral

portion corresponding to the ventral pallium in the pallio-

subpallial zone (see below). In contrast, fragmentary data

from elasmobranchs (Scyliorhinus canicula; Ferreiro-Galve

et al., 2008) and lungfishes (Protopterus dolloi; Gonz�alez

and Northcutt, 2009) suggest that abundant Pax6 expres-

sion remains in pallial cells after development. Interest-

ingly, both elasmobranchs and lungfishes are character-

ized by the presence of remarkably abundant TH-

immunoreactive cells in the pallium, unlike other verte-

brate groups (Smeets and Gonz�alez, 2000; Gonz�alez and

Northcutt, 2009). As with the dopaminergic cells in the ol-

factory bulbs, it would be interesting to investigate the

relationship of the catecholaminergic and the Pax6 cells

in the pallium in these groups of vertebrates.

A special part of the pallium, close to the palliosubpallial

boundary (psb) in several vertebrates (mice, domestic

chicks, turtles, and frogs), distinctly shows Pax6 expression

but lacks the Emx1 expression found in all other pallial

regions (Smith-Fern�andez et al., 1998). This pallial region is

considered the ventral pallium, currently recognized in

most vertebrate groups (Puelles, 2000; Medina et al.,

2004; Brox et al., 2004; Moreno and Gonz�alez, 2004; Lind-

say et al., 2005). The psb in mammals is a complex bound-

ary zone that guides the migratory route of radially migrat-

ing subpallial cells (Marın and Rubenstein, 2003); it might

influence cell migration between the subpallium and pal-

lium, and it controls the migration of pallial cells ventrally to

the striatum (Fishell et al., 1993; Chapouton et al., 1999;

Carney et al., 2006). Analysis of this zone in vertebrates

with different pallial and subpallial features is therefore of

great interest from a comparative perspective, because dif-

ferences in the organization of the psb could be implicated

in the evolution of pallial differences. Interestingly, Pax6

expression in this region is present late in development and

in adult zebrafish (Wullimann and Rink, 2001), dogfish (Fer-

reiro-Galve et al., 2008), Xenopus (Moreno et al., 2008), tur-

tles (Moreno et al., 2010), and mouse (Carney et al., 2006).

The lateral amygdala in amphibians is a derivative of

the ventral pallium, characterized by its own developmen-

tal gene expression patterns, hodology, and neurochemi-

cal features (Brox et al., 2004; Moreno and Gonz�alez,

2004, 2006). Since the Pax6 expression observed in this

region in adult Pleurodeles can be better related to the

amygdaloid complex (Moreno and Gonz�alez, 2007), it will

be discussed below.

SeptumIn the last few years the septal region has been analyzed

in genoarchitectonic terms, since it is now considered one

of the multiple origin forebrain centers (Medina, 2008), as

opposed to its classical designation as a subpallial struc-

ture. In amniotes, it essentially comprises a pallial compo-

nent in the most dorsal portion, a pallidal component com-

prising the medial septum and the diagonal band of Broca,

and a striatal component in the lateral septum (Puelles

et al., 2000; Moreno et al., 2010). In Pleurodeles, Pax6

expressing cells are present from rostral to caudal levels in

the subventricular zone of the most dorsal septal compo-

nent, which has been called dorsal septum in comparison

with the same characterized region in turtles (Moreno

et al., 2010); the ventricular zone of the lateral septum also

reveals Pax6 expression. In addition, Pax6 cells occupy the

region of the diagonal band of Broca in urodeles (Gonz�alez

et al., 1996; Moreno et al., 2002). By comparison, in adult

mice moderate expression was observed in the lateral sep-

tal nucleus, whereas strong signal was detected in the

medial septal nucleus and in the horizontal and vertical

limbs of diagonal band of Broca (Stoykova and Gruss,

1994; Duan et al., 2012). In the turtle Pseudemys scripta

Pax6 expression in ventricular and mantle zones was used

to identify a rostrocaudal dorsal septal region, included in

GABA-expressing territory and located dorsal to the TH ter-

minal field of the lateral septum generally identified as a

striatal septal subdivision (Moreno et al., 2010), and there-

fore comparable to the situation observed in Pleurodeles

(present results). In mammals, the septal region adjacent

to the psb is continuous with the lateral ganglionic emi-

nence and shares some characteristics with it, such as

expression of Pax6 and lack of Nkx2.1 transcripts (Flames

et al., 2007), as in Xenopus (Moreno et al., 2008a). In Pleu-

rodeles in this region the double Pax6/Nkx2.1 staining

showed that both transcription factors were intermingled

but no double-labeled cells were observed.



Basal gangliaThe basal ganglia share a common pattern of organiza-

tion in tetrapods, including the presence of the striatum,

nucleus accumbens, and the pallidum (Marın et al.,

1998b). The extension and the boundaries of these

regions in Pleurodeles were proposed on the basis of

hodological and chemoarchitectonic results (Gonz�alez

and Smeets, 1991; Marın et al., 1997c,d, 1998a) and

later confirmed by genoarchitectonic data (Moreno and

Gonz�alez, 2007). The Pax6 expression pattern in adult

Pleurodeles can therefore be considered in the context of

these regions. Thus, numerous Pax6 cells occupy the pro-

posed nucleus accumbens, in the rostral dopaminergic

neuropil in the ventral telencephalon (Gonz�alez and

Smeets, 1991). In addition, strikingly abundant Pax6 cells

are distributed throughout the striatal cell layer, medial to

the dopaminergic terminal field in adult Pleurodeles. In

chicks, turtles, and mice, the primordium of nucleus

accumbens is immediately caudal to the olfactory bulb

and expresses Dlx2 and Pax6 in cells entering the mantle

zone radially (Puelles et al., 2000; Moreno et al., 2010).

Adult mice appear to lack Pax6 cells in the striatum (Stoy-

kova and Gruss, 1994; Duan et al., 2012), whereas in

chickens Pax6 neurons persist at the lateral edge of the

ventral striatum, forming a distinct cell mass postnatally

(Puelles et al., 2000). In adult turtles, Pax6 expression

was observed in the striatum in migrated cells located

near the pial surface (Moreno et al., 2010). In striking

contrast with the results of the present study, adult Xeno-

pus revealed no Pax6 cells in striatal regions, although

only in situ hybridization was used (Moreno et al., 2008).

Therefore, among tetrapods, urodeles appear to show the

most abundant Pax6 expression in striatal regions in

adults. In amniotes, the region that gives rise to the stria-

tum, i.e., the lateral ganglinic eminence (LGE), has differ-

ent progenitor domains for the differential expression of

transcription factors, neurotransmitters, and neuropepti-

des, including Pax6 (Flames et al., 2007). Thus, Pax6

defines the most dorsal domain: pLGE1 and pLGE2 in

mammals (Flames et al., 2007); the dorsal striatal domain

in birds (Abell�an and Medina, 2009); and reptiles (Moreno

et al., 2010). Developmental studies in Pleurodeles are

needed to determine if Pax6 is expressed in a similar sub-

domain in the striatal region.

As in other tetrapods, the pallidal derivatives in Pleuro-

deles express Nkx2.1 (Moreno and Gonz�alez, 2007; Mor-

eno et al., 2009), and double labeling for Pax6 reveals

that the cells expressing each of these markers consti-

tute independent populations, as no double-labeled cells

have been observed. In adults, only scattered Nkx2.1-

expressing cells are present in the striatum, the Pax6-

expressing territory, suggesting a comparable migratory

process between adjacent pallidal areas and the striatum

in urodeles (Marın and Rubenstein, 2000; Moreno et al.,

2008b).

Amygdaloid complexThe amygdaloid complex has been intensely reeval-

uated recently, mainly in terms of nuclei origin and speci-

fication in all tetrapods (Moreno and Gonz�alez, 2006,

2007; Martınez-Garcıa et al., 2012). It has a very con-

served multimodal structure formed by different nuclei

with different embryonic origins, including pallial, subpal-

lial, and extratelencephalic components. In Pleurodeles a

multimodal/multiorigin amygdaloid complex is comprised

of three components: the lateral amygdala (LA), a ventro-

pallial derivative which receives olfactory information; the

medial amygdala (MeA), which is of subpallial origin and

is the main secondary vomeronasal center, projecting

strongly to the ventral hypothalamus; and the medial

amygdala (CeA), a striatal area that constitutes the auto-

nomic amygdaloid subdivision (Moreno and Gonz�alez,

2007). In this context, Pax6 cells in adult Pleurodeles are

present only in the LA and the CeA. Both areas were pre-

cisely characterized with double immunohistochemistry

for NOS (Moreno et al., 2002), CB (Morona and Gonz�alez,

2008), GABA, and Nkx2.1 (Moreno and Gonz�alez, 2007).

During development in mice, a stream of Pax6 cells

appear to arise at the psb, extends toward prospective

amygdaloid regions, and later occupies the dorsal portion

of the anterior amygdala and the central amygdala (Tole

et al., 2005). In the case of the pallial portion of the amyg-

daloid complex, analysis of Pax6 mutants demonstrated

that Pax6 is selectively required for the specification of

the lateral, basolateral, and, to a lesser extent, basome-

dial amygdaloid nuclei (Tole et al., 2005). Therefore, the

formation of the pallial and subpallial nuclei appears to

depend on the level of Pax6 expression in the neuroepi-

thelial domain from which the different structures arise

(Tole et al., 2005).

The amygdaloid complex has been interpreted as a

continuum through the basal forebrain (extended amyg-

dala) including other structures such as the bed nucleus

of the stria terminalis (BST; for review see Martınez-Gar-

cıa et al., 2012). In adult Pleurodeles, Pax6 expression is

lacking in the region of the BST (Moreno and Gonz�alez,

2007a) and is restricted to adjacent striatal zones, thus

suggesting that the whole BST has a pallidal origin, as

recently described in anurans (Moreno et al., 2012a). In

the turtle Pseudemys, BST also has a pallidal origin, but

with components from adjacent areas, such as Pax6-posi-

tive cells of striatal origin, and Tbr1-positive cells that

appear to constitute the rostral continuation of the pre-

thalamic eminence (Moreno et al., 2010). In birds, ventral

to the caudal lateral striatal area, Pax6 cells are located

in the dorsolateral part of the BST and the globus palidus,

Joven et al.


most likely originating in the dorsal striatum (Abell�an and

Medina, 2008, 2009). The situation in birds resembles

that in mammals, as the striatal domain in mice produces

the CeA and contributes to the BST (Nery et al., 2002;

Garcıa-L�opez et al., 2008). Therefore, the analysis of the

Pax6 expression suggests that in amphibians (anam-

niotes) the BST lacks a striatal component, whereas this

component is present in amniote tetrapods.

HypothalamusThe regionalization and subdivisions of the hypothala-

mic region have been reanalyzed in tetrapods, mainly on

the basis of the distribution patterns of sets of develop-

mental regulatory transcription factors and neuronal

markers in relation to actual topological relationships in

the forebrain (Shimogori et al., 2010; Domınguez et al.,

2013; Moreno et al., 2012b; Puelles et al., 2012a). Essen-

tially, alar hypothalamic regions include the supraoptope-

duncular and suprachiasmatic subdivisions, whereas ba-

sal regions are the tuberal and mammillary subdivisions

(for review, see Medina, 2008; Moreno and Gonz�alez,

2011; Puelles et al., 2012a). In addition, it is currently

accepted that the preoptic area, classically included in

the hypothalamus (Butler and Hodos, 2005), actually

belongs to the telencephalon (for review, see Puelles

et al., 2012a). In Pleurodeles, the preoptic area is a large

territory that expresses Nkx2.1, which allows the identifi-

cation of a commissural preoptic area, as in other verte-

brates (Flames et al., 2007; Abell�an and Medina, 2008,

2009; Moreno et al., 2008c, 2010). However, Pax genes

were not detected in the whole preoptic area, in agree-

ment with results in anurans (Moreno et al., 2008a;

Domınguez et al., 2013), turtles (Moreno et al., 2012b),

and chickens (Abell�an and Medina, 2009), but in contrast

to mammals (mice), where Pax6 has been described in

the lateral portion of the preoptic area in adults (Duan

et al., 2012).

In the present study of adult Pleurodeles, in the hypo-

thalamus, only scattered Pax7 cells were seen distributed

in the mammillary region. In amniotes, Pax6 has also

been described in the supraoptoparaventricular region

(Medina, 2008; Moreno et al., 2012b), but this expression

seems to be absent in anamniotes (Murakami et al.,

2001; Moreno et al., 2008a; Moreno and Gonz�alez, 2011;

Domınguez et al., 2013). However, Pax6 has been demon-

strated to be necessary in urodeles, not only for eye mor-

phogenesis but also for hypothalamic formation, in partic-

ular the suprachiasmatic region (Eagleson et al., 2001).

Therefore, developmental studies in urodeles will most

likely reveal increased Pax6 expression than exists in

adults where it seems no longer needed.

The discrete Pax7 cell subpopulation observed in the

mammillary area is situated close to the catecholaminer-

gic (TH-positive) cell subpopulation described in this area

(Gonz�alez and Smeets, 1991) and to the serotonergic

cells of the nucleus of the periventricular organ (present

results), but no double-labeled cells were detected. The

precise localization of these Pax7 cells in the mammillary

region was assessed by combined labeling for calbindin

(Morona and Gonz�alez, 2008), and by comparison with

recent data in Xenopus (Domınguez et al., 2013). Accord-

ingly, we found cells double-labeled for Nkx2.1/Pax7 in

the mammillary region of adult Pleurodeles (present

results), as in Xenopus (Domınguez et al., 2013). Pax7

expression has been reported in chick Nkx2.1-positive

hypothalamic basal progenitors during development

(Ohyama et al., 2008), and in the subthalamic nucleus of

mice during development and in postnatal life (Stoykova

and Gruss, 1994).

Most cells in the intermediate lobe of the hypophysis

showed Pax7 expression in adult Pleurodeles. The precise

location in the intermediate lobe was corroborated by the

innervation of catecholaminergic (TH-positive) fibers, pri-

marily arising in the suprachiasmatic nucleus, as previ-

ously revealed in urodeles (Corio et al., 1992). Therefore,

the maintenance of the Pax7 expression in the intermedi-

ate lobe in adult urodeles suggests that this transcription

factor is needed in the cells that release the a-melano-

phore-stimulating hormone, which at least in anurans

activates skin melanophores to darken when the animal

is placed on a dark background and is controlled from the

hypothalamus (Tuinhof et al., 1994a,b; Roubos et al.,

2010). In the hypophysis of other vertebrates, such as

zebrafish, Pax7-expressing cells have been described in a

comparable pars intermedia, and Pax7 blockage was

demonstrated to selectively impair formation of the pars

intermedia (Guner et al., 2008). Also in mammals, Pax7

constitutes a specific marker of the melanotrope cells of

the intermediate lobe of the hypophysis (Budry et al.,

2011), and it was demonstrated that a subpopulation of

Pax7 postnatal cells constitutes a progenitor cell popula-

tion in the intermediate lobe (Hosoyama et al., 2010).

Therefore, the Pax7 expression in the cells of the interme-

diate lobe of the hypophysis might be a well-conserved

feature throughout vertebrates.

DiencephalonThe diencephalon is the caudal part of the forebrain

and comprises three segmental units: prosomeres p3, p2,

and p1, from rostral to caudal, which are clearly defined

by gene expression patterns (Puelles and Rubenstein,

2003). The alar regions of the three prosomeres repre-

sent the prethalamus (PTh), thalamus (Th), and pretectum

(PT). respectively, whereas the smaller basal components

form an underlying tegmental region in each case (Puelles

and Rubenstein, 2003; reviewed in Puelles et al., 2012b).



p3Prosomere 3 comprises the roof plate, which contains

the prethalamic eminence, the alar component that forms

the prethalamus, and the basal p3 portion (reviewed in

Puelles et al., 2012b). The nature of the paraphysis (par),

the structure that evaginates from the ventricle, is contro-

versial. For some authors, this constitutes a telencephalic

derivative, whereas for others it belongs to the roof of p3.

Specifically in amphibians, it has been described as a poste-

rior telencephalic organ adjacent to the choroid plexus of

the third ventricle (Ari€ens-Kappers, 1956), but unlike the

plexus it lacks the uptake mechanisms for cerebrospinal

fluid (Jansen and Diederen, 1987). In urodeles a type of argi-

nine vasotocin receptor was observed in the paraphysis,

related to the osmotic and/or ionic regulation of the cere-

brospinal fluid (Hasunuma et al., 2010). Strong Pax7 expres-

sion was observed in the paraphysis of adult Pleurodeles,

and not in the adjacent choroid plexus, suggesting that it

likely constitutes a derivative of the telencephalic roof. Simi-

lar expression in the paraphysis has been reported only dur-

ing development in the chick (Nomura et al., 1998).

In adult Pleurodeles the ventricular zone of the pretha-

lamic eminence shows Pax6 expression, and the prethala-

mus is filled with Pax6 expressing cells in the subventricu-

lar and mantle zones, as reported in all other vertebrates

studied (Puelles et al., 2000; Wullimann and Rink, 2001,

2002; Moreno et al., 2008a, 2010, 2012b; Pritz and

Ruan, 2009; Duan et al., 2012; Domınguez et al., 2013).

Pax6 staining in the prethalamus of Pleurodeles

decreases ventralwards, suggesting that Pax6 cells origi-

nate dorsally within p3 to migrate through more basal ter-

ritories. In the mouse diencephalon, the alar–basal plate

boundary was defined as the ventral extent of alar Pax6

expression (Hauptmann and Gerster, 2000; Mastick and

Andrews, 2001; Hauptmann et al., 2002; Ferr�an et al.,

2007, 2008). In the alligator Alligator mississipiensis,

Pax6 expression was analyzed in the diencephalon, and

the boundary between p3 and the secondary prosence-

phalon was defined by Pax6 expression, while Pax6 cells

were also reported in the basal part of p3 (Pritz and Ruan,

2009). In the turtle Pseudemys scripta, scattered Pax6

cells were seen to invade the basal portion of p3 (Moreno

et al., 2012b). In Pleurodeles the basal p3 was defined by

the complementary Pax6/Pax7 expression pattern in this

segment, and in addition by the double labeling for Pax6

and Nkx2.2. A Pax7 group is present in the p3 basal plate

and defines the alar/basal portions of this segment in all

vertebrates studied so far (Moreno et al., 2012). Support-

ing the basal nature of this subpopulation, we found

some double Pax7þ/Nkx2.1þ cells, as also occur in Xen-

opus, in contrast to the neighboring tuberal hypothala-

mus, which lacks Pax7 expression (Domınguez et al.,

2013). The rostral boundary of the basal p3 can be

observed by double labeling for 5-HT/Pax7, which allows

a distinction between the hypothalamus, rich in 5-HT

cells, and the basal p3, with its presence of Pax7 cells. In

addition, expression in the ventricular zone was very

scarce in adults, but still present, supporting the dience-

phalic origin of the Pax7 cells, as previously proposed for

anuran amphibians and chelonians (Domınguez et al.,

2013; Moreno et al., 2012). In mammals, it was demon-

strated that Pax6 regulates the expression of Nkx2.2,

which is restricted to progenitors that intriguingly do not

express Pax6 in the ventral neural tube (Ericson et al.,

1997). In the diencephalon, Nkx2.2 is directly or indi-

rectly repressed by Pax6 (Pratt et al., 2000; Kiecker and

Lumsden, 2005), and the progenitor population was

defined by reciprocal overlapping expression gradients of

both transcription factors (Mastick and Andrews, 2001),

suggesting that a primary action of Pax6 is to generate

correct dorsoventral patterning in the diencephalon (Pratt

et al., 2000). In this context, Pax6 has been linked to the

formation of A13 dopaminergic THþ neurons in the

medial zona incerta, where double-labeled cells were

found in mouse development (Mastick and Andrews,

2001). In adult Pleurodeles, cells expressing Pax6 and

Pax7 in p3 are adjacent to the TH cell population, but no

double-labeled cells were detected, although develop-

mental studies might reveal a different situation.

p2The intermediate prosomere of the diencephalon con-

sists of the roof derivatives, the epiphysis or pineal com-

plex and the habenula, the alar thalamic nuclei, and the

basal tegmental component (for review, see Puelles et al.,

2012b). In adult Pleurodeles, Pax6 expression is present

only in the epiphysis, and scattered Pax6 and Pax7 cells

are distributed in the habenula.

In adult urodeles the epiphysis was described as a

small, flattened, simply lobulated epithelial vesicle,

entirely detached from the brain except for a few fibers of

the parietal nerve (Herrick, 1948). In adult Pleurodeles,

Pax6 is strongly expressed in the cells of this vesicle,

whose function has been related to the regulation of cir-

cadian and other biological rhythms, as in other verte-

brates. In addition, in newts the epithelial vesicle has

been implicated in an animal’s orientation to the mag-

netic compass by a response to long-wavelength light (for

review, see ten Donkelaar, 1998; Butler and Hodos,

2005). The importance of Pax6 in the epiphysis has also

been reported in mammals, where mutants lacking Pax6

also lack a pineal gland (Mitchell et al., 2003).

The habenular complex occupies the dorsal part of p2

and is asymmetrical in Pleurodeles (Morona and

Gonz�alez, 2008). Only a few Pax6 and Pax7cells were

seen, mainly across the central part of the habenula

Joven et al.


among fibers that cross in the habenular commissure and

in close proximity to the labeled cells in the epiphysis. In

comparison, cells expressing Pax6 in the lateral and

medial habenular nuclei during mouse development per-

sist until adulthood (Duan et al., 2012).

The thalamus did not show any Pax expression in adult

Pleurodeles. Actually, the boundary of the thalamus with

the prethalamus (rostrally) and the pretectum (caudally)

can be defined by the striking lack of Pax expression in

the thalamus, in contrast to the alar parts of p3 and p1,

as also revealed by Pax6 expression in mammals (Walther

and Gruss, 1991; Stoykova and Gruss, 1994; Duan et al.,

2012). Pax6 appears to be important for this boundary

formation, as demonstrated in null mutants (Mastick

et al., 1997) and early expression of Pax6 in the thalamus

needs to be downregulated, in order to produce a normal

thalamus (Grindley et al., 1997).

p1Prosomere 1 is situated between the midbrain and p2,

and includes in its alar portion the subcommissural organ

and the pretectal nuclei. In general the neuromeric organi-

zation of this prosomere in vertebrates is deformed by the

particular expansion of the optic tectum; however, the

course of the retroflex fascicle highlights the rostral bound-

ary of p1 with p2, the posterior commissure marks the pre-

tecto-mesencephalic border, and in the basal region the

oculomotor nucleus indicates the rostral boundary of the

midbrain (reviewed in Puelles et al., 2011b). It is within the

pretectum where abundant Pax6, Pax7, and Pax3 cells are

located in Pleurodeles. In adults, the tripartite anteroposte-

rior subdivision of the pretectum into precommissural, jux-

tacommissural, and commissural domains exists, as in

other tetrapods (Ferr�an et al., 2007, 2008, 2009; Morona

and Gonz�alez, 2008; Morona et al., 2011). In Pleurodeles,

the roof plate neuroepithelium of p1, including the sub-

commissural organ, expressed Pax3, Pax6, and Pax7. In

the alar domain, Pax6 and Pax7 cells were seen accumu-

lated in the juxtacommissural part and, to a lesser extent,

in the commissural subdivision, in contrast to the precom-

missural part (identified by its calbindin-containing cells;

Morona and Gonz�alez, 2008), which is almost devoid of

Pax3, Pax6, and Pax7 cells. A role of Pax6 in the develop-

ment of dopaminergic pretectal neurons has been pro-

posed in zebrafish (Wullimann and Rink, 2001), where a

detailed confocal analysis showed no double-labeled

Pax6/TH cells. A similar situation is present in Pleurodeles.

In Xenopus, Pax3 and Pax7 help to delineate the precom-

missural alar subdomain, establishing its difference in mo-

lecular identity from the two other subdomains (Morona

et al., 2011). Also in Xenopus, Pax7 expression was

observed in the juxtacommissural domain (Morona et al.,

2011), as in Pleurodeles (present results), in contrast to

the lack of expression described in chickens and mice

(Ferr�an et al., 2007, 2008). During development in mam-

mals, it was described that Pax3, whose expression do-

main coincides with Pax7, positively controls Pax7 expres-

sion in the brain, in parallel with the general development

of the alar plate (Maczkowiak et al., 2010). In the alar plate

of Pleurodeles and Xenopus, as in other vertebrates, the

caudal boundary of the pretectal region is defined molecu-

larly mainly by the expression of Pax6 and Pax7 (Ferr�an

et al., 2008, 2009; Moreno et al., 2008a; Morona et al.,

2011; present results). Pax6 function is known to define

this boundary in mammals by repression of the midbrain-

centered markers En2 and Pax2 (Matsunaga et al., 2000).

Finally, Pax6 expression has been noted in the basal plate

of p1 in representatives of all vertebrate groups studied

(Agarwala et al., 2001; Murakami et al., 2001; Wullimann

and Rink, 2001; Scubert and Lumsden, 2005; Ferr�an et al.,

2007, 2008; Ferreiro-Galve et al., 2008; Pritz and Ruan,

2009; Merch�an et al., 2011; Morona et al., 2011; Duan

et al., 2012). The dorsal portion of the basal plate also has

Pax6 in Pleurodeles, in cells that extend caudally into the

neighboring mesencephalon and lack ventricular expres-

sion (present results), as they probably reflect a rostral

migration occurring during development.

MesencephalonThe topological analysis of cell groups in the mesen-

cephalon is complicated, because in adults the apex of

the cephalic flexure is located under the basal midbrain,

deforming this area to form a wedge that in classical

transverse series appears with ‘‘ventral’’ parts that are

actually adjacent rostral regions of the diencephalon or

caudal regions of the isthmus. Recent molecular data in

amniotes necessitated reconsideration regarding the

presence of two neuromeres (m1 and m2) in the mesen-

cephalon (reviewed in Puelles et al., 2012c). The m1 is

rostral, forms the boundary with the diencephalon, and it

comprises the optic tectum and torus semicircularis (or

homologous structures) as alar derivatives and the rostral

part of the midbrain tegmentum, which contains the ocu-

lomotor nucleus in the basal plate. In turn, the m2 domain

is a thin transverse segment of the caudal midbrain,

located in front of the isthmus and its trochlear nucleus.

In Pleurodeles the alar plate derivatives are character-

ized by the absence of Pax6 expression and widely distrib-

uted Pax7 cells. In amniotes, Pax6 expressed rostral to the

midbrain in the alar diencephalon contributes jointly with

other molecular signals to the establishment of the mesen-

cephalic–diencephalic boundary (Matsunaga et al., 2000),

and the absence of Pax6 in the alar midbrain has been

used extensively to discriminate between p1 and the mes-

encephalon in all vertebrates studied so far (Murakami

et al., 2001; Wullimann and Rink, 2001; Scholpp and



Brand, 2003; Thompson et al., 2007; Moreno et al., 2008a;

Pritz and Ruan, 2009; Morona et al., 2011). Thus, in adult

Pleurodeles the most outstanding feature in the alar mes-

encephalon is the presence of Pax7 expression, extending

in the alar derivatives, as corroborated here with double

labeling and in agreement with previous studies (Naujoks-

Manteuff et al., 1994; Gonz�alez et al., 1996; Morona and

Gonz�alez, 2009). The optic tectum has frequently been

considered primitive in urodeles, in comparison to other

amphibians; however, extensive analysis demonstrated

that it possesses very similar functional and morphological

cell types, but its lower frequency of migratory processes

give the tectum urodeles a juvenile appearance (Roth

et al., 1990). In this context, Pax7 cells are distributed in

both the deep and superficial zones of the tectum. Compa-

rable results have been described in other vertebrates,

such as Xenopus, where abundant Pax7 cells are present

in the midbrain during development (Chen et al., 2006)

and in adults (unpubl. obs.). In adult chickens, Pax7 was

found in neurons located mostly in the outer layers of the

optic tectum (Shin et al., 2003). Also in chickens the cru-

cial role of Pax7 in tectal development was demonstrated,

as the ectopic Pax7 expression in the diencephalon was

proved to induce the formation of an ectopic tectum (Mat-

sunaga et al., 2001). Further, during development Pax7 is

involved in establishing tectal polarity (Thomas et al.,

2004) and the retino-tectal topography (Thomas et al.,

2006). In adult rodents, Pax7 expression is concentrated

in neurons located in the retino-recipient laminae (Thomas

et al., 2004), and it likely has a role in retinotopic mapping

(Thompson et al., 2007). In addition, a faint signal was

described in the dorsal part of the midbrain central gray in

adult mice (Stoykova and Gruss, 1994).

In the mesencephalic basal plate, Pax6 cells form a lon-

gitudinal band, distinct from the well-characterized cho-

linergic, dopaminergic, nitrergic, and calbindin-containing

cell groups characterized in the basal plate of adult Pleu-

rodeles (Gonz�alez and Smeets, 1991; Gonz�alez et al.,

1996; Marın et al., 1997b; Morona and Gonz�alez, 2009).

These cells were located ventral to the midbrain alar–ba-

sal boundary that is visualized by morphological land-

marks (lateral mesencephalic sulcus) and also by the

Nkx2.2 cells situated slightly dorsal to the Pax6 cell popu-

lation. The presence of a comparable Pax6 cell subpopu-

lation in the basal mesencephalon is widespread among

vertebrates (Stoykova and Gruss, 1994; Vitalis et al.,

2000; Wullimann and Rink, 2001; Ahsan et al., 2007;

Bayly et al., 2007; Pritz and Ruan, 2009; Duan et al.,

2012).

HindbrainThe hindbrain is the large brain region rostrally continu-

ous with the midbrain and caudally continuous with the

spinal cord. It comprises segmental components: the

isthmus, also defined as r0, and rhombomeres 1–8. In

this context the cerebellum is an outgrowth of the dorsal

parts (the rhombic lip region) of r1 (Aroca and Puelles,

2005; Watson, 2012). In Pleurodeles, only scattered Pax7

cells are seen in the alar plate of the isthmus, whereas

Pax3 and Pax6 cells are absent. The precise isthmic loca-

tion of the labeled cells in the upper hindbrain of Pleuro-

deles was assessed by the presence of the ChAT-positive

trochlear and isthmic nuclei in r0 (Marın et al., 1997b;

L�opez et al., 2003), the lack of GABAergic and serotoner-

gic cells in the isthmic tegmentum (Clairambault et al.,

1994; Naujoks-Manteuffel et al., 1994), and the distribu-

tion of isthmic calretinin-containing cells (Morona and

Gonz�alez, 2009). Recently, an important role for Pax7

was described in Xenopus in the maintenance of the mid-

brain–hindbrain boundary (prospective isthmus) and pre-

vention of the posterior expansion of forebrain and mid-

brain fates during early development (Maczkowiak et al.,

2010). In the brain of adult chickens, Pax7-immunoreac-

tive cells were detected in the nucleus isthmo-opticus

(Shin et al., 2003) and in mice they were seen in the dor-

somedial tegmental area surrounding the posterior dorsal

tegmental nucleus (expressing Pax6), as well as in cells of

the oral part of the pontine reticular nucleus (Stoykova

and Gruss, 1994). The formation of the mid-hindbrain

boundary has been shown to be critically dependent on

several transcription factors and certain genes expressed

in this region, such as Pax2/5/8, can be used as markers

to delineate the MHB (Wada et al., 1998; Fritzsch and

Glover, 2006), but the expression of these genes should

be studied in urodeles. In Pleurodeles, the Pax7 cell popu-

lation of the interpeduncular nucleus that extends to r1

expands into the ventral portion of r0. The position of

these cells has been confirmed by double labeling with

calbindin (Morona and Gonz�alez, 2009), GABA (Naujoks-

Manteuffel et al., 1994), serotonin (Clairambault et al.,

1994), and ChAT (Marın et al., 1997b). Interestingly,

recent studies of Pax7 expression in chickens and other

experimental evidence demonstrates that the interpedun-

cular nucleus has a dual origin in r0–r1 (Lorente-C�anovas

et al., 2012).

In the cerebellum of adult Pleurodeles, Pax6 immuno-

histochemistry labels the granule cell layer, whereas

Pax7 expression only lines the ventricle. The Purkinje

cells, positive for calbindin (Morona and Gonz�alez, 2009;

Joven et al., 2012), were not positive for any Pax member

under study. In adult chickens, Pax7 marks some types of

cells located around the Purkinje cells, while the Purkinje

cells are negative (Shin et al., 2003). During mouse devel-

opment, Pax6 is strongly expressed in the rhombic lip.

Postmitotic neurons generated at the rhombic lip migrate

long distances to become widely dispersed in different

Joven et al.


destinations, giving rise to cerebellar granule cells and

the precerebellar nuclei (Engelkamp et al., 1999; Fink

et al., 2006). Pax6 is also expressed in cerebellar granule

cell precursors in chicks (Gilthorpe et al., 2002), zebrafish

(Wullimann et al., 2001), and the shark Scyliorhinus cani-

cula (Rodrıguez-Moldes et al., 2008). The absence of

rhombic lip-derived cerebellar and precerebellar systems

in lampreys has been related to the lack of Pax6 expres-

sion in the rhombic lip (Murakami et al., 2005). Thus,

Pax6 is a general developmental marker of these cell

types in gnathostomes and likely drives the origin of the

cerebellum in their evolution.

Also in r1, Pax7 expression is located in Pleurodeles in

cells of the reticular superior nucleus, whose caudal por-

tion intermingles with the central gray, as revealed by

GABA and calretinin staining (Morona and Gonz�alez, 2009;

present results). In the laterodorsal tegmental nucleus of

Pleurodeles, identified by its nitrergic (Gonz�alez et al.,

1996; Moreno et al., 2002) and cholinergic (Marın et al.,

1997b; L�opez et al., 2003) nature, Pax expression was not

observed, in contrast to mammals, where Pax6-positive

cells have been described (Duan et al., 2012). In Pleuro-

deles we found that Pax6 and Pax7 are closely situated

around the TH cells of the locus coeruleus (Gonz�alez and

Smeets, 1995), but double-labeled cells were not

detected. In chickens, double-labeling experiments with

locus coeruleus markers and Pax7 suggested that these

cells migrate tangentially ventralwards from their origin in

the alar plate to a final position in the lateral basal plate

(Aroca et al., 2006). In addition, in r1 we found a small

group of Pax3-expressing cells that are readily comparable

to the Pax3 cells described in adult mice in the dorsal teg-

mental nucleus of Gudden (Stoykova and Gruss, 1994),

which projects to the mammillary bodies and is involved in

angular head velocity, learning, and navigation (Morest,

1961; Bassett and Taube, 2001; Saunders et al., 2012).

A peculiar feature of the ventricular zone of the alar

plate in adult Pleurodeles is the maintenance of Pax7

expression all along the hindbrain. This concurs with de-

velopmental data from chickens, where Pax7 generally

characterizes the alar ventricular zone throughout the

hindbrain (Aroca et al., 2006; Lorente-C�anovas et al.,

2012). Pax6 cells, however, were seen to migrate from

the ventricle and form a column along the hindbrain that

coincided in many cases with the localization of the bra-

chiomotor nuclei. Interestingly, it has been suggested

that Pax6 is involved in the specification of subtypes of

hindbrain neurons in mammals (Osumi et al., 1997).

Finally, in Pleurodeles, as in mice (Stoikova and Gruss,

1994), Pax6 cells are also present in the rhombence-

phalic alar plate in regions equivalent to the cochlear/

vestibular nuclei. In addition, in Pleurodeles, sparse cells

are also positive for Pax6 in the nucleus of the solitary

tract and in the dorsal column nucleus, but cells in corre-

sponding areas in mice could only been seen in the devel-

oping brain (Duan et al., 2012).

Spinal cordOur data confirm the expression pattern previously

described for the spinal cord in an adult urodele, Ambys-

toma mexicanum (Schnapp et al., 2005; McHedlishvili

et al., 2007), except that ventricular expression of Pax7

was observed in the axolotl and not detected in Pleuro-

deles. The described Pax7 cells in the dorsal horn subven-

tricular zone in the axolotl are also present in Pleurodeles.

The expression of Pax6 was highly comparable in both spe-

cies, restricted to the cells located above the motoneurons

in the upper part of the ventral gray and in the intermediate

zone of the ependymal layer. In the axolotl, it has been sug-

gested that Pax-positive cells probably represent a progeni-

tor cell population in the mature spinal cord (McHedlishvili

et al., 2007). Comparatively, in Xenopus Pax7 has also

been described in the rostral spinal cord (Markitantova

et al., 2004; Maczkowiak et al., 2010).

RetinaThe retina in adult Pleurodeles shows different levels of

intensity for Pax6 immunoreactivity in subsets of cells in

the deep part of the inner nuclear layer and in the gan-

glion cell layer. Abundant displaced amacrine cells have

been described in the ganglion cell layer (Morona et al.,

2007), however, and we therefore cannot conclude the

real nature of the Pax6-expressing cells in this layer. Pax6

continues to be expressed in adult retinal ganglion cells

in zebrafish and lizards, where a Pax6 role in the forma-

tion and refinement of topographic projections during

optic nerve regeneration has been demonstrated (Rodger

et al., 2006). The functional conservation of the regula-

tory mechanisms governing Pax6 transcription in the ret-

ina of vertebrates has been studied extensively (for

review, see Erclik et al., 2009). Finally, in the retina Pax6

is present in the developing and the regenerating events,

and thus when a urodele is not capable of lens regenera-

tion (axolotl) Pax6 is only expressed at embryonic stages,

whereas in adult newts, which are capable of lens regen-

eration, a decline in Pax6 expression does not occur (Del

Rio-Tsonis et al., 1995), as is the case in P. waltl (Mita-

shov et al., 1995).

ACKNOWLEDGMENT

We thank Mary Sue Northcutt for assistance with the

English writing. The Pax7 and Pax6 monoclonal antibod-

ies, developed by Atsushi Kawakami, and the Pax3 mono-

clonal antibody, developed by C.P. Ordahl, were obtained

from the Developmental Studies Hybridoma Bank,



developed under the auspices of the NICHD and main-

tained by the University of Iowa, Department of Biological

Sciences, Iowa City, IA. We thank Mr. Jorge Perlado for

help during the elaboration of the article.

CONFLICT OF INTERESTThe authors declare that they have no conflict of interest.

ROLE OF AUTHORSAll authors had full access to all the data in the study and

take responsibility for the integrity of the data and the

accuracy of the data analysis. AG and NM devised the

study. AJ performed all of the experiments, with the

exception of the western blot analysis carried out by RM. AJ

and NM were the primary contributors to the data analysis.

AJ and AG led the figure preparation. AJ and NM wrote most

of the article, further completed and edited by AG and all

authors approved the article.

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