Development 112, 397-406 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

397

Expression of the fibroblast growth factor-5 gene in the mouse embryo

OLIVIA HAUB and MITCHELL GOLDFARB*

Columbia University College of Physicians and Surgeons, Department of Biochemistry and Molecular Biophysics, 630 West 168th Street,New York, NY 10032

* To whom reprint requests should be addressed

Summary

Fibroblast growth factors (FGFs) are structurallyrelated mitogens that can regulate the differentiation ofa wide variety of cells. As a step towards elucidating thedevelopmental roles played by one of these factors, wehave used in situ hybridization methods to examineexpression of the murine Fgf-5 gene during embryogen-esis. Fgf-5 RNA was detected at seven distinct sites in thedeveloping mouse embryo: (1) postimplantation epiblast(embryonic day 5|-7i), (2) lateral splanchnic mesoderm(E9i-10i), (3) lateral somatic mesoderm (E10±-12i), (4)

myotomes (E10i-12±), (5) mastication muscle(Ell|-14§), (6) limb mesenchyme (E12i-14i), and (7)acoustic ganglion (E12i-14i). At several of these sites,expression is spatially restricted within the tissues. Weoffer several hypotheses regarding the roles of FGF-5 inmurine development.

Key words: mouse embryo, FGF-5, fibroblast growthfactor-5, RNA, in situ hybridization.

Introduction

Fibroblast growth factors (FGFs) are structurallyrelated mitogenic proteins encoded by at least sevendistinct genes in mammals (Goldfarb, 1990). In additionto their growth-promoting activities towards a broadspectrum of cell types, FGFs are suspected of playingvarious roles in the control of cellular differentiation(reviewed in (Burgess and Maciag, 1989)). In vitro, theprototypic FGFs (acidic and basic FGF) can promoteneuronal survival and neurite outgrowth (Hatten et al.1988; Lipton et al. 1988; Morrison et al. 1986; Unsickeret al. 1987; Walicke et al. 1986; Walicke and Baird,1988), commit sympathoadrenal progenitor cells to thesympathetic neural lineage (Birren and Anderson,1990), regulate the development of skeletal muscleprecursor cells (Lathrop et al. 1985; Seed andHauschka, 1988), and induce mesodermal differen-tiation in amphibian blastula ectoderm (Kimelman andKirschner, 1987; Slack etal. 1987). FGF in vivo implantscan promote angiogensis (Folkman and Klagsbrun,1987), promote the formation of new nerve fiber tracts(Thompson et al. 1989) and prevent retrograde de-generation of neurons in the central nervous system(Anderson et al. 1988). Consistent with their potentialroles in development, several FGF genes are expressedat various stages of embryogenesis (reviewed in(Goldfarb, 1990)).

FGF-5 was first identified as the product of a humanproto-oncogene detected by DNA transfection assays(Zhan et al. 1988). The growth factor is a secreted

glycoprotein (Bates et al. 1991) that is mitogenictowards fibroblasts in vitro (Zhan et al. 1988). As a steptowards understanding the native functions of thisgrowth factor in vivo, we have studied the profile ofmouse Fgf-5 gene expression in adult tissues and duringembryogenesis. We have previously reported thepresence of Fgf-5 RNA at low levels in the adult centralnervous system and its localization within certainneurons (Haub et al. 1990). In this paper, we present ananalysis of Fgf-5 gene expression in the mouse embryo,as detected by in situ hybridization.

Materials and methods

Enzymes and radionucleotid.esEnzymes for nucleic acid biochemistry were purchased fromNew England Biolabs, Boehringer Mannheim and Strata-gene. Radiolabelled nucleotides were purchase fromDuPont/New England Nuclear.

cDNA isolation and RNA filter blot hybridizationA 2.2 kbp cDNA clone was isolated by screening a Ell±mouse embryonic cDNA library (Clonetech) with a murineFgf-5 third exon fragment (Haub et al. 1990). The cDNAclone extends from approximately the 50th codon in the Fgf-5open reading frame to near the 3 end of the message. RNAwas isolated from cells following guanidine isothiocyanateextraction (Chirgwin et al. 1979). CCE embryonic stem (ES)cells were provided by Dr E. Robertson and were maintainedin the undifferentiated state by passage on STO fibroblastfeeder layers, or were used to make embryoid bodies by

398 O. Haub and M. Goldfarb

standard procedure (Martin and Evans, 1975). E6f-E7 eggcylinders were dissected from their decidua and teased free ofectoplacental cone, which is reddish in appearance. E. coliRNA was added as carrier during egg cylinder RNApurification. To quantitate egg cylinder RNA, one tenth ofrecovered material was analyzed by RNA 'northern' filterblotting (Thomas, 1980) in comparison to a 10-1000nanagram range of ES RNA, using rat glyceraldehydephosphate dehydrogenase (GAPDH) cDNA (Fort etal. 1985)as hybridization probe. DNA probes were P-radiolabelledby random hexamer-primed DNA synthesis (Feinberg andVogelstein, 1983).

In situ hybridizationRadiolabelled RNA riboprobes were synthesized in standardreactions (Melton et al. 1984) using SP6, T3, or T7 RNApolymerase, depending upon vector DNA template. Uri-dine(a35S-thio)triphosphate and uridinefa^PJtriphosphatewere incorporated to specific activities of 7X108 and2xl09ctsmin~1 ng~l, respectively. The Pstl-Sacl fragmentof Fgf-5 DNA (Haub et al. 1990) (see Fig. 1) was used astemplate for antisense- and sense-strand riboprobes (antiPSand sensePS), and the Pstl-Pstl cDNA fragment was used tomake antisense probe (antiPP) to corroborate hybridizationdata. A plasmid containing 120 base pairs of non-coding firstexon sequence of the murine tf-cardiac actin gene (Sassoon etal. 1988) and pINT2fg, containing part of the murine int-2gene (Wilkinson et al. 1988), were also used as vectors forriboprobe synthesis.

Embryos (E3i, 5i, 5f, 6}, 6i, 7, 7i, 8, 8i, and dailythereafter through E15i) were dissected from pregnant MF1mice, fixed overnight at 4°C in phosphate-buffered saline plus4 % paraformaldehyde, and embedded in paraffin. E5i to E9iembryos were left in their decidua. E3i blastocysts wereflushed from uteri, prefixed and loaded into dissectedampulae of pseudopregnant females before processing. Serialmicrotome sections (7-8 microns thick) were deparaffinizedand hybridized essentially according to the procedure ofWilkinson (Wilkinson et al. 1987). The only modificationswere in the content of the hybridization mix, which wasprepared as previously described (Haub et al. 1990), andcontained 3 nanograms probe in 0.2 ml per slide. Afterposthybridization RNAase treatment and washes (Wilkinsonet al. 1987), slides were dipped in NTB2 emulsion (E. Kodak)diluted 1:1 with 2% glycerol, and autoradiographed (14-20days for 32P, 6 weeks for 35S). Exposed slides were developedin D19 (Kodak), stained with hematoxylin and eosin, and

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Fig. 1. Mouse Fgf-5 cDNA and riboprobes. The barrepresents the 2.2 kbp Fgf-5 cDNA cloned from a mouseE1U cDNA library. The coding region is filled in, while 3'untranslated sequence is unfilled. Pstl (P), Sacl (S),Hindlll (H), and Kpnl (K) cleavage sites are indicated.Riboprobes are shown with arrows. The Pstl-Sacl 450 bpregion all derives from exon 3, and has been usedpreviously to make antisense (antiPS) and sense (sensePS)ribroprobes (Haub et al. 1990). The more 5' Pstl-PstlcDNA fragment was used to make a corroboratingriboprobe (antiPP).

mounted with Permount (Fisher Scientific). Bright- and dark-field images were photographed from a Nikon Microphot-FXA microscope. All embryonic stages were exhaustivelyanalyzed; multiple embryos were hybridized with both senseand antisense Fgf-5 riboprobes, using a complete series ofsections for each probe for E34-E8i embryos, and alternatinggroups of three sections for both probes with older, largerembryos. Anatomical assignments were made with the aid ofseveral texts on embryonic anatomy (Gasser, 1975; Rugh,1968; Theiler, 1989).

Computer-aided three-dimensional modellingCamera-lucida drawings on transparencies were made frombright- and dark-field images of thirty hybridized sectionsspaced 96 microns apart across an E10i embryo. The drawingswere manually aligned, and then traced into a Sun Viewcomputer using CARP software (Biographies, Inc.). Super-imposed objects were 'built' from the contours of wholeembryo, dorsal aorta and arches, primary head and cardinaltrunk veins, Fgf-5 signal in myotomes, and Fgf-5 signal insomatic mesoderm, and visualized in various combinations fordirect photography from the computer's screen.

Results

In order to localize Fgf-5 expression during embryogen-esis, deparaffinized sections of prefixed mouse embryosat various stages of development were hybridized insitu, using radiolabelled antisense and sense Fgf-5 RNAprobes. Two different antisense probes, transcribedfrom nonoverlapping segments of Fgf-5 cDNA (Fig. 1),detected identical profiles of gene expression. Hybridiz-ation to deparaffinized sections gives signals 3-4 foldweaker than to post-fixed, fresh frozen sections (ourunpublished observations); in fact, our previous in situanalysis of Fgf-5 expression in adult mouse brainrequired the use of fresh frozen sections to detect thevery low abundance of Fgf-5 RNA in this tissue (Haubet al. 1990). However, morphology in sections of freshfrozen embryos is very poor, mandating our use ofparaffin-embedded material in these studies. With thistechnique, we have detected seven distinct sites of Fgf-5RNA expression in the mouse between embryonic days3£ to 154 post-conceptus1.

Expression of Fgf-5 RNA in early embryogenesisEmbryonic Fgf-5 expression first occurs soon afteruterine implantation. As can be seen in Fig. 2, there isno detectable expression in preimplantation blastocysts(panels E,F). Fgf-5 RNA was detected in embryos atthe next stage examined, E5J (panels A,B), while nosignal was seen with the negative control (sense-strand)probe (panels C,D). The failure to detect Fgf-5expression in blastocysts (embryonic day 3i), cannot beattributed to the small size of the blastocyst; E5iexpression was readily observed in tangential embry-onic slices containing very few cells (data not shown).The postimplantation induction of expression is consist-

1We use standard nomenclature to define conception asmidnight preceding the morning of vaginal plug detection.E3£=embryonic day 34 postconceptus.

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Fig. 2. Fgf-5 RNA in early postimplantation embryos. AntiPS and sensePS Fgf-5 35S-riboprobes were hybridized to eggcylinders in decidua and to blastocysts manually loaded into ampulae of pseudopregnant mice. Dark-field imaging (panelsB,D,F,H,J,K) makes exposed silver grains from NTB2 emulsion luminesce. Panels C,D used sensePS probe, while all otherpanels used the antiPS probe. Panels A,B and C,D, E5| egg cylinder; E,F, E3j blastocyst; G,H, E53 egg cylinder, showingexpression in embryonic ectoderm and adjacent visceral endoderm (arrows); I,J, cross-section through E7 egg cylinder;K,L, transverse section through E7 egg cylinder, showing expression in embryonic ectoderm, but not mesoderrn. Embryo(em), deciduum, (dec), blastocyst inner cell mass (icm), trophectoderm (te), ampulla (amp), embryonic ectoderm (erne),extraembryonic ectoderm (eee), visceral endoderm (ve), ectoplacental cone (epc), mesoderm (mes), primitive streak (ps),microns (um).

Fig. 4. Three-dimensional computer models of Fgf-5 expression in an E10| embryo. Thirty sections along an E10£ embryohybridized with Fgf-5 antiPS probe were used for modelling. For each section, a camera-lucida drawing of the embryo'scontour, dorsal aorta and aortic arches, head vein and cardinal veins, and regions of Fgf-5 expression in myotomes andsomatic mesoderm was traced by computer 'mouse' into a SunView computer containing CARP (Biographies) software.The data was used to construct superimposed objects: embryo (tan), Fgf-5 RNA in myotomes (pink), Fgf-5 RNA insomatic mesoderm (yellow), arterial vasculature (red), and venous vasculature (blue). Panel A shows a dorsolateral view ofthe embryo rendered 'transparent' to reveal sites of Fgf-5 expression. Panel B is the same view of Fgf-5 expression alongwith vascular structures, with the embryo's exterior removed. Panel C is viewed dorsally. Eye (e), nasal pit (np), branchialarches (ba), forelimb bud (fl), hindlimb bud (hi), heart bulge (hb), tail (t), head vein (hv), cardinal vein (cv), dorsal aorta(da), aortic arches (aa). Sections from the top of the head (diencephalon) were not included in the analysis, giving theembryo a flat-top distortion. The use of one section per 96 microns (12 sections) and difficulties in section alignments alsocaused distortions and the artefactual fusion of some Fgf-5-positive myotomes.

Expression of Fgf-5 gene in mouse embryo 399

ent with the observed rapid induction of Fgf-5 mRNAlevels when embryonal carcinoma cells or totipotentembryonic stem (ES) cells differentiate as embryoidbodies in vitro ((Hebert et al. 1990), and see below).

Fgf-5 expression in E5J embryos is restricted to theembryonic ectoderm and adjacent visceral endoderm(Fig. 2, panels G,H). By contrast, extraembryonicectoderm its flanking visceral endoderm and otherextraembryonic tissues are negative. This same ex-pression profile is evident through the onset ofgastrulation at E6| (data not shown). While expressionin the embryonic ectoderm of the egg cylinder persistsat E7, newly formed mesoderm is negative (panelsK,L). A cross-section through the E7 egg cylinderillustrates Fgf-5 RNA expression throughout theepiblast, without substantial lateral/medial or anterior/posterior variation (panels I,J). The E7i embryo showsweaker Fgf-5 RNA signals, and expression is undetect-able in the E8 embryo.

The pattern of early embryonic expression for theFgf-5 gene contrasts with that for another gene in theFGF family, int-2. int-2 is expressed in parietalextraembryonic endoderm and also in newly formedmesoderm leaving the primitive streak, commencing atE7i (Wilkinson et al. 1988). By hybridizing adjacent E7iembryo sections with Fgf-5 and int-2 antisense RNAprobes, we have observed Fgf-5 expression in embry-onic ectoderm adjacent to int-2 expression in mesoderm(data not shown).

Expression of Fgf-5 RNA in Later embryogenesisBetween embryonic days 9i and 14i, six additional sitesof Fgf-5 expression are evident. Two of these sites are inderivatives of lateral mesoderm, two others are skeletalmuscle precursors derived from paraxial mesoderm,and the other two sites are limb mesenchyme and acranial ganglion (see below). On embryonic day 15i, nosites of Fgf-5 expression are identifiable in situ. SinceFgf-5 RNA can still be detected by northern blotanalysis at this developmental stage (Hebert et al.1990), our in situ hybridization assays are failing todetect very low levels of expression in unidentifiedtissues. We estimate such expression to be <10 copiesmRNA/cell (discussed later).

Fgf-5 RNA in lateral mesodermOn embryonic day 9i, the Fgf-5 gene is expressed in onesmall region of splanchnic mesoderm ventral to theportion of the foregut bearing the hepatic bud (Fig. 3,panels A-C). Expression in this mesenchyme is lessreadily detectable on ElOi (data not shown), andundetectable thereafter. The onset of expression isapproximately coincident with that of rapid livergrowth, which proceeds by the invasion of hepatic cordsfrom the hepatic bud into the underlying mesenchyme.Whether the apparent fall-off of splanchnic mesoder-mal Fgf-5 expression from E9i to ElOi representsdown-regulation or merely the dilution of signal due tomesenchyme thinning and intermingling with hepaticcells cannot be ascertained.

Fgf-5 RNA is expressed in a region of somatic

mesoderm between ElOi and E12i. On embryonic day10i, this region extends rostrocaudally from the level ofthe newly formed sixth aortic arch to approximately thelevel of the liver primordium. Fig. 3 shows thisexpression in a transverse section through a ElOiembryo (panels F,G) and a parasagittal section througha El l i embryo (panels H,I). Fgf-5 expression alsooccurs in myotomes at these times (see below). Thesomatic mesoderm signal is readily distinguished fromthat of skeletal muscle precursors by hybridization ofadjacent sections with a probe for ^-cardiac actinmRNA (Sassoon et al. 1988), which is expressed instriated muscle cells and their precursors. This isillustrated in Fig. 3, panel J, where the actin probedetects myotomes and cardiac muscle in the El l iembryo, but does not hybridize to somatic mesoderm.

In order to illustrate the overall region of somaticmesoderm expressing Fgf-5 RNA in the ElOi embryoand the proximity of this region to vascular structures,the data in thirty transverse sections spaced 96 micronsapart were used for three-dimensional computer model-ling. CARP software (Computer-Aided ReconstructionPackage, Biographies Inc.) was used to generatemodels of the entire embryo, dorsal aorta and aorticarches, primary head veins and cardinal trunk veins,and sites of somatic mesoderm and myotome Fgf-5expression (Fig. 4). The broad anterior regions of Fgf-5-positive somatic mesoderm (panels A-C) lie bothlateral and just caudal to the newly formed sixth aorticarch. It is worth noting that this approximate region ofexpression sees the enlargement of the sixth arch andthe emergence of a caudally projecting vascular branch(the future pulmonary artery) during the ensuing 36 h ofdevelopment. The caudal 'tails' of expression in theElOi embryo run adjacent to the paired cardinal veins(panels B,C).

Fgf-5 expression in skeletal muscle precursor cellsSkeletal muscles of the trunk and limbs are descendantsof the somites, which arise by segmentation of paraxialmesoderm in an anterior-to-posterior sequence begin-ning at E8i. Each maturation phase of anterior somitesprecedes that of posterior ones owing to their differenttimes of birth. The precursor cells for limb muscleemigrate from the newly formed myotome derivative ofthe somite and enter limb buds shortly after theirformation on E9i (forelimb) and E10 (hindlimb)(Sassoon et al. 1989). The trunk muscle precursor cellsremain in the segmented myotomes and commencemigration on ElOi to Ell i .

Fgf-5 RNA is expressed in myotomes starting onElOi (Fig. 3, panels D-G). This induction occurs laterthan the morphological appearance of myotomes (E8i)and their expression of o--cardiac actin mRNA(E8i-E9i) ((Sassoon et al. 1989), and our obser-vations). On embryonic day 10i, only myotomesanterior to the middle of the hindlimb bud are Fgf-5positive, as shown in the 3-D model (Fig. 4, panel A).More caudal myotomes also express Fgf-5 RNA a daylater, reflecting their delayed development, but tailregion myotomes never express Fgf-5 (data not shown).

400 O. Haub and M. Goldfarb

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Fig. 3. Fgf-5 RNA in lateral mesoderm and myotomes. Hybridizations were with antiPS Fgf-5, sensePS Fgf-5, anda-cardiac actin 32P-riboprobes. Panels B,C,E,G,I,J are dark field images. Panels A-C, adjacent E9i sections hybridizedwith sensePS (A,B) or antiPS (C) with Fg/-5-positive lateral splanchnic mesoderm (arrow); panels D,E, ElOi longitudinalsection through rostral somites, antiPS probe with Fg/-5-positive myotomes (arrows); panels F,G, ElOi transverse sectionthrough rostral tip of heart bulge, antiPS probe, with Fg/-5-positive myotomes (thin arrows) and lateral somatic mesoderm(thick arrows); panels H-J, adjacent El l i parasagittal sections hybridized with antiPS (H,I) and a--cardiac actin (J) probes.Hepatic bud (hp), foregut (f), lateral splanchnic mesoderm (sp), neural tube (nt), notocord (n), dermatome (d), myotome(m), dermamyotome (dm), somite (so), sclerotome (s), dorsal aorta (a), cardinal vein (v), heart bulge (hb), lateral somaticmesoderm (Ism), branchial arch (ba), dorsal root ganglia (dg).

Myotomal cells continue to express Fgf-5 RNA on E l l i(Fig. 3, panels H-J) and E12i (data not shown) duringtheir ventral and lateral migration. Expression in trunkmuscle precursor cells is barely evident in E13J embryosand is undetectable by E14i. By contrast to trunkmuscle differentation, precursors of limb muscles,identifiable by a--cardiac actin RNA expression (seebelow), do not express Fgf-5 RNA at any stage in theirdevelopment.

Cranial skeletal muscles (tongue, mastication, extrin-sic ocular and facial) all derive from paraxial mesodermin birds (Noden, 1988), and similar origins in mammalsare suspected. A prominent site of Fgf-5 RNAexpression in the head of the E13| embryo (Fig. 5,

panel F) corresponds to the mastication muscleprecursor cells. Hybridization of a nearly adjacentsection with the tr-cardiac actin probe (Fig. 5, panelsD,E) reveals expression in mastication muscles as wellas in the extrinsic ocular muscles. Facial and tonguemuscle lineages, as recognized by cr-cardiac actinhybridization, are also Fgf-5 negative (data not shown).The strong Fgf-5 mastication muscle signal persists onE14J, but is completely absent 24h later (data notshown). This site of Fgf-5 RNA expression originateson embryonic day 111 as bilateral patches, each situatednear the juncture of the maxillary process and themandibular arch (Fig. 5, panels A,B). Curiously,cv-cardiac actin RNA expression is very weak at this site

Expression of Fgf-5 gene in mouse embryo 401

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These cranial expression data are open to radicallydifferent interpretations. Mastication muscle precursorcells may strongly express Fgf-5 RNA in advance ofsubstantial o--cardiac actin RNA expression. This wouldrepresent the opposite temporal order of gene ex-pression from that which occurs in myotomes. Alterna-tively, the cranial Fgf-5 RNA expression at El l i may bein mesenchyme of neural crest or lateral mesodermorigin which neighbors or is intermingled with musclecell precursors.

Fgf-5 RNA in developing limbsFgf-5 RNA has been detected in developing limbs fromembryonic days 12| through 14i. Expression is limitedto a patch of cells near the base of each limb, and whileexpression is readily detected in hindlimbs, it is far lessevident in forelimbs. Panels A and B of Fig. 6 shows theexpression patch in a transverse section through theE12| hindlimb. In the E13| embryo, parasagittalsections, which effectively represent cross-sectionsthrough the base of the limb, show that the mesenchy-mal region of Fgf-5 expression (panel E) is ventral tothe femur (panel D), which is undergoing chondrifi-cation. The expression site is distinct from the variousdeveloping muscle groups, which are visualized in

Fig. 5. Fgf-5 RNA in masticationmuscle. Sections were hybridizedwith Fgf-5 antiPS and a--cardiac actin32P-riboprobes. Panels B,C,E,F aredark-field images. Adjacent El l itangential sagittal sections throughthe head hybridized with antiPS(panels A,B) and a-cardiac actin (C)probes. Arrows denote strong Fgf-5and very weak a--cardiac actinsignals. Nearly adjacent E13iparasaggital sections through thehead hybridized with cv-cardiac actin(panels D,E) and antiPS (F) probes.While the actin probe detectsextrinsic ocular and masticationmuscles, the Fgf-5 probe detects onlythe latter. Maxillary process (mx),mandibular arch (ma), eye (e),diencephalon (d), snout (sn),extrinsic eye muscles (em),mastication muscles (mm), trigeminalganglion (tg), inner ear (ie).

adjacent sections by their expression of a-cardiac actinRNA (panel E). The tracing in panel G illustrates thespatial relationship of Fgf-5 positive mesenchyme todeveloping muscle and cartilage. The weaker Fgf-5RNA signal in forelimbs is positioned similarly to thatin hindlimbs (data not shown).

Fgf-5 RNA in the acoustic ganglionFgf-5 RNA has been detected in the acoustic branch ofthe eighth cranial ganglion within the inner ear onembryonic days 121 through 14? (Fig. 6, panels G-J).All other cranial ganglia lack detectable Fgf-5 RNA,including the vestibular branch of the eighth ganglion(Fig. 6, panels G,H) and the trigeminal fifth ganglion(Fig. 5, panels D,F). The cochlea is another develop-mental site where Fgf-5 and int-2 RNAs are expressedin neighboring cells, int-2 is expressed in portions of thecochlear sensory epithelium and underlying mesen-chyme (Wilkinson etal. 1989), which are innervated byprojections from acoustical neurons.

The seven sites of Fgf-5 embryonic expression arecatalogued in Table 1.

Levels of Fgf-5 RNA in the egg cylinderThe measurement of intracellular Fgf-5 RNA concen-tration is only possible in tissues where dissection allowsisolation of expressing cells to a known degree of purity.The one site amenable to RNA quantitation is the

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Fig. 6. Fgf-5 RNA in hindlimband acoustic ganglion. Sectionswere hybridized with Fgf-5antiPS, antiPP, and o--cardiacactin 32P-riboprobes. PanelsB,D,E,H,J are dark-field images.Panels A,B, hybridization of Fgf-5 antiPP probe to E12i transversesection through hindlimb, showingpositive patch near base of limb(arrow). Panels C-E, adjacentE13j parasagittal sections throughbase of hindlimb hybridized withFgf-5 antiPS (C,D) and a-cardiacactin (E) probes. Arrows in Dand E denote Fg/-5-positivemesenchyme and <x-cardiac actin-positive limb muscle groups,respectively. Panel F, E13|hindlimb schematic derived bytracings from panels C-E,showing femur cartilage (spotted),a--cardiac actin-positive muscleprecursors (light shaded), andFgf-5-positive mesenchyme (darkshaded). Panels G,H, longitudinalsection through head of E12|embryo hybridized with Fgf-5antiPP probe, showing expressionin acoustical branch of eighthganglion (bold arrow), but not inmore lateral vestibular branch(thin arrow). Panels I,J,parasagittal section through headof E14| embryo hybridized withantiPS probe. Arrow denotesexpression in acoustic ganglion.Hindlimb (hi), neural tube (nt),femur (fe), acoustic ganglion (ag),vestibular ganglion (vg), brain(b), spiral canal (sc), other canalsof inner ear (iec), otic capsule(oc), head vein (v).

Expression of Fgf-5 gene in mouse embryo 403

Table 1. Embryonic sites of FGF-5 gene expression

Expression siteDays ofgestation Comments

Epiblast and surrounding visceral endoderm 5.3-7.5

Lateral splanchnic mesoderm adjacent to 9.5-10.5hepatic bud

Region of lateral somatic mesoderm 10.5-12.5Myotomal muscle lineages 10.5-12.5

weak at 13.5Mastication muscle lineage 11.5-14.5

Mesenchyme near base of limb 12.5-14.5(hind>fore)

Acoustic ganglion 12.5-14.5

Negative in E3.5 blastocyst;Negative in egg cylinder mesodecm and

extraembryonic tissuesL a t e r al other splanchnic mesoderm

Prominent expression near arterial vesselsNegative in limb muscle lineages and tail

myotomesNegative in other cranial skeletal musclesEll.5 expression in myoblasts or mesenchymeNegative in limb muscle and cartilage

Negative in other cranial ganglia

postimplantation egg cylinder. On embryonic day 61-7, embryonic ectoderm accounts for —70% of the cellsin the embryonic compartment (embryonic ectoderm,mesoderm, flanking visceral endoderm) (Snow, 1977),and this compartment is larger than the extraembryoniccompartment of the egg cylinder. Hence, the embry-onic ectodermal cells expressing Fgf-5 RNA constitutes40-50 % of the cells in the egg cylinder.

Thirty egg cylinders (embryonic day 6f-7) weredissected from decidua, teased free of ectoplacentalcone tissue and dissolved in guanidinium isothiocyanatefor the isolation of RNA. An aliquot of the RNA wasanalyzed by northern blotting for GAPDH mRNA, aconstitutively expressed message, as compared toGAPDH mRNA levels in known amounts of ES cellRNA. The analysis demonstrated a recovery of onemicrogram total RNA from the egg cylinders (data notshown). The egg cylinder RNA was then analyzed forFgf-5 mRNA by northern blot hybridization, and thesignal compared to those obtained for several quantitiesof Fgf-5 cDNA. As shown in Fig. 7, one microgram

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Fig. 7. Northern blot quantitation of Fgf-5 RNA in eggcylinders. Total RNA (one microgram) from E6|—7 eggcylinders (lane e), undifferentiated ES cells (lane f), andES cell-derived embryoid bodies (three days in suspension)(lane g) were analyzed by formaldehyde-agarose gelelectrophoresis and filter blot hybridization, using murineFgf-5 3iP-labelled cDNA as probe. Denatured 2.2kbpmurine Fgf-5 cDNA (0.5 pg, lane a; 2pg, b; 10 pg, c; 50pg,d) was also analyzed to allow quantitation of Fgf-5 RNA.The hybridization signal from 1 fig egg cylinder RNA (lanee) equalled that from 2pg Fgf-5 cDNA (lane b).

RNA from 30 egg cylinders gave a 2.9 kb Fgf-5 RNAsignal (lane e) comparable to 2 picograms 2.2 kb Fgf-5cDNA (lane b). From this, we calculate that Fgf-5mRNA represents 2.5x10 of the total egg cylinderRNA. Since the embryonic compartment of eachE6|-7 egg cylinder contains -3000 cells (Snow, 1977),the estimated level of egg cylinder Fgf-5 expression is30-40 copies/embryonic ectodermal cell.

The Fgf-5 RNA expression level in the egg cylinder(lane e) is similar to that in ES cells allowed todifferentiate into simple embryoid bodies followingthree days in suspension (lane g), and is dramaticallyhigher than the level expressed in undifferentiated EScells, which is undetectable at the exposure lengthshown (lane f). These simple embryoid bodies havemorphological features comparable to the egg cylinderprior to gastrulation (Martin et al. 1977). Hence, theinduction of Fgf-5 expression in embryoid bodiesquantitatively and temporally parallels the Fgf-5 gene'sin vivo induction postimplantation.

The intensity of the Fgf-5 in situ hybridization signalin egg cylinders was consistently equal to or strongerthan those seen locally at the later sites of Fgf-5 geneexpression. We estimate local expression in laterembryogenesis to be at 10-30 copies mRNA/cell. Dueto the constraints of signal versus background, we donot feel that our assay conditions could detect <10copies RNA/cell. Hence, sites of very weak expressioncould be missed in our analysis. This presumablyexplains our failure to define the sites of Fgf-5expression in the E15i day embryo. Additionally, ourpreliminary in situ analysis of E13| embryos, using freshfrozen tissue for higher sensitivity, has revealed weaksites of Fgf-5 expression (dorsal root ganglia, myentericganglia) in addition to the more prominent hindlimb,mastication muscle and acoustic ganglion signals de-scribed earlier.

Discussion

Embryonic expression of the Fgf-5 gene is regulated asa function of time, tissue type and position withintissue. This lattermost characteristic has defined subdiv-isions of splanchnic mesoderm, somatic mesoderm and

404 O. Haub and M. Goldfarb

limb mesenchyme that were not morphologicallyevident nor previously appreciated by other molecularcriteria. The spatial restriction of expression may, inpart, be governed by combinations of transcriptionfactors, such as homeobox proteins, which themselvesare expressed within spatial boundaries. For example,the expression boundaries of various homeobox genesin vertebrate limbs define a 'grid' along both anterior-posterior and proximal-distal axes (Smith et al. 1989),and this grid may help dictate Fgf-5 expression in limbmesenchyme. Another manifestation of spatially re-stricted expression is the presence of Fgf-5 RNA insome skeletal muscle groups and cranial ganglia, butnot in others.

The structurally related int-2 gene is also expressed ina complex spatiotemporal pattern, although the sites ofexpression differ from those for the Fgf-5 gene(Wilkinson et al. 1989; Wilkinson et al. 1988). At certainstages in development, Fgf-5 and int-2 are expressed inclose proximity: Fgf-5 in visceral endoderm versus int-2in parietal endoderm, Fgf-5 in E7i embryonic ectodermversus int-2 in E7£ embryonic mesoderm, and Fgf-5 inacoustic ganglion versus int-2 in cochlear sensoryepithelium. Since both of these growth factors aresecreted proteins (Bates et al. 1991; Dixon et al. 1989),we suspect that FGF receptors expressed in theseregions differentially react with or respond to these twoFGFs. While the known spectra of in vitro biologicalactivities are similar among FGFs, there are contrasts(Finch et al. 1989; Lipton et al. 1988; Valles et al. 1990)which are presumably mediated by differences inligand-receptor interactions.

FGF-5 is likely to mediate a diverse set of eventsduring embryogenesis. The Fgf-5 expression profilealong with known biological effects of other FGFs in invitro and in vivo assays allow for speculation regardingFGF-5's native functions. Such conjecture merelyserves as a guide for further studies.

Uterine implantation of the blastocyst is followed byrapid growth of the egg cylinder and rapid growth of theuterine deciduum. Fgf-5 expressed in embryonic ecto-derm postimplantation could serve as an autocrine orparacrine factor to mediate these growth events.Several FGFs have been shown to possess mesoderm-inducing activity when assayed on cultures of amphib-ian animal cap ectoderm (Kimelman and Kirschner,1987; Paterno et al. 1989; Slack et al. 1987). Theexpression of Fgf-5 RNA at the onset of mousegastrulation suggests that this growth factor maycontribute to mesoderm formation, although two factsargue against a direct role for FGF-5 in this process: (1)the onset of gene expression (^E5i) far precedes thestart of gastrulation (E6£), and (2) Fgf-5 RNA is notspatially restricted within the embryonic ectoderm.Future localization of Fgf-5 protein and receptors in thedeveloping egg cylinder is of obvious importance.

Acidic and basic fibroblast growth factors aremitogenic for vascular endothelial cells in vitro and areangiogenic when applied to tissues in vivo (Burgess andMaciag, 1989). FGF-5 is most likely an endothelial cellmitogen, since conditioned medium containing secreted

FGF-5 will stimulate endothelial cell growth (Zhan etal. 1988). FGF-5 might act as an angiogenic factor inlateral mesoderm, which becomes more highly vascu-larized than dorsal mesoderm (Le Douarin, 1975;Sherer, 1975). In splanchnic mesoderm, local Fgf-5expression might promote vascularization required forthe induction of neighboring hepatic cord proliferation(Sherer, 1975). In somatic mesoderm, local Fgf-5expression could contribute to the ongoing remodellingof the arterial vasculature (Rugh, 1968).

FGFs have well-documented effects upon the differ-entiation of skeletal muscle progenitor cells. Fibroblastgrowth factors can inhibit the terminal differentiation ofmyoblasts, as monitored by biochemical markers andby myotube formation (Lathrop et al. 1985; Seed andHauschka, 1988). Fgf-5 expression commences inmyotomes on ElOi well after their formation (E8£) andcommitment to muscle lineage, as gauged by the onsetof a-cardiac actin (E8i-9£) and MyoD (E9£) geneexpression (Sassoon et al. 1989). Fgf-5 expressiondiminishes by E13i, before the first appearance ofmyotubes on E15 (Rugh, 1968). FGF-5 could inhibit theterminal differentiation of myotomal myoblasts duringtheir migration through the trunk. Limb muscledevelopment differs from that of trunk muscle byseveral molecular criteria. The onset of ar-cardiac actinand MyoD expression in limb muscle precursors occurslater in embryogenesis (Sassoon et al. 1989), althoughappearance of myofibrils occurs at similar times for limband trunk muscle (Rugh, 1968); i.e. the later stages ofmuscle differentiation transpire within a more com-pressed time frame in limb versus trunk. A lack of Fgf-5expression in limb muscle myoblasts might account fortheir more rapid terminal differentiation.

In vitro culture of dissociated chicken limb bud cellshas revealed an FGF-dependent subcomponent ofmyoblasts which require treatment with basic FGF inorder ultimately to form colonies of terminally differen-tiated muscle (Seed and Hauschka, 1988). It is possiblethat E12£-14i Fgf-5 expression in limb mesenchymeacts to induce the development of FGF-dependent limbmyoblasts. Furthermore, if the mandibular site of E l l iFgf-5 expression consists of mesenchymal as opposed tomyoblastic cells, such expression could act to inducelocal myoblasts to develop into the mastication musclegroups.

Several lines of experimentation are required toelucidate FGF-5's roles in embryogenesis. FGF'sreceptors and their sites of expression need to beidentified. Alternatively spliced transcripts of the FLGand BEK genes encode a family of related FGFreceptors (reviewed in (Goldfarb, 1990)), but theiraffinities toward FGF-5 await characterization. A moreprecise cellular assignment of Fgf-5 expression is clearlydesirable, particularly as it relates to mesenchymalversus myoblastic gene expression in the mandibularregion. Transgenes Unking an enzymatic reporter toFgf-5 transcriptional regulatory elements may providesuch insights. Lastly, the use of in vivo gene targettingto disrupt FGF-5 production partially or completelymay generate informative embryonic phenotypes.

Expression o/ Fgf-5 gene in mouse embryo 405

We thank Beverly Drucker for help in several experiments,and Matthew Buttrick for assisting in paraffin embedding andsectioning. We are most grateful to David Sassoon (BostonUniversity) and to Godon Peters (Imperial Cancer ResearchFund, London) for cr-cardiac actin and int-2 clones, respect-ively, to Elizabeth Robertson for providing ES cells, toFrancoise Poirier for instruction in egg cylinder dissection, toJames Lee for help with blastocyst preparation, to ConnieCepko (Harvard Medical School) for access to her Sun View/CARP 3-D imaging system, and to Lisa Jepeol and ElizabethRyder for instruction in its use. We also thank VassilusPachnis, Jane Dodd, Taube Rothman, David Sassoon,Elizabeth Taparowsky (Purdue University) and Cliff Tabin(Harvard Medical School) for helpful discussions. This workwas supported by PHS research grant CA48054. O.H. issupported by NIH Training Grant DK07328.

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(Accepted 14 March 1991)