Origins of the insect enteric nervous system: differentiation …...0.01 % insulin, supplemented...

Development 113, 1115-1132 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

1115

Origins of the insect enteric nervous system: differentiation of the enteric

ganglia from a neurogenic epithelium

PHILIP F. COPENHAVER1 •• and PAUL H. TAGHERT2

1 Department of Cell Biology and Anatomy L215, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, Oregon97201, USA2Department of Anatomy and Neurobiology, Box 8108, Washington University Medical School, St Louis, Missouri 63110, USA

* Author for correspondence

Summary

The enteric nervous system (ENS) of the moth Manducasexta is organized into two distinct cellular domains: ananterior domain that includes several small ganglia onthe surface of the foregut, and a more posterior domainconsisting of a branching nerve plexus (the entericplexus) that spans the foregut-midgut boundary.Previously, we showed that the neurons of the posteriordomain, the enteric plexus, are generated from a largeplacode that in vagina tes from the caudal lip of theforegut; subsequently, the cells become distributedthroughout the enteric plexus by a sequence of activemigration. We now demonstrate that the neurons of theanterior domain, the cells of the enteric ganglia, arise viaa distinct developmental sequence. Shortly after theforegut has begun to form, three neurogenic zonesdifferentiate within the foregut epithelium and give riseto chains of cells that emerge onto the foregut surface.The three zones are not sites of active mitosis, asindicated by the absence of labelling with a thymidineanalogue and by clonal analyses using intracellularlyinjected dyes. Rather, the zones serve as loci throughwhich epithelial cells are recruited into a sequence ofdelamination and neuronal differentiation. As they

emerge from the epithelium, the cells briefly becomemitotically active, each cell dividing once or twice. Inthis manner, they resemble the midline precursor classof neural progenitors in the insect central nervoussystem more than neuroblast stem cells. The progeny ofthese zone-derived precursors then gradually coalesceinto the ganglia and nerves of the anterior ENS.Although this reorganization results in some variabilityin the precise configuration of neurons within theganglia, the overall morphology of the ganglia is highlystereotyped, consisting of cortical layers of cells thatsurround a ventral neuropil. In addition, a number ofthe neurons within the frontal and hypocerebral gangliaexpress identifiable phenotypes in a manner that issimilar to many cells of the insect central nervoussystem. These observations indicate that the differen-tiation of the enteric ganglia in Manduca involves anunusual combination of features seen during theformation of other regions of the nervous system and, assuch, constitutes a distinct program of neurogenesis.

Key words: enteric nervous system, neurogenic zones,enteric ganglion, Manduca sexta, cell lineage.

Introduction

The assembly of the nervous system involves both theproliferation of undifferentiated cells and their commit-ment to express particular morphological and biochemi-cal characteristics. In many invertebrates, these twoaspects of neurogenesis are closely linked. During theformation of the insect CNS, for example, mostembryonic neurons are derived from identified stem cellneuroblasts, and the phenotypes that they subsequentlyexpress may be determined by the mitotic sequence inwhich they arise (Bate, 1976; Thomas et al. 1984;Taghert and Goodman, 1984; Doe et al. 1988).Moreover, the locations of most neurons within thedeveloping ganglia are determined primarily by the

spatial arrangement of the stem cells from which theyarise, with only minor repositioning of neighboring cellsas development proceeds (Goodman et al. 1984;Campos-Ortega and Hartenstein, 1985). Quite a differ-ent sequence of events occurs during retinal formationin Drosophila, however, in which the proliferativephase of neurogenesis produces a field of uncommittedprecursor cells, while neuronal differentiation is sub-sequently directed by a series of position-specificinductive interactions (Ready et al. 1976; Basler andHafen, 1989; Baker et al. 1990).

A third neurogenic program has recently beencharacterized during the formation of the entericnervous system (ENS) of the moth, Manduca sexta(Copenhaver and Taghert, 1990). As in other insects,

1116 P. F. Copenhaver and P. H. Taghert

the ENS of Manduca spans the length of the alimentarytract and provides innervation to the visceral muscu-lature and several related structures. Within the ENS ofManduca, two distinct domains can be identified:anteriorly, the enteric neurons are organized intoseveral small ganglia on the foregut surface, while moreposteriorly, a distinct set of cells is distributedthroughout a branching nerve plexus (the entericplexus) that spans the foregut-midgut boundary. Thislatter population has been named the EP cell group(Copenhaver and Taghert, 1989a; see Fig. 1). In aprevious report (Copenhaver and Taghert, 1990), weshowed that the EP cells arise en masse from aneurogenic placode within the foregut epithelium, butdelay their differentiation until after they have migratedacross the gut surface and reached their maturepositions (Copenhaver and Taghert, 19896). Moreover,at least some aspects of their differentiation (includingneurotransmitter expression) are delayed until aftermigration is complete, and then are expressed in aposition-specific manner (Copenhaver and Taghert,1989a). In this regard, differentiation of the EP cellpopulation resembles patterns of neurogenesis seen indeveloping vertebrate systems, in which both centraland peripheral neurons undergo extensive migratorydisplacements and only later express phenotypes thatare correlated with specific regions of the nervoussystem (Rakic and Sidman, 1973; Le Douarin, 1982;Sanes, 1989; Austin and Cepko, 1990).

To complete our characterization of neurogenesis inthe embryonic ENS in Manduca, we have examined thedevelopmental events by which the second major groupof enteric neurons, those constituting the frontal andhypocerebral ganglia, arise and become committed toexpress their mature phenotypes. We now report thatthe neurons of the enteric ganglia arise from threeneurogenic zones in the foregut epithelium that,surprisingly, are not mitotically active. Rather, thesethree zones produce chains of undifferentiated cells thatdelaminate from the epithelium onto the foregutsurface and only subsequently undergo a limited phaseof mitotic activity prior to their differentiation. Unlikethe large neuroblasts of the insect central nervoussystem, each of these zone-derived precursors gives riseto 2-4 presumptive neurons of approximately equalsize, a pattern of mitosis that is reminiscent of themidline precursor (MP) class of neural progenitors inthe insect CNS (Bate and Grunewald, 1981; Goodmanet al. 1981). Subsequently, the zone-derived neuronsundergo extensive reorganization and delay theirdifferentiation as the primordial ganglia are formed,similar to the delayed sequence of differentiation seenin the enteric plexus. Once the ganglia are assembled,however, a number of the neurons can be recognized onthe basis of cell-specific phenotypes, while many othercells can be categorized as members of a particularsubtype. Thus in the ENS of Manduca, a novel blend ofneurogenic and differentiative events that have beenpreviously associated with other regions of the nervoussystem gives rise to the enteric ganglia.

Materials and methods

Characterization of neuronal phenotypesAnimal rearing and embryonic staging was performed aspreviously described (Copenhaver and Taghert, 1989a, 1990)and by reference to published schedules of external andinternal markers (Dorn et al. 1987; Broadie et al. 1991). Thedeveloping ENS was visualized by dissecting embryos in thefollowing medium (vol/vol): 50% Schneider's Drosophilamedium, 40% Eagle's basic salts, 9.9% heat-inactivated fetalcalf serum, 0.09% penicillin-streptomycin (Sigma #P-0906),0.01 % insulin, supplemented with Manduca hemolymph(Copenhaver and Taghert, 1990; after Chen and Levi-Montalcini, 1969; and Seecof et al. 1971). For cell counts, theenteric ganglia of embryos and postembryonic larvae werestained in whole-mount with toluidine blue (after the methodof Fahrbach and Truman, 1987). Structural features ofindividual neurons were revealed by iontophoresis of the dyelucifer yellow CH (5% in 2 M LiCl; Sigma), followed by theapplication of an anti-lucifer yellow antiserum (1:400; Taghertet al. 1982).

Whole-mount immunohistochemical staining was per-formed as previously described (Copenhaver and Taghert,1989a), using 2 % paraformaldehyde or a modified Zamboni'sfixative (4 % paraformaldehyde, 15 % saturated picric acid, insodium phosphate buffer, pH7.2). Ascites fluid containingmonoclonal antibody TN-1, which recognizes a cell-surfacemolecule that may be related to fasciclin II (Nardi, 1990), wasused to visualize the developing ENS throughout the period ofganglion formation (at dilutions of 1:20000 to 1:40000;Copenhaver and Taghert, 19896). Rabbit antisera madeagainst commercially synthesized FMRFamide (Phe-Met-Arg-Phe-NH2; Sigma) were used at concentrations of 1:4000to 1:8000. Controls for this antiserum included the applicationof pre-immune serum in place of the immune serum and pre-absorption of the antisera with the conjugated antigen, bothof which resulted in an inhibition of positive staining.

Antisera to a number of other candidate transmittersubstances were used at the following concentrations: anti-adipokinetic hormone (AKH; gift of Dr H. Schooneveld) sera#241 and #433 at 1:400 and 1:1000, respectively; anti-proctolin (gift of Dr M. O'Shea) at 1:500; anti-Substance P(serum R5; gift of Dr J. Krause) at 1:5000; anti-cholineacetyltransferase (CHAT; gift of Dr M. Forte) at 1:400; anti-g-aminobutyric acid (GABA; gift of Dr J. Hildebrand) andanti-[Dro5op/ii/a]-dopa decarboxylase (DDC; gift of Dr R.Hodgetts) were both used at 1:1000 to 1:2000. Commerciallyavailable antisera to serotonin (Immunonuclear Inc) was usedat 1:400 to 1:1000. Monoclonal antibodies to gastrin/CCK(gift of Drs A. Strack and A. Lowey) and to the molluscansmall cardioactive peptide (SCPB; gift of Drs B. Masinovskyand A. O. D. Willows) were used at dilutions of 1:200 and1:20, respectively. A monoclonal antibody against [Dros-op/i/TaJ-choline acetyl transferase (gift of Drs M. Forte and W.Wolfgang) was used at 1:50 concentration. For most of theimmunohistochemical reactions described above, we did notperform all of the appropriate controls needed to characterizethe transmitter-related epitopes; rather, the various antiserawere used as a means of distinguishing individual phenotypeswithin the enteric ganglia, as a prelude to more extensiveanalyses of specific cell lineages in the developing embryo.

Mitotic relationships in the developing ENSStaged embryos were selected at specific times throughout theperiod of ganglion formation (24-40% of development; 1 %of development equals ~1 h of real time) and labelled with the

Neurogenesis in the insect ENS 1117

thymidine analogue, 5-bromo-2'-deoxyuridine (BrdU;Sigma) using previously described methods (Truman andBate, 1988; Bodmer et al. 1989; Copenhaver and Taghert,1990). After 2h of incubation with BrdU (SO^gml"1), thepreparations were fixed and stained with an antibody to BrdUat concentrations of 1:30 to 1:50 (Becton-Dickinson; seeGratzner, 1982). Cell lineage relationships within the devel-oping enteric ganglia were examined using intracellularinjections of fluorochrome-coupled dextran amines (Copen-haver and Taghert, 1990; after Gimlich and Braun, 1985, andWetts and Fraser, 1988). Lysinated tetramethylrhodaminedextran amine (LRD; lOxlO3^) or lysinated fluoresceindextran amine (FRD; lOxlO 3 ^; from Molecular Probes,Inc.) was injected into individual zone-derived cells at timesthroughout this same period of development, and the numberand distributions of labelled progeny were examined after 24 hin embryonic culture. Preparations were also routinelycounterstained with TN-1 to visualize the overall morphologyof the ENS.

Results

Neuroanatomy of the anterior ENSAs in other insect species, the ENS of Manduca spans

the length of the alimentary tract (Fig. 1A), supplyinginnervation to the visceral musculature of the foregut,midgut and hindgut, and to several adjacent structures.The gut also receives innervation from the brain andterminal abdominal ganglia, but most of its innervationis supplied by discrete populations of enteric neuronsthat are localized within specific regions of the ENS onthe gut surface. Previously, we have described thedifferentiation of the EP cell group, which occupies abranching nerve network (the enteric plexus) that spansthe foregut-midgut boundary (Fig. IB; Copenhaverand Taghert, 19896, 1990). In this report, we havefocussed on the developmental origins of the secondmajor group of enteric neurons that form the anteriorenteric ganglia and associated neural structures.

Most of the neurons in the anterior ENS areorganized into two contiguous ganglia, the frontal andhypocerebral ganglia (FG and HG) that lie superficiallyon the dorsal surface of the pharyngeal region of theforegut (Fig. IB). Typical of all insects, the frontalganglion in Manduca is the largest structure of the ENS.It lies just rostral to the brain and is connected to the

A. B.

Fig. 1. Dorsal view of the enteric nervoussystem (ENS) of Manduca sexta(postembryonic larval stage).(A) Schematic overview of the threeprincipal divisions of the alimentary tract(foregut, midgut and hindgut) with thecomponents of the ENS highlighted inblack. The thickened regions of the ENSindicate the distribution of enteric neuronsof the foregut and midgut surface. Thebrain (BR) is shown in its normal positionabove the foregut, while the terminalabdominal ganglion (AT) has been rotatedlaterally from its mid-ventral position.(B) An enlarged view of the anteriorportion of the alimentary tract to show theganglionic regions of the ENS. The brainhas been omitted to show the frontalganglion (FG), hypocerebral ganglion(HG) and frontal ganglion connectives(FGC) which normally terminate in thetritocerebral lobes of the brain. Therecurrent nerve (RN) runs along thesurface of the foregut from thehypocerebral ganglion to the enteric plexus(EP), which spans the foregut-midgutboundary. The neurons of the ganglia andrecurrent nerve (filled profiles) arise by adifferent sequence of developmental eventsfrom the cells of the enteric plexus(unfilled profiles). The neuroherhalcomplex of the brain, consisting of thebilaterally paired corpora cardiaca (CC)and corpora allata (CA), are connected tothe recurrent nerve by the 'nervicardiostomatogastrici'(cardiostomatogastric nerves).Scale=0.1mm.


B

' * • • ! • •

0\

rn

rn

N* • * %

^'A

tritocerebral brain lobes via the bilaterally pairedfrontal ganglion connectives (FGC). The generalmorphology of the frontal ganglion is analogous to theorganization of ganglia of the insect CNS: its ~70neurons are segregated in the form of a cortical dome(Fig. 2A), 2-4 cell layers thick, that surrounds a ventralneuropil. Immediately caudal to the frontal ganglion isthe hypocerebral ganglion (Fig. IB), so named becauseit sits directly below the brain on the gut surface. Thiselongate ganglion contains —40 neurons that arearrayed in loose columns along the length of theganglion.

Posteriorly, the hypocerebral ganglion is continuouswith the recurrent nerve, which in Manduca alsocontains a small population of enteric neurons that arevariably dispersed along the length of the nerve. Therecurrent nerve is also connected to the majorneurohemal organs of the brain (the corpora cardiaca-corpora allata complex, or CC-CA), via bilaterally

Fig. 2. Photomicrographs of the anterior enteric gangliaand related structures on the foregut surface.(A) Toluidine-blue-stained preparation of the frontalganglion taken from a 5th instar larva, shown in dorsalview. The neurons form a cortical dome that covers theunderlying neuropilar region of the ganglion. (B) Lateralview of the frontal ganglion (same age as in A) stainedimmunohistochemically with an anti-FMRFamideantiserum. Two large neurons in the dorsolateral quadrantof the ganglion are intensely stained; these cells haveprocesses that run through the ventral neuropil of theganglion and into the recurrent nerve (rn, out of focus inpanel D). (C) Lateral view of the frontal ganglion from a1st instar larva, stained with antisera to AKH. Two dorsalneurons and two ventrolateral neurons show positiveimmunoreactivity, and one pair sends immunoreactiveprocesses into the recurrent nerve (out of view).(D) FMRFamide-immunoreactive processes in the NCSnerves (clear arrows) that run from the corpora cardiaca tothe recurrent nerve. These processes originate fromprotocerebral neurosecretory cells in the brain andterminate in the vicinity of the recurrent nerve (see text).(E) Two FMRFamide-immunoreactive neurons near theposterior boundary of the hypocerebral ganglion; thesecells are bipolar and often are separated along therecurrent nerve. (F) FMRFamide-immunoreactive terminalprocesses (arrowheads) that are derived from the pair offrontal ganglion neurons shown in panel B. The positivelystained axons of these cells are clearly visible within therecurrent nerve in this figure. Scale=100/xm.

paired nerves (Fig. IB). These nerves have been givena variety of names (Fraser and Pipa, 1977; Copenhaverand Truman, 1986), but will be described in this paperas the 'nervi cardiostomatogastrici' (NCS; after Penzlin,1985). Near the foregut-midgut boundary, the recur-rent nerve is continuous with the apex of the entericplexus, from which the plexus nerves bifurcate andextend onto adjacent regions of the visceral muscu-lature.

Neuronal phenotypes in the postembryonic ENSWithin the enteric ganglia of Manduca, cell-specificphenotypes could be distinguished both by anatomicaland biochemical criteria. The relative positioning of theneuronal somata varied somewhat from animal toanimal and was often asymmetric across the midline ofthe ganglia (e.g. Fig. 2A, 2B). Nevertheless, a numberof the cells could be routinely identified in postembryo-nic larvae on the basis of morphology, transmitterphenotype, and approximate soma position. Forexample, two of the largest neurons (~40^xm) in therostral region of the frontal ganglion stained with anantiserum against the molluscan neuropeptide FMRFa-mide (Fig. 2B). The axons of these cells could beroutinely traced along the recurrent nerve and ontospecific muscle fibers near the foregut-midgut bound-ary (Fig. 2F). A second set of four neurons in thefrontal ganglion reacted positively to an antiserumagainst the C-terminus of the insect neuropeptide,AKH (Fig. 2C), and these cells possessed axons thatextended through the enteric plexus and onto themuscle bands of the midgut (not shown).


Many other neurons in the frontal ganglion could notbe uniquely identified but could be categorized into oneof several distinct subtypes: for example, severalsmaller cells (2-4) were stained with anti-serotoninantibodies and a variable number of cells (10-14) werestained weakly with anti-dopa decarboxylase (notshown). Consistent patterns of transmitter expressionwere also observed in the hypocerebral ganglion andrecurrent nerve, including one pair of bipolar neuronsthat were immunoreactive to anti-FMRFamide(Fig. 2E), and an additional subset of cells that stainedmore weakly with anti-DDC (not shown). Severalantisera that label structures in the ENS in other studies(e.g. Homberg et al. 1987; Davis et al. 1989) did notstain any of the enteric neurons in Manduca, includingantisera to proctolin and GABA, although immuno-reactive processes projecting to the frontal ganglia fromthe brain were detected with antisera to GABA andCHAT (not shown). In addition, FMRFamide-immu-noreactive processes could be detected within the NCSnerves that join the recurrent nerve with the CC-CA(Fig. 2D). These fibers originate from a set of cerebralneurosecretory cells within the protocerebrum of thebrain (a subset of the 'type Ila' cells; Copenhaver andTruman, 1986; see also Homberg et al. 1991). Antiseraagainst AKH also stained the intrinsic neurons of thecorpora cardiaca, some of which sent immunoreactiveprocesses into the NCS (not shown). As discussedbelow, the anterior ENS and the corpora cardiaca aredevelopmentally related; these observations suggestthat the two structures may share overlapping func-tions, as well.

The morphological phenotypes of cells within theenteric ganglia were examined in more detail bysystematically injecting individual cells in differentregions of the ganglia of mature embryos with luciferyellow. The results of 72 such preparations revealed anumber of representative cell types (each identified in>3 fills) that could be grouped by their primaryprojection patterns, as shown in Fig. 3. In both thefrontal and hypocerebral ganglia, several morphologi-cal subtypes were found to possess unilateral (orasymmetric) projection patterns (e.g. Fig. 3A, D, I),but the vast majority of dye-injected cells had bilaterallysymmetrical processes. Within the frontal ganglion,neurons could be classified on the basis of whether theirprocesses were confined to the ENS (Fig. 3A-C) orwhether they also projected to the CNS (Fig. 3D-E). Inthe latter case, they usually terminated in the tritocere-bral lobes of the brain, although a few cells projectedinto the protocerebrum or through the brain connec-tives and into the subesophageal ganglion (not shown).An additional subset of cells sent processes both to theCNS and to the gut surface, providing innervationeither to the nearby pharyngeal musculature (Fig. 3F)or to other foregut musculature (Fig. 3G). Similarlywithin the hypocerebral ganglion, one subset of cellswas found that projected only to peripheral regions ofthe ENS and gut surface (Fig. 3H-J), while anothersubset sent bilateral projections to the CNS, as well(Fig. 3K-L). These morphological classes are similar to

the subtypes of enteric neurons that have beendescribed in orthopteran species (Gundel and Penzlin,1978; Kirby et al. 1984).

It should be emphasized that while Fig. 3 showsrepresentative examples of the different cell classes inthe enteric ganglia, these drawings do not indicate all ofthe anatomical variations that we have observed. Forexample, both the FMRFamide-positive and AKH-positive cell types shown in Fig. 2 have morphologiesthat are generally similar to the cell class illustrated inFig. 3C, although both sets of peptidergic neurons gaverise to much more extensive peripheral terminations, asalready described. It was also not possible to examineevery neuron unambiguously, given the variability insoma positions that occurred in both the frontal andhypocerebral ganglia. Nevertheless, these observationsindicate that the anterior enteric ganglia of Manducaare organized in a manner that is similar to the insectCNS: within the enteric ganglia, cells form discretecortical layers that are segregated from the underlyingneuropilar regions. In addition, at least some of theneurons are individually identifiable by anatomical andbiochemical criteria, while many of the smaller neuronsmay be classified on the basis of their transmitterphenotypes or shared morphological characteristics. Asdescribed below, the variability that we observed in therelative positions of particular neurons could be tracedto the initial assembly of the enteric ganglia duringembryogenesis.

Developmental origins of the enteric gangliaTo characterize the formation of the enteric ganglia inManduca, we visualized the embryonic ENS at pro-gressive stages of development using the monoclonalantibody TN-1 (Taghert et al. 1983; Carr and Taghert,1988), which selectively labels the neuronal componentsof the ENS as they differentiate on the gut surface(Copenhaver and Taghert, 19896). The earliest indi-cation of neurogenesis in the ENS was detected at about24% of embryogenesis, with the appearance of threeneurogenic 'zones' in the mid-dorsal epithelium of thestomodeum (Fig. 4, 5A). These three zones, which wedesignated Z1; Z2 and Z3, differentiated at about thesame time, each consisting of a small number ofepithelial cells (6-10 cells) that were weakly TN-1positive and that had begun to delaminate from theepithelial layer. When viewed dorsally (Fig. 5E), thezones initially appeared as rounded structures thatcould be distinguished from the surrounding epi-thelium; this image is reminiscent of the 'clear areas'reported by Baden (1936) in the foregut of theembryonic grasshopper. A sagittal view of this process(Fig. 5A, B) showed that the appearance of the zonesresulted from a marked change in the shape of the zonecells, the basal ends of each cell becoming increasinglyenlarged with a corresponding narrowing of the apicalportions. As development progressed, the clustering ofthese cells gave each zone a distinctive cone-shapedappearance, which tapered at its apical margin andexpanded onto the basal epithelial surface (Fig. 5B, C).

Over the next 15 % of development (25-40 %), these

c

X,

Fig.

3.

Rep

rese

ntat

ive

mor

phol

ogie

s of

ind

ivid

ual

neur

ons

in t

he f

ront

al a

ndhy

poce

rebr

al g

angl

ia (

take

n fro

m c

amer

a lu

cida

dra

win

gs o

f dy

e-in

ject

ed c

ells

).T

he d

iffer

ent

cell

type

s ha

ve b

een

arra

nged

on

the

basi

s of

the

ir p

rim

ary

proj

ectio

n pa

tter

ns.

(A-C

) Fr

onta

l ga

nglio

n ne

uron

s w

hose

pro

cess

es a

reco

nfin

ed t

o th

e E

NS

and

adja

cent

gut

mus

cula

ture

. (D

-E)

Fron

tal

gang

lion

neur

ons

that

hav

e pr

oces

ses

exte

ndin

g in

to t

he C

NS

via

the

fron

tal

gang

lion

conn

ectiv

es (

FG

C);

the

pro

ject

ions

of

thes

e ce

lls i

nto

the

trito

cere

bral

bra

inlo

bes

are

indi

cate

d by

the

bro

ken

proc

esse

s. A

and

D s

how

exa

mpl

es o

fne

uron

s w

ith u

nila

tera

l pr

ojec

tions

; m

ost

of t

he c

ells

in t

he f

ront

al g

angl

ion

have

bila

tera

l pr

oces

ses.

E s

how

s a

rare

cel

l ty

pe t

hat

has

bila

tera

l pr

oces

ses

runn

ing

into

the

CN

S bu

t no

det

ecta

ble

term

inat

ions

with

in t

he E

NS

. T

hem

ajor

ity o

f ne

uron

s in

the

fro

ntal

gan

glio

n co

ntri

bute

to

the

vent

rally

situ

ated

neur

opil

of t

he g

angl

ion.

F a

nd G

sho

w n

euro

ns t

hat

have

ter

min

atio

ns i

n bo

thth

e C

NS

and

EN

S, a

nd s

end

mul

tiple

pro

ject

ions

ont

o th

e ad

jace

nt g

utm

uscu

latu

re.

(H-J

) N

euro

ns i

n th

e hy

poce

rebr

al g

angl

ion

that

inn

erva

te t

head

jace

nt f

oreg

ut m

uscu

latu

re.

(K a

nd L

) E

xam

ples

of

hypo

cere

bral

gan

glio

nne

uron

s th

at a

lso

have

pro

cess

es r

unni

ng t

hrou

gh t

he f

ront

al g

angl

ion

and

into

the

brai

n.


three zones gave rise to columns of cells that extendedalong the foregut surface (Fig. 4). Individual cellsemerged from the zones, gradually losing continuitywith the epithelial layer and assuming a roundedmorphology. As additional cells were generated fromthe zones, those cells that had already emerged becamedisplaced anteriorly, so that three distinct chains of cellscould soon be distinguished on the foregut surface(Fig. 5B, C). These three chains overlapped, the cellsfrom zone 3 extending over the cells of zone 2 and thenover zone 1 (Fig. 5C). As the stomodeum continued toelongate, cells from all three zones became increasinglyintermingled, forming a loose cellular ridge thatsubsequently became reorganized into all of the majorstructures of the anterior ENS. Those cells nearest theanterior margin of the foregut aggregated into abulbous cluster (Fig. 4, 30-39%), which graduallycondensed into the incipient frontal ganglion (Fig. 4,5D). At the same time, adjacent cells coalesced to formthe hypocerebral ganglion, while more posteriorly, theresidual cell columns became progressively thinner andelongated to form the recurrent nerve (Fig. 5F). Othercells from the anterior cluster migrated laterally androstrally, establishing the major nerve roots associatedwith the enteric ganglia (Fig. 4, 36-42%).

While the initial formation of the three neurogeniczones occurred more or less simultaneously (around24 %), the contributions of the different zones variedboth in duration and in the number of cells that theyelaborated onto the foregut surface. Zone 3, the mostposterior zone (Fig. 4, 5A), persisted for the shortestperiod. As with the other zones, zone 3 initially gaverise to a continuous chain of cells that extended onto theforegut surface (Fig. 5A). Starting around 27%,however, this zone underwent a transformation, duringwhich all of the residual cells within the zone graduallyemerged onto the foregut surface (Fig. 4). As a result,by 30% of development, the zone was no longerapparent in the gut epithelium (Fig. 5B). This samesequence of events subsequently occurred in zones 1and 2, as well. The residual cells of zone 2 graduallyemerged from the epithelium between 36 and 38 %(Fig. 4, 5C), while the cells of zone 1 maintained apersistent connection with the epithelial layer until38-40 % (see Fig. 5D, F). By 42 %, all three zones haddisappeared, leaving only the mid-dorsal ridge of cellson the foregut for the continued differentiation of theENS.

Concurrent with this sequence of zone-derivedneurogenesis, a second major program of neurogenesiscommenced and gave rise to the EP cell population ofthe enteric plexus. As previously described (Copen-haver and Taghert, 1990), the EP cells are derived froman epithelial placode that invaginates into the bodycavity from the protruding lip of the dorsal foregut(invaginating from the apical to the basal surface of theepithelium). In the present study, we observed that theneurogenic zones of the anterior ganglia and thisneurogenic placode were spatially related but tem-porally distinct. As shown in Figs 4 and 5, the EP cellplacode first differentiated at about 30 % of develop-

ment, encompassing the narrow strip of epithelium thathad initially given rise to the zone 3 cells. However, theneurogenic placode appeared only after zone 3 hadbeen obliterated. During the subsequent invaginationof the placode, the last of the zone 3-derived cellsremained closely apposed to the emerging cell packet;these residual zone 3 cells were clearly distinguishableas a small cluster of rounded cells at the apex of the EPcell group (Fig. 5D,F, arrowhead). In this manner, themore anterior domains of the ENS established conti-nuity with the components of enteric plexus from theearliest stages of their differentiation, a relationshipthat was maintained throughout all subsequent phasesof development.

Patterns of DNA synthesis during zone neurogenesisTo characterize the mitotic patterns that were associ-ated with the three neurogenic zones, we next mappedthe distributions of cells that were undergoing activeDNA synthesis throughout the period of entericganglion formation, using the BrdU-antiBrdU tech-nique (see methods). Surprisingly, during the initialphases of neurogenesis (24-30%), we could detect nolabelled cells either within the three zones themselvesor in the delaminating cell groups (Fig. 6), althoughthere was substantial labelling in the surroundingepithelium. Only after individual cells had completelyleft the epithelial layer could labelled nuclei be detectedwithin the zone-derived groups. In any particulardevelopmental stage, only a small number of zone-derived cells were found to be immunoreactive forBrdU (typically between 4-10 cells per preparation),and these were scattered along the length of the foregutsurface (Fig. 6, 32-40%). At no time could we detectlabelled nuclei within the neurogenic zones themselves;rather, immunoreactive cells were often found justoutside of the zones, presumably having recentlyemerged from the epithelial layer. Scattered examplesof labelled cells could also be found in more rostralregions of the developing ENS, as well.

In general, we saw no evidence for organizedpatterns of mitosis during the subsequent coalescenceof the zone-derived cells, although synthetically activenuclei occasionally were seen within the cell groups ofboth the frontal and hypocerebral ganglia. In rarepreparations, a coordinated pattern of labelled nucleiwas detected in the vicinity of individual zones just priorto their obliteration (Fig. 6, 36 %): in these cases, manyof the residual cells that were completing the process ofdelamination became immunoreactive for BrdU simul-taneously, forming a 'halo' of labelled nuclei around theremnants of the zone. The fact that this pattern wasonly seen in a few preparations (3 out of 50) suggestseither that this coordinated mitotic pattern occurredwithin an extremely narrow developmental window, orthat this pattern was simply due to the chancesynchronization of cells in the same phase of theirdifferentiative cycle.

During the subsequent period of enteric ganglionformation, a scattered number of zone-derived cellswere invariably labelled (38-40%), suggesting that at


24

27

30

33

36

39

42°/c


Fig. 4. Origins of the enteric ganglia from threeneurogenic zones in the foregut epithelium. Panels showsequential stages of development (from 24 to 42 %; 1 % ofdevelopment=lh) as visualized by staining with TN-1 andredrawn from camera lucida images. The elongation of theforegut into the body cavity is shown (A) in mid-sagittalsection, with anterior to the left; and (B) in dorsal view,with anterior at the top of the page. The reflected layer ofepithelium at the posterior end of the foregut is shown inA, while the foregut-midgut boundary is indicated in B.Zone cells that are still within the epithelial layer of theforegut tube are highlighted in black. The three neurogeniczones first differentiate in the foregut epithelium at about24 % (Zi, Z2, Z3i arrows) and generate columns of cells(shown as open profiles) that delaminate onto the foregutsurface. Cells from all three zones intermingle and shiftanteriorly as the foregut continues to elongate posteriorly(27-33%). Cells at the anterior end of the foregut coalesce(36-42%) to form the incipient frontal ganglion (FG),while cells that are situated more posteriorly coalesce toform the hypocerebral ganglion (HG) and recurrent nerve(RN). Each of the zones is active for a limited period ofdevelopment before it is extruded from the foregut layer(arrowheads): zone 3 has disappeared by 30%, zone 2 by38%, and zone 1 by 40% of embryogenesis. Soon afterthe extrusion of zone 3 (30%), the neurogenic placode thatwill form the EP cells also begins to differentiate,encompassing the epithelial region in which zone 3originally appeared. The residual zone 3 cells remain incontact with the invaginated packet of EP cells(arrowhead, 30%), thereby establishing continuity betweenthe foregut and midgut domains of the ENS.

least some of the zone-derived cells underwent morethan one round of mitosis. However, by 42-43 %, thegeneration of neurons in the enteric ganglia wasessentially complete: numerical analyses of toluidine-blue-stained preparations at these stages indicated thatthe mature complement of frontal ganglion cells hadalready been attained (68.6±7.8 cells, «=16), althoughthe ganglion continued to expand in size throughout theremainder of embryonic and postembryonic develop-ment. Also during subsequent development, specificsubsets of cells derived from zones 2 and 3 did continueto be mitotically active, producing a large number ofglial-like cells that populated the branches of the entericplexus as well as the recurrent nerve (to be described ina subsequent report).

Clonal analysis of the neurogenic zone cellsThe results of the foregoing experiments indicated thatwhile the neurogenic zones gave rise to the progenitorsof the enteric ganglia, the zones themselves were notsites of active cell proliferation. To gain insight into thecontributions of individual progenitors to the develop-ing ENS, we next examined the clonal relationships ofcells within the enteric ganglia using intracellularinjections of fluorochrome-coupled dextran amines inembryonic culture. While individual cells within aparticular zone could be labelled by this technique,variability in cellular positions within the zones prec-luded us from determining whether specific precursorcells from a particular zone could be linked with unique

cell lineages. Nevertheless, these experiments revealedthe replicative patterns involved in the formation of theenteric ganglia and provided insight into the patternsand distribution of progeny that were derived from thethree different zones.

When individual zone cells were injected during theinitial phases of neurogenesis (25-28 %), a small subsetof labelled neurons were typically labelled within thecoalescing ganglia (Figs 7,8). The progeny of individualcells were usually clustered in either the frontal orhypocerebral ganglion (e.g. Fig. 8C,F), althoughlabelled neurons were occasionally distributed acrossboth regions (Fig. 7A). The number of labelled neuronsin these preparations was consistently low (Fig. 9), witha maximum of four cells being labelled following theinjection of a progenitor cell near one of the zones(Figs 7A, 8C,F). More often, we subsequently detectedonly one or two labelled neurons within the developingganglia (Fig. 7B), regardless of the position occupied bythe progenitor cell at the time of injection. In manycases, labelled neurons could be seen extendingneurites in patterns reminiscent of the phenotypesthat we observed in the mature ganglia (compareFigs 3 and 7). When more than one labelled cell wasdetected, the cells were typically similar in size andextent of outgrowth. Not all of the neurons derivedfrom an individual zone cell acquired identical morpho-logical characteristics, however, indicating that indi-vidual progenitor cells could give rise to more than oneneuronal subtype within the enteric ganglia.

During subsequent phases of development(29-36%), the average number of cells that werelabelled in this manner gradually declined (Fig. 9):injections resulting in as many as four labelled neuronsbecame relatively rare, while more frequently only oneor two fluorescent neurons were detected (Figs 7,8B,E). By the time the enteric ganglia had begun tocoalesce (37-40%), most of the injected cells hadalready begun to differentiate (as indicated by the onsetof neurite outgrowth), and typically these cells did notundergo any additional rounds of mitosis (Figs 7,8A,D). At all developmental ages examined, a fewpreparations resulted in no labelled neurons (indicatedin Fig. 9 as '0' labelled progeny), indicating that theinjected cell may have died. We have not yet detectedany evidence for significant patterns of stage-specific orsystematic cell death, however.

We also used these experiments to examine therelative contributions of the three zones to the differentstructures of the ENS. During the initial phases ofneurogenesis (25-28%), cells derived from all threezones were found to contribute progeny to either thefrontal or hypocerebral ganglion and occasionally toboth, as already mentioned. These results are inagreement with our observations of TN-1 stainedpreparations (Figs 3, 4), in which we observed thechains of cells from all three zones streaming anteriorlyin an intermingled fashion prior to the formation of theenteric ganglia. With continued development, however,the positions of labelled progeny became increasingly


mV

rn

42.1

i "fg< * * -

restricted in a predictable manner: cells derived fromthe most anterior zone (zone 1) were most likely to beincorporated into the coalescing frontal ganglion, whilecells from the more posterior zones typically produced

cells that occupied the hypocerebral ganglion orrecurrent nerve. Thus there was a gradual restriction inthe contribution of the neurogenic zones to thedifferent regions of the ENS.


Fig. 5. Photomicrographs of zone neurogenesis and entericganglion formation in preparations stained with TN-1.A-D show mid-sagittal views (anterior is to the left) andE-F show dorsal views (anterior is towards the top of thepage). (A) The developing foregut at 25-26% ofembryogenesis; the three neurogenic zones (Zi, Z2, Z3)have differentiated within the medial region of the dorsalforegut surface and have begun to generate rounded cellsonto the foregut surface. Several large cells that are in theprocess of leaving the epithelial layer are visible in eachzone. (B) By 32-33%, zones 1 and 2 are still visible, butzone 3 has been obliterated; the last of the zone 3 cells(arrowhead) can be seen at the apex of the EP cellplacode. (C) By 36-37%, the last of the zone 2 cells areleaving the epithelial layer. Three distinct chains of cells(one from each of the original zones) can also be seen atthis stage: cells from zone 2 extend over zone 1 and thezone 3-derived cells extend over the other two zones. Thelast of the zone 3 cells (arrowhead) are still positioned nextto the invaginating EP cell packet. (D) 40-41 % (lowermagnification than A-C), an overview of the entire foregutregion; zone 1 has almost disappeared, and the EP cellpacket is completely invaginated. Cells derived from allthree zones have begun to aggregate into the frontalganglion (fg), while more posterior groups will form thehypocerebral ganglion and recurrent nerve (ra). (E) Dorsalview of a preparation at a similar stage as in panel A(25-26%). Zones 1 and 2 are clearly visible in the dorsalforegut epithelium; zone 3 is partially obscured by thereflected foregut lip. Structures labelled lb are thedeveloping labral lobes that will contribute to the larvalmouthparts. (F) Same age as panel D (40-41%), dorsalview. The last of the zone 1 cells are still visible beneaththe migrating cell columns that will form the recurrentnerve. The connection between the zone-derived cells andthe EP cell packet is clearly visible; the frontal ganglion ispartially out of focus. Scale=50/an in A-C, E; 100/an inD and E.

Subsequent differentiation of the anterior ENSWhile most of the experiments in this paper focussed onthe neurogenic phase of enteric ganglion formation(25-40% of development), the differentiation of theanterior ENS occupied a substantially longer period ofembryogenesis. As previously mentioned, some of thezone-derived cells associated with the enteric gangliaalso grew out to form nerve roots (Fig. 4). In addition, anumber of other cell groups were detected with TN-1staining that were not zone-derived but that delami-nated from more lateral regions of the foregutepithelium during the formation of the ganglia (Figs 10,11). These additional cell groups subsequently partici-pated in the differentiation of the major nerves of thefrontal ganglion, hypocerebral ganglion and corporacardiaca.

The initial differentiation of these lateral groupscommenced soon after the neurogenic zones had begunto elaborate neuronal precursors: as early as 30% ofdevelopment, bilaterally paired sets of epithelial cellson either side of the neurogenic zones had begun todelaminate from the foregut epithelium. The earliestand most prominent of these groups subsequentlyaggregated on either side of the frontal ganglion to help

form the frontal ganglion connectives (Fig. 10). Othercell groups differentiated next to the hypocerebralganglion and gradually formed nerves projecting to thebuccal and pharyngeal musculature (Figs 10,11). At thesame time, paired groups of cells also emerged fromventrolateral regions of the foregut, posterior to thelevel of the hypocerebral ganglion (Fig. 10, 35%; seecurved arrows). Cells from these groups graduallyshifted both medially towards the recurrent nerve andlaterally towards the paired corpora allata (structuresthat invaginate from the ectodermal layer of the bodywall), forming the NCS nerves. In addition, a smallnumber (5-8) of cells became incorporated into thedistal portions of the NCS nerves adjacent to thecorpora allata, forming the intrinsic neurons of thecorpora cardiaca. Thus while all of the ganglionicneurons of the ENS are derived from the threeneurogenic zones, an independent population of epi-thelial cells contributes substantially to the differen-tiation of ancillary portions of the ENS in Manduca,including the intrinsic neurons of the neurohemalcomplex of the brain.

Discussion

A model for neurogenesis in the anterior ENSThese experiments show that the enteric ganglia ofManduca arise via a neurogenic sequence that is distinctfrom the patterns of differentiation seen in otherregions of the developing ENS (Copenhaver andTaghert, 1990). As summarized in Fig. 12, threeneurogenic zones appear simultaneously within thecolumnar epithelium of the foregut (at about 24 % ofdevelopment; panel A) and give rise to a progression ofneuronal precursor cells. Without undergoing mitosis,epithelial cells in proximity to these three zones areapparently recruited into the zones and undergo anepithelial-to-mesenchymal transformation, duringwhich the cells delaminate onto the foregut surface(Fig. 12, panel B). Then, only after they have emergedfrom the epithelial layer do the cells enter a limitedphase of mitotic activity. Both the distribution ofmitotically active cells (revealed by the BrdU tech-nique) and the lineage relationships among the entericneurons (as indicated by clonal analysis) have shownthat each precursor produces a maximum of 2-4progeny of approximately equal size and similardevelopmental potentials, the majority being destinedto assume neuronal phenotypes.

As precursor cells continue to be generated from thethree zones, the cells and their progeny shift forward,intermingling as they aggregate into the rostral clustersthat will form the enteric ganglia (Fig. 12, panel C). Theneurogenic zones remain active for only a limited phaseof development: starting with the most posterior zone(Z3), each zone gradually disappears from the epitheliallayer as all of its residual cells emerge onto the foregutsurface (Fig. 12, panels C and D). At the same time, theneurogenic placode that will produce the EP cells of theenteric plexus (Copenhaver and Taghert, 1990) begins


30% 32 34 36a

Fig. 6. Patterns of anti-BrdU antibody staining (taken fromcamera lucida drawings) at progressive developmental stagesduring zone-derived neurogenesis. Synthetically active nucleiare indicated in black. Labelled nuclei occur throughout theforegut epithelium at this time (see Fig. 6D, G) but haveonly been drawn within the zone-derived cell groups and inthe vicinity of the neurogenic placode. Three differentpreparations are shown at 36 % to illustrate rare examples ofsynchronous zone cell labelling (arrowheads; see text).

to form, incorporating the epithelial region from whichthe zone 3 cells initially emerged (Fig. 12, panel D). Asthe ganglia coalesce, some variability occurs in therelative positioning of individual neurons within theganglia (as manifested by the variable positions ofspecific cells observed in post-embryonic animals).Nevertheless, at least some of the neurons subsequentlyexpress individually identifiable characteristics (Fig. 12,panel E), including specific transmitter phenotypes andmorphological features, while many other ganglionicneurons can be classified by subtype. Zone cells alsocoalesce to form the recurrent nerve that serves as apersistent link between the enteric ganglia and moreposterior domains of the ENS, including the developingenteric plexus. By 40% of development, essentially all

of the neurons that will be incorporated into the entericganglia have been generated, and the proliferativephase of neurogenesis is complete.

In several respects, the origins of the enteric gangliain Manduca resembles patterns of neurogenesis thathave been described for the insect CNS. For example,the generation of most central neurons in insects beginswith the differentiation of uniquely identifiable stemcells (neuroblasts) from the neuroepithelium, a processthat is regulated by inhibitory interactions amongequipotent ectodermal cells (Doe and Goodman, 1986).The initial specification of the neurogenic zones in theinsect stomodeum may be regulated in an analogousmanner. Each zone differentiates at a specific (mid-dorsal) position from within a field of apparently

Fig. 8. Clonal analysis experiments in which individual zone-derived precursor cells were injected with a fluorescent dyeand the distribution of their labelled progeny photographed after 24h of development in embryo culture. Panels A, C, D,and E show progeny labelled with lysinated rhodamine dextran (LRD); panels B and F show injections using lysinatedfluorescein dextran. In all panels but B, the preparations were also counterstained with TN-1 and the appropriatefluorochrome to complement the injected dye. A-C show examples of labelled cells that were incorporated into the frontalganglion; D-F show examples of labelled cells in the vicinity of the developing hypocerebral ganglion. (A and D)Examples of single neurons (arrows) that were labelled after injection of individual zone cells at 38% of development.Arrowheads indicate processes from these cells that are in the same plane of focus. (B and E) Examples of pairs oflabelled neurons following precursor injections at 30% of development. (C and F) Examples of 4 labelled neuronsfollowing precursor injections at 25% of development. (The two circled cells in C are out of the plane of view).Scale=50jan.

A. B.Neurogenesis in the insect ENS 1127

c.25%

25%

30%

25% 30%

T35% 40%

Fig. 7. Patterns of labelled progeny in the anterior ENS following fluorescent dye injections of individual zone-derivedcells. The stage at which the original injections were performed is indicated in percent (of development). Labelled progenyhave been redrawn from camera lucida images. (A) Examples of preparations in which four labelled neurons weredetectable following the injection of a single precursor; (B) examples of pairs of labelled neurons; (C) examples of solitarylabelled neurons. The examples shown were chosen to illustrate representative numbers and distributions of progenydetected by this technique (see Fig. 9). The approximate boundaries of the developing enteric ganglia were deduced fromTN-1 counterstaining.

uniform cells, in the absence of any obvious prespecifi-cation of the epithelial layer. As with neuroblasts in theinsect CNS, individual zone cells then lose theircontinuity with neighboring cells and round out ontothe epithelial surface. However, while most neuroblastssubsequently undergo an extended sequence of asym-metric divisions and produce many neuronal progeny

cCDCDO

T3_CD

I)_Q

O

3 -

2 -

1 -

0 -

25-28 29-32 33-36 37-40

Time of Initial Injection (% dev't)

(Bate, 1976; Goodman et al. 1984; Taghert andGoodman, 1984), the zone-derived precursors aremitotically active for only a short period of time. Atmost, individual precursors were found to produce fourprogeny and more typically gave rise to only one or twodifferentiated neurons. In this regard, the zone-derivedprecursors more closely resemble the midline precursor(MP) class of neural progenitors in the insect CNS, eachof which undergoes a single, symmetric division andgives rise to a pair of identified neurons (Bate andGrunwald, 1981; Goodman et al. 1981). Thus one view

Fig. 9. Numerical analysis of the number of labelledprogeny that were observed in the ENS, 24 h afterindividual zone-derived precursors were injected with afluorescent lineage tracer. (1 h equals ~1 % ofdevelopment). The developmental stage of the initialinjection is indicated on the horizontal axis. Each filledcircle represents the result of a single injection (81preparations are included). Only preparations in which anindividual zone-derived cell was unambiguously labelled atthe beginning of the experiment were included in thisanalysis. Preparations in which no labelled cells couldsubsequently be detected are indicated in the '0' column.


35 45%

55% 65%

FGC

of the neurogenic zones in the ENS is that they eachprovide a series of MP-like precursors, whose progenyassume a variety of mature phenotypic characteristicsonly after they have migrated into the developingenteric ganglia.

Cellular identity and the regulation of specificphenotypesAn important aspect of zone neurogenesis is the processby which the neurons of the enteric ganglia becomecommitted to express cell-specific phenotypes. Indi-vidual neurons may differentiate according to theirmitotic ancestry or they may be regulated by positionalinformation encountered during subsequent phases oftheir development. For example, in each neuromere ofthe developing insect CNS, ~7 MP cells emerge fromdistinct regions of the neurectoderm and give rise tounique sets of neurons whose phenotypes are correlatedwith the initial precursor positions (Bate and Grun-wald, 1981; Goodman et al. 1981). In contrast, theprogenitors of the enteric ganglia generate a greatervariety of cell types (Figs 2 and 3) but themselves arisefrom just one of three positions (zones Z!-Z3) within

Fig. 10. Contribution of additionalgroups of foregut epithelial cells to theENS. Drawings were taken from cameraludda images of TN-1 stainedpreparations to show the origins anddifferentiation of cells that delaminatefrom the lateral regions of the foregutand form neural structures. Preparationsfrom 35-65 % show only the anteriorportion of the foregut (indicated by theposition of the black bars at 30%),which continues to expand posteriorlyinto the body cavity throughout thisperiod of development. The first of theancillary cell groups appear on both sidesof the rostral end of the foregut at about30% (large arrowheads). These cellsdelaminate and contribute to theformation of the frontal ganglionconnectives (FGC) and rostral nerveroots of the enteric ganglia (35-65 %,large arrowheads). Additional cell groupssoon appear more posteriorly, including abilateral pair that emerge from theventrolateral regions of the foregut(35%, curved arrows). These cellsextend laterally off the gut surfacetowards the developing corpora allata(CA; stippled structures in 45-65 %stages) and form paired nerves (the nervicardiostomatogastrici; NCS) that connectthe recurrent nerve to the corporacardiaca (CC). A subset of these cellsalso differentiate into the intrinsicneurons of the corpora cardiaca. Othercell groups delaminate in a similarfashion between 35-45 % (smallarrowheads) and form the major nerveroots of the hypocerebral ganglion (HG);by 65 %, all of these ancillary regionshave established continuity with the ENS.

the stomodeal epithelium. If a lineage-based mechan-ism regulates the differentiation of these neurons, thenthe properties of the zones must change with time sothat the precursor cells that emerge from each zone canbe assigned distinct fates. Alternatively, a lineage-independent model of neuronal differentiation in theENS would involve regulatory interactions among thezone-derived progeny during their incorporation intothe developing enteric ganglia. In this scenario,heterogeneity of neuronal phenotype would be regu-lated by a stepwise series of cellular interactions, as hasbeen documented during the formation of the insectretina (Tomlinson and Ready, 1987; Baker et al. 1990).A more thorough examination of cell lineage relation-ships within the ENS (in terms of specific neuronalphenotypes) and an analysis of the developmentalpotential of individual precursor cells will be needed todiscriminate between these possibilities.

It should be noted that our characterization oftransmitter and morphological phenotypes within theenteric ganglia did not provide an exhaustive index ofthe different cell types found in the ENS but wasintended to demonstrate the nature of cellular identities


r fgc

Fig. 11. Photomicrographs of the anterior foregut, stained with TN-1 to show the further development of the entericganglia. (A) Preparation at 40%, in which several additional cell groups adjacent to the frontal and hypocerebral gangliahave begun to stain and delaminate (arrowheads); the incipient frontal ganglion connectives have already formed but areout of focus in this picture (above the top set of arrowheads). (B) At 50%, the frontal ganglion has enlarged substantially,and the laterally positioned cell groups (arrowheads) have begun to form connections with the hypocerebral ganglion andrecurrent nerve. (C) At 60%, both the frontal and hypocerebral ganglia have assumed their mature proportions, althoughthey will continue to expand in size. Most of the major nerve roots from the lateral cell groups have become integratedinto the ganglionic regions of the ENS (arrowheads). The developing recurrent nerve (rn) is indicated by the open arrow.Same scale as in Fig. 5A.

within the ganglia, and to provide markers for futureinvestigations into the regulation of neurogenesis. Anumber of the observations reported in this paper aresimilar to features of the insect ENS that have beenreported elsewhere, including cells (or processes) thatare immunoreactive for GAB A (Homberg et al. 1987),serotonin (Radwan et al. 1989), AKH (Schooneveld etal. 1985; Homberg etal. 1991), and FMRFamide (Whiteet al. 1986; Carroll et al. 1986; Myers and Evans, 1987;Homberg et al. 1990).

Regional differences in the formation of the ENSThe neurogenic sequence that we have described forthe anterior ENS differs markedly from the sequencethat gives rise to the other major group of entericneurons in Manduca, namely the EP cells of the entericplexus (Copenhaver and Taghert, 1989a,b). Whereasthe zone-derived precursors delaminate sequentiallyfrom the foregut epithelium and then become mitoti-cally active, the EP cells arise en masse from anepithelial placode and become mitotically inactive asthey leave the columnar layer (Copenhaver andTaghert, 1990). While at least some of the zone-derivedneurons assume cell-specific phenotypic traits, the EPcells do not assume uniquely identifiable characteristics

but constitute several distinct subtypes that are inter-mingled within the enteric plexus (Copenhaver andTaghert, 1989a). In addition, while both the zone-derived and plexus-derived cell groups undergo sub-stantial rearrangements prior to their terminal differen-tiation, the outcome of migration in the two regions ofthe ENS is strikingly different: anteriorly, the zone cellscoalesce into discrete ganglia, while posteriorly, the EPcells become progressively dispersed throughout thenerves of the plexus.

Despite these differences between the two domainsof the ENS, the neurogenic zones and the neurogenicplacode are developmentally related: the EP cellplacode appeared immediately after zone 3 (the mostposterior zone) had disappeared, and its boundarieswere larger, encompassing the region of epitheliumpreviously denned as zone 3 cells (shown schematicallyin Fig. 12). Thjs relationship is maintained duringsubsequent stages of embryogenesis, as well, with theresidual zone 3-cells eventually forming the neuralpathway through which neurons from both the gangliaand the enteric plexus send their axonal processes(Copenhaver and Taghert, 1989a and unpublishedobservations). Thus the zones and the EP placode arisefrom overlapping regions of the foregut but aretemporally distinct, suggesting that similar mechanisms


ooooooooriQpocR<-g3oooc.qpoo8*©< ^oooog*©*]

Y

may control the initial commitment of the gut epi-thelium into these two programs of neuronal differen-tiation.

In comparing the differentiation of the ENS indifferent insects, at least two (and usually three) 'clearareas' or imaginations have been detected in thedeveloping foregut of other species, which have beenascribed a neurogenic function (Heider, 1889; Poulson,1965; Anderson, 1972; Kobayashi and Ando, 1983).Undoubtedly, these invaginations are equivalent to thethree neurogenic zones that we have characterized inManduca. In Drosophila, few (if any) mitoses havebeen detected within the invaginations themselves, butsporadic divisions have been reported within thedelaminated cell groups (Campos-Ortega and Harten-stein, 1985). These observations corroborate our ownanalysis of the neurogenic sequence that gives rise to

Fig. 12. Schematic model for zone-derived neurogenesis inthe ENS. (A) The neurogenic zones (Zi, Z2, Z3)differentiate within the foregut epithelium in a position-specific manner. (B) The proliferative phase ofneurogenesis involves the recruitment of cells into thezones from the surrounding epithelium without anyregional increase in mitotic activity. Recruited cells arethen induced to undergo a morphological transformation,rounding out of the epithelial layer onto the foregutsurface. Only after individual cells have delaminated dothey enter a limited phase of mitotic activity, giving rise toa maximum of four progeny. (C) Cells that are generatedfrom all three zones shift progressively forward asdevelopment proceeds, intermingling and aggregating intoa bulbous cluster at the rostral end of the foregut. Thezones are active for only a limited period, however, anddisappear sequentially from the foregut. In this panel, thelast of the zone 3 cells are emerging from the epitheliallayer, while zones 1 and 2 are still actively generatingprecursors. (D) With continued development, the residualcells of zone 2 and then zone 1 also emerge from theepithelial layer, while post-mitotic neurons coalesce to formthe frontal and hypocerebral ganglia. Concurrently, theepithelial region that initially gives rise to zone 3 isincorporated into the neurogenic placode that will generatethe enteric plexus cells (EP). (E) Despite the extensivemixing and migration of zone-derived cells during theproliferative phase of neurogenesis, the formation of theenteric ganglia proceeds in an ordered fashion, so thatmany of their neurons can be identified on the basis ofmorphology, transmitter phenotype and relative positionwithin the ganglia. Zone-derived cells also coalesce to formthe recurrent nerve (RN) between the anterior entericganglia and the EP cell group, a connection that persistsduring the subsequent dispersal of the EP cells throughoutthe different regions of the developing enteric plexus. Thusthe two major programs of neurogenesis in the ENS arespatially linked and may be regulated by similar types ofcellular and molecular interactions.

the insect enteric ganglia. With respect to the laterstages of development, we have shown that a number ofcell groups besides the three neurogenic zones delami-nate from the foregut epithelium and contribute to theformation of the ENS. The observation that the corporacardiaca arise in this fashion is novel, in that thederivation of these structures in other insects has beenbeen attributed to one or more of the neurogenic zones(Schoeller, 1964; Kobayashi and Ando, 1983; Campos-Ortega and Hartenstein, 1985). Our observationssuggest that in Manduca, the neurogenic potential ofthe foregut epithelium may be more widespread thanwas previously appreciated.

We wish to thank Drs H. Schooneveld, M. O'Shea, J.Krause, J. Hildebrand, R. Hodgetts, A. Strack, A. Lowey, B.Masinovsky, A. O. D. Willows, M. Forte, and W. Wolfgangfor gifts of the antisera used in this work. We also thank MsMarisa LaGrange for excellent technical assistance, and DrsJ. C. Weeks and S. Matsumoto for critical readings of thismanuscript. This research was supported by NIH grant #NS-21749 to P.H.T., NIH fellowship #F32NS07957 to P.F.C., anda research initiation grant from the Medical ResearchFoundation of Oregon (to P.F.C.).


References

ANDERSON, D. T. (1972). The development of holometabolousinsects. In Developmental Systems: Insects Vol. 1 (ed. S.J.Counce and C.H. Waddington), pp. 166-242. New York:Academic Press.

AUSTIN, C. P. AND CEPKO, C. L. (1990). Cellular migrationpatterns in the developing mouse cerebral cortex. Development110, 713-732.

BADEN, V. (1936). Embryology of the nervous system in thegrasshopper, Melanoplus differentially (Acrididae: Orthoptera)./. Morph. 60, 159-188.

BAKER, N. E., MLODZIK, M. AND RUBIN, G. M. (1990). Spacingdifferentiation in the developing Drosophila eye: a flbrinogen-related lateral inhibitor encoded by scabrous. Science 250,1370-77.

BASLER, K. AND HAFEN, E. (1989). Ubiquitous expression ofsevenless: position-dependent specification of cell fate. Science243, 931-934.

BATE, C. M. (1976). Embryogenesis of an insect nervous system I.A map of the thoracic and abdominal neuroblasts in Locustanugratoria. J. Embryol. exp. Morph. 35, 107-123.

BATE, C. M. AND GRUNEWALD, E. B. (1981). Embryogenesis of aninsect nervous system II: a second class of precursor cells andthe origin of the intersegmental connectives. J. Embryol. exp.Morph. 61, 317-330.

BODMER, R., CARRETTO, R. AND JAN, Y. N. (1989). Neurogenesisof the peripheral nervous system in Drosophila embryos: DNAreplication patterns and cell lineages. Neuron 3, 21-32.

BROADIE, K. S., BATE, M. AND TUBLITZ, N. J. (1991). Quantitativestaging of embryonic development of the tobacco hawkmoth,Manduca sexta. Roux's Arch, devl Biol. 149, 327-334.

CAMPOS-ORTEGA, J. A. AND HARTENSTEIN, V. (1985). Theembryonic development of Drosophila melanogaster. New York:Springer-Verlag.

CARR, J. N. AND TACHERT, P. H. (1988). Formation of thetransverse nerve in moth embryos. I. A scaffold of non-neuronalcells prefigures the nerve. Devi Biol. 130, 487-499.

CARROLL, L. S., CARROW, G. M. AND CALABRESE, R. L. (1986).Localization and release of FMRFamide-like immunoreactivityin the cerebral neuroendocrine system of Manduca sexta. J. exp.Biol. 126, 1-14.

CHEN, J. S. AND LEVI-MONTALCINI, R. (1969). Axonal outgrowthand cell migration in vitro from nervous systems of cockroachembryos. Science 166, 631-632.

COPENHAVER, P. F. AND TAGHERT, P. H. (1989a). Development ofthe enteric nervous system in the moth I. Diversity of cell typesand the embryonic expression of FMRFamide-relatedneuropeptides. Devi Biol. 131, 70-84.

COPENHAVER, P. F. AND TAGHERT, P. H. (19896). Development ofthe enteric nervous system in the moth II. Stereotyped cellmigration precedes the differentiation of embryonic neurons.Devi Biol. 131, 85-101.

COPENHAVER, P. F. AND TAGHERT, P. H. (1990). Neurogenesis inthe insect enteric nervous system: generation of pre-migratoryneurons from an epithelial placode. Development 109, 17-28.

COPENHAVER, P. F. AND TRUMAN, J. W. (1986). Metamorphosis ofthe cerebral neuroendocrine system in the moth Manduca sexta.J. comp. Neurol. 249, 186-204.

DAVIS, N. T., VELLEMAN, S. G., KINGAN, T. G. AND KESHISHIAN,H. (1989). Identification and distribution of a proctolin-likeneuropeptide in the nervous system of the Gypsy moth,Lymantria dispar, and in other Lepidoptera. J. comp. Neurol.283, 71-85.

DOE, C. Q. AND GOODMAN, C. S. (1986). Neurogenesis ingrasshopper and fushi tarazu Drosophila embryos. Cold SpringHarbor Symp. quant. Biol. 50, 891-903.

DOE, C. Q., HIROMI, Y., GEHRING, W. J. AND GOODMAN, C. S.(1988). Expression and function of the segmentation gene fushitarazu during Drosophila neurogenesis. Science 239, 170-175.

DORN, A., BISHOFF, S. T. AND GILBERT, L. I. (1987). Anincremental analysis of the embryonic development of thetobacco hornworm, Manduca sexta. Int. J. Invert. Reprod. Dev.11, 137-158.

FAHRBACH, S. E. AND TRUMAN, J. W. (1987). Possible interactionsof a steroid hormone and neural inputs in controlling the deathof an identified neuron in the moth Manduca sexta. J.Neurobiol. 18, 497-508.

FRASER, J. AND PIPA, R. (1977). Corpus allatum regulation duringthe metamorphosis of Periplaneta americanus: Axon pathways.J. Insect Physiol. 23, 975-984.

GIMUCH, R. L. AND BRAUN, J. (1985). Improved fluorescentcompounds for tracing cell lineage. Devi Biol. 109, 509-514.

GOODMAN, C. S., BASTIANI, M. J., DOE, C. Q., DU LAC, S.,HELFUND, S. L., KUWADA, J. y. AND THOMAS, J. B. (1984). Cellrecognition during neuronal development. Science 225,1271-1279.

GOODMAN, C. S., BATE, C. M. AND SPITZER, N. C. (1981).Embryonic development of identified neurons: origins andtransformation of the H cell. J. Neurosci. 1, 94-102.

GRATZNER, H. G. (1982). Monoclonal antibody to 5-bromo- and 5-iodo-deoxyuridine: a new reagent for detection of DNAreplication. "Science 218, 474—475.

GUNDEL, M. AND PENZUN, H. (1978). the neuronal connections ofthe frontal ganglion of the cockroach Periplaneta americana. CellTissue Res. 193, 353-371.

HEIDER, K. (1889). Die Embryonalentwicklung von Hydrophiluspiceus L. Gustave Fischer, Jena.

HOMBERG, U., DAVIS, N. T. AND HILDEBRAND, J. G. (1991).Peptide-immunocytochemistry of neurosecretory cells in thebrain and retrocerebral complex of the sphinx moth Manducasexta. J. comp. Neurol. 303, 35-52.

HOMBERG, U., KINGAN, T. G. AND HILDEBRAND, J. G. (1987).Immunocytochemistry of GABA in the brain and subesophagealganglion of Manduca sexta. Cell Tissue Res. 248, 1-24.

HOMBERG, U., KINGAN, T. G. AND HILDEBRAND, J. G. (1990).Distribution of FMRFamide-like immunoreactivity in the brainand suboesophageal ganglion of the sphinx moth Manduca sextaand colocalization with SCPB-, BPP-, and GABA-likeimmunoreactivity. Cell Tissue Res. 259, 401-419.

KIRBY, D., BECK, R. AND CLARKE, K. U. (1984). Thestomatogastric nervous system of the house cricket, Achetadomesuca L. I. The anatomy of the system and innervation ofthe gut. J. Morph. 180, 81-103.

KOBAYASHI, Y. AND ANDO, H. (1983). Embryonic development ofthe alimentary canal and ectodermal derivatives in the primitivemoth, Neomicropteryx nipponensis issiki (Lepidoptera)./. Morph. 176, 289-314.

LE DOUARIN, N. M. (1982). The Neural Crest. (Cambridge,England: Cambridge Univ. Press).

MYERS, C. M. AND EVANS, P. D. (1987). An FMRFamideantiserum differentiates between populations of antigens in thebrain and retrocerebral complex of the locust, Schistocercagragaria. Cell Tissue Res. 242, 109-114.

NARDI, J. B. (1990). Expression of a surface epitope on cells thatlink branches in the tracheal network of Manduca sexta.Development 110, 681-688.

PENZUN, H. (1985). Stomatogastric nervous system. InComprehensive Insect Physiology, Biochemistry, andPharmacology Vol. 5 (G.A. Kerkut and L.I. Gilbert, eds), pp.371—406. Pergammon Press, Oxford.

POULSON, D. F. (1965). Histogenesis, organogenesis, anddifferentiation in the embryo of Drosophila melanogasterMeigen. In Biology of Drosophila (ed. M. Demeric), pp.168-274. New York: Hafner Press.

RADWAN, W. A., GRANGER, N. A. AND LAUDER, J. M. (1989).Development and distribution of serotonin in the centralnervous system of Manduca sexta during embryogenesis I. thebrain and frontal ganglion. Int. J. Dev. Neurosci. 7, 27-41.

RAKIC, P. AND SIDMAN, R. L. (1973). Weaver mutant mousecerebellum: defective neuronal migration secondary toabnormality of Bergmann glia. Proc. natn. Acad. Sci. U.S.A.70, 240-244.

READY, D. F., HANSON, T. E. AND BENZER, S. (1976).Development of the Drosophila retina, a neurocrystalline lattice.Devi Biol. 53, 217-240.

SANES, J. R. (1989). Analysing cell lineage with a recombinantretrovirus. Trends Neurosci. 12, 21-28.


SCHOELLER, J. (1964). Recherches descriptives et experimentalessur la cephalogenese de Calliphora erythroccphala (Meigen), aucourse des developpements embryonaire et postembryonnaire.Arch. Zoo!, exp. gen. 103, 1-216.

SCHOONEVELD, H . , ROMBERG-PRIVEE, H . M. AND VEENSTRA, J. A .(1985). Adipokinetic hormone-immunoreactive peptide in theendocrine and central nervous system of several insect species: acomparative immunocytochemical approach. Gen. comp.Endocrinol. 57, 184-194.

SEECOF, R. L., ALLEAUME, N., TEPLITZ, R. L. AND GERSHON, I.(1971). Differentiation of neurons and myocytes in cell culturesmade from Drosophila gastrulae. Expl Cell Res. 69, 161-173.

TAGHERT, P. H., BASTIANI, M., HO, R. K. AND GOODMAN, C. S.(1982). Guidance of pioneer growth cones: filopodial contactsand coupling revealed with an antibody to Lucifer Yellow. DeviBiol. 94, 391-399.

TAGHERT, P. H. AND GOODMAN, C. S. (1984). Cell determinationand differentiation of identified serotonin-immunoreactiveneurons in grasshopper embryos. J. Neurosci. 4, 989-1000.

TAGHEJTT, P. H., TUBLJTZ, N. J., TRUMAN, J. W. AND GOODMAN,C. S. (1983). Monoclonal antibodies against insect peptidergicneurons. Soc. Neurosci. Abst. 9, 314.

THOMAS, J. B., BASTIANI, M. J., BATE, M. AND GOODMAN, C. S.(1984). From grasshopper to Drosophila: a common plan forneuronal development. Nature 310, 203-207.

TOMLINSON, A. AND READY, D. F. (1987). Neuronal differentiationin the Drosophila ommatidium. Devi Biol. 120, 366-376.

TRUMAN, J. W. AND BATE, M. (1988). Spatial and temporalpatterns of neurogenesis in trie central nervous system ofDrosophila melanogaster. Devi Biol. 125, 145-157.

WETTS, R. AND FRASER, S. E. (1988). Multipotent precursors cangive rise to all major cell types of the frog retina. Science 239,1142-1145.

WHITE, K., HURTEAU, T. AND PUNSAL, P. (1986). NeuropeptideFMRFamide-like immunoreactivity in Drosophila: developmentand distribution. J. comp. Neurol. 247, 430-438.

(Accepted 13 September 1991)

Origins of the insect enteric nervous system: differentiation …...0.01 % insulin, supplemented...

Documents

Transcript of Origins of the insect enteric nervous system: differentiation …...0.01 % insulin, supplemented...