INTRODUCTION

Segmentation is a feature characteristic of the axial skeleton,musculature and nervous system of vertebrates (Keynes andStern, 1984; Lumsden and Keynes, 1989; Puelles and Ruben-stein, 1993). Craniofacial structures such as the branchialarches, pharyngeal pouches and cranial nerve ganglia aretypically arranged into serially repeated patterns in mammalianembryos (Hunt and Krumlauf, 1991; Hunt et al., 1991a;Thorogood, 1993). The craniofacial tissues are composed prin-cipally of neuroectodermally derived neural crest cells and thecranial paraxial mesoderm, both of which are segmentallyorganised during early embryogenesis (Couly et al., 1993;Noden, 1986, 1988). Recent studies on the ontogeny of cranialneural crest cells have revealed that the neuromeric organisa-tion of the neural tube has a significant impact on neural crestdifferentiation (Wilkinson et al., 1989; Figdor and Stern, 1993;Fraser, 1993; Puelles and Rubenstein, 1993). In essence, neuralcrest cells destined for different branchial arches or cranialnerve ganglia always arise from specific neuromeres and theirmigration pattern faithfully follows their original metameric

order in the neural tube (Tan and Morriss-Kay 1986; Chan andTam, 1988; Lumsden, 1990; Lumsden et al., 1991; Serbedzijaet al., 1992; Wilkinson and Krumlauf, 1990; Hunt et al.,1991a,b,c). Furthermore, neural crest cells that originate fromspecific segments of the hindbrain (e.g. rhombomeres r2, r4and r6) express a similar set of

Hoxb genes characteristic oftheir rhombomeric origins (Hunt et al.,1991a). The emulationof rhombomeric Hox gene expression by neural crest cellssuggests that the early segmental organisation of the hindbrainmay have long-term effects on cell differentiation and pattern-ing of the craniofacial structures.

Presently it is not known whether the cranial paraxialmesoderm also exerts a similar segmental influence on cranio-facial morphogenesis. A meristic pattern of somitomeres(Meier, 1979) has been described in head mesoderm of eightvertebrate embryos, including the mouse (Tam and Trainor,1994). Somitomeres are clusters of loosely packed mesenchy-mal cells that form in the paraxial mesoderm along the lengthof the embryo (Meier and Tam, 1982). The somitomeres in thesegmental plate or presomitic mesoderm have been regardedas the primordial structures for somites (reviewed by Tam and

2397Development 120, 2397-2408 (1994)Printed in Great Britain © The Company of Biologists Limited 1994

A combination of micromanipulative cell grafting and flu-orescent cell labelling techniques were used to examine thedevelopmental fate of the cranial paraxial mesoderm of the8.5-day early-somite-stage mouse embryo. Mesodermalcells isolated from seven regions of the cranial mesoderm,identified on the basis of their topographical associationwith specific brain segments were assessed for their con-tribution to craniofacial morphogenesis during 48 hours ofin vitro development. The results demonstrate extensivecell mixing between adjacent but not alternate groups ofmesodermal cells and a strict cranial-to-caudal distributionof the paraxial mesoderm to craniofacial structures. A two-segment periodicity similar to the origins of the branchialmotor neurons and the distribution of the rhombencephalicneural crest cells was observed as the paraxial mesodermmigrates during formation of the first three branchialarches. The paraxial mesoderm colonises the mesenchymalcore of the branchial arches, consistent with the location of

the muscle plates. A dorsoventral regionalisation of cell fatesimilar to that of the somitic mesoderm is also found. Thissuggests evolution has conserved the fate of the murinecranial paraxial mesoderm as a multiprogenitor populationwhich displays a predominantly myogenic fate. Heterotopictransplantation of cells to different regions of the cranialmesoderm revealed no discernible restriction in cellpotency in the craniocaudal axis, reflecting considerableplasticity in the developmental fate of the cranialmesoderm at least at the time of experimentation. The dis-tribution of the different groups of cranial mesodermmatches closely with that of the cranial neural crest cellssuggesting the two cell populations may share a commonsegmental origin and similar destination.

Key words: cell fate, somitomere, craniofacial development,branchial arches, mouse embryo

SUMMARY

Cranial paraxial mesoderm: regionalisation of cell fate and impact on

craniofacial development in mouse embryos

Paul A. Trainor1, Seong-Seng Tan2 and Patrick P. L. Tam1,*1Embryology Unit, Children’s Medical Research Institute, Locked Bag 23, Wentworthville NSW 2145, Australia2Embryology Laboratory, Department of Anatomy and Cell Biology, University of Melbourne, Parkville, VIC 3052, Australia

*Author for correspondence

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Trainor, 1994). In the mouse, the cranial mesoderm consists ofseven pairs of contiguous somitomeres, which maintain a con-sistent topographical relationship with specific regions of theneural tube (Tam and Meier, 1982; Meier and Tam, 1982;Jacobson and Meier, 1986). Somitomere I (Sm-I) underlies theprosencephalon. Somitomeres II and III (Sm-II and Sm-III)reside lateral to the mesencephalon. Somitomere IV (Sm-IV)is associated with the metencephalon. Somitomeres V, VI andVII (Sm-V, Sm-VI and Sm-VII) underlie the myelencephalon(Fig. 1). However, to this date, lineage restriction andmolecular heterogeneity of cells in different somitomeres com-parable to those displayed by the neuromeres (Fraser et al.,1990; Puelles and Rubenstein, 1993) have not been found.

In the chick, different groups of voluntary cranial musclesand some components of the chondrocranium are found to bederived from different portions of the cranial paraxialmesoderm (Noden, 1983b, 1988; Couly et al., 1992). Suchregionalisation of cell fate has been attributed to the partition-ing of the different myogenic and skeletogenic precursors tothe cranial somitomeres. The chimaeric avian embryosanalysed in these studies had developed beyond the stages ofbranchial arch and facial primordia formation such that fusionand growth of these structures had already occurred. The earlypattern of distribution and the fate of individual cranial somit-omeres were not examined, leaving unresolved the question ofwhether the difference in the myogenic fates between somito-meres is a reflection of the regionalisation of cell fate or arestriction in cell potency. What is also lacking in previousstudies of cephalic mesodermal organisation is a clear appre-ciation of the precise somitomeric origins of the branchial archmesenchyme. It remains to be seen if, like the neural crest cells,there is a segmental allocation of paraxial mesoderm to cran-iofacial structures. In the present study, we have taken the firststep to analyse the role of somitomeres in craniofacial mor-phogenesis by testing if there is any regionalisation of cell fatealong the craniocaudal axis of the cranial paraxial mesodermregarding its contribution to serially repeated craniofacialstructures of the mouse embryo. Specifically, we aimed (i) totrace the distribution of cells in different segments of cranialmesoderm as defined by the somitomeres and (ii) to determineif the craniocaudal order of cells in the cranial mesoderm istransposed into patterning of craniofacial structures such as thebranchial arches. We have also examined the cranial mesodermfor evidence of restriction of cell fate by heterotopic trans-plantation of lacZ-tagged cells and by differential labelling ofdifferent parts of the paraxial mesoderm.

MATERIALS AND METHODS

Experimental strategyFor tracking the fate of the cranial mesoderm, small groups of meso-dermal cells were isolated from specific regions of the paraxialmesoderm (Figs 1, 2A-C), and were then transplanted to an equiva-lent site in isochronic 8.5-day host embryos. The transplanted cellswere marked by the

β-galactosidase activity encoded by a lacZtransgene, which is ubiquitously expressed during development (Tamand Tan, 1992). Alternatively, the mesodermal cells were labelled insitu with fluorescent carbocyanine dyes. Heterotopic transplantationsof mesoderm to different sites in host embryos were also performedto assess the prospective potency and state of commitment of the cells.In the absence of regional specification, the transplanted cells would

develop according to their site of transplantation rather than theirorigin. Transplantations and labelling of cells were performed bymicromanipulating embryos that were explanted from the uterus.Experimental embryos were cultured in vitro for the assessing the fateof the cells.

Recovery and in vitro culture of host embryosEarly-somite-stage embryos were obtained from pregnant femalemice of ARC/S strain at 8.5 days post coitum (p.c.), Whole concep-tuses were dissected out from the uterus. The parietal yolk sac wasremoved leaving the embryo proper with an intact visceral yolk sac,amnion and ectoplacental cone. Previous studies on the migration ofcranial neural crest cells in the mouse embryo have established thatthe first population of neural crest cells to leave the neural tube arethose of the mesencephalon and it happens at about 5- to 6-somitestage (Jacobson and Tam, 1982; Chan and Tam, 1986, 1988; Serbedz-ija et al., 1992). To ensure that only the paraxial mesoderm and noneural crest cells were labelled or grafted, we applied a stringentcriterion that only embryos having ≤5 pairs of somites were used forthe experiments. After cell grafting or labelling, embryos werecultured in vitro for 48 hours (Sturm and Tam, 1993).

Isolating donor transgenic tissue and grafting 8.5-day embryos were obtained from matings of transgenic H253mice, which express a bacterial lacZ transgene under the control ofthe 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) reductasepromoter (Tam and Tan, 1992). Donor H253 embryos were firstbisected along their midline (Fig. 2A). Wedge-shaped fragments con-taining the paraxial mesoderm and the associated neuroectoderm andsurface ectoderm (Fig. 2B) were isolated by cutting the embryo trans-versely at various neuromeric junctions (Fig. 2A).

P. A. Trainor, S.-S. Tan and P. P. L. Tam

I

II

III

IV

V

VI

VII 21

HT

ProMes

Met

Mye

Fig. 1. The subdivision of the cranial paraxial mesoderm into seven(I to VII) somitomeres (Meier and Tam, 1982) in accordance to theirtopographical relationship to major segments of the prosencephalon(Pro), mesencephalon (Mes), Metencephalon (Met) andMyelencephalon (Mye). The short dashes demarcate the boundariesbetween brain segments. The first 2 somites are numbered and HTdenotes the heart.

2399Fate of cranial somitomeres

The embryonic fragments were then incubated in a solution of 0.5%trypsin, 0.25% pancreatin, 0.2% glucose and 0.1% polyvinylpyroli-done in PBS for 20 minutes at 37°C to loosen the mesoderm andectoderm layers. Finely polished alloy and glass needles were used toseparate the ectoderm from the underlying mesoderm (Fig. 2C). Themesoderm was dissected into fragments containing approximately 30cells which were then grafted into the cranial mesoderm of the hostembryo (Fig. 2D). For the purpose of this study, the mesodermisolated from different cranial fragments and the sites to which theywere transferred are referred to by their somitomeric designation, i.e.Sm I-VII (Fig. 1).

Transplantation experiments1. Orthotopic transplantations of cranial mesoderm toisochronic hostsDonor mesoderm derived from Sm I-VII was transplanted to theequivalent somitomere in host embryos (Fig. 2E). Seven types oftransplantation were performed, designated I→I, II→II, III→III,IV→IV, V→V, VI→VI or VII→VII, to denote the somitomericorigin of donor cells and the site of transplantation, respectively.

2. Heterotopic transplantations of somitomeric mesoderm toisochronic hostsIn the first set of experiments, donor mesoderm from somitomeres I,II, III, IV, V and VI was transplanted to different somitomeres in hostembryos. In the second set, somitomeres I, II, IV, V and VI werechosen to demonstrate how tissues both rostral and caudal to somito-mere III develop when ectopically grafted to somitomere III. Nineexperiments designated III→I, III→II, III→IV, III→V, I→III, II→III,IV→III, V→III and VI→III referring to the source and destination of

the donor mesoderm were performed. Somitomere III was chosenrespectively as a source of donor mesoderm and a site for transplan-tation because it is centrally located within the head. This allowed usto assess its development in ectopic positions in the head both rostraland caudal to its normal axial position.

Analysis of the results of transplantation experimentsHost embryos were harvested after 48 hours of in vitro culture andgrossly abnormal embryos were discarded. Embryos were fixed in4% paraformaldehyde for 30 minutes and stained by X-gal histo-chemistry to reveal the β-galactosidase activity (Tam and Tan,1992). Embryos containing blue X-gal-stained cells were processedfor wax histology and counterstained with nuclear fast red. Thelocation of labelled cells in structures such as the branchial archesand craniofacial mesenchyme was determined on serial sections ofthe embryo.

Preparation of fluorescent dyes for injection0.05% solutions (w/v) of DiI (1,1-dioctadecyl-3,3,3′,3′-tetra-methylindocarbocyanine perchlorate) and DiO (3,3′-dioctadecyloxa-carbocyanide perchlorate, Molecular Probes) were made from a 0.5%stock ethanolic solutions. The stock was diluted 1:7.5 in 0.3 M sucroseimmediately before injection. Micropipettes were backfilled with dyesolution and injection was done under positive pressure generated bya deFonbrune oil pump (Alcatel).

Dye labelling of individual somitomeresThe same topographical landmarks employed for locating somito-meres in the transplantation experiments were also used for targetingthe dye injection. Following insertion of the micropipettes into the

Table 1. Summary of the patterns of tissue colonisation in transplantation and labelling experimentsSomitomere No. performed No. analysed FNM POM MXP MDP FM SA CV PTM TA FA

a. Dye InjectionI 22 17 + + − − − − − − − −II 30 25 − + + − − − − − − −III 27 21 − + + + + − − − − −IV 25 22 − − − − + + + − − −V 26 21 − − − − − + + + − −VI 20 15 − − − − − − − + + −VII 22 18 − − − − − − − + + +

b. Orthotopic graftsI

➞I 15 11 + + − − − − − − − −II➞II 13 10 − + + − − − − − − −III➞III 40 33 − + + + + − − − − −IV➞IV 32 26 − − + + + − − − − −V➞V 15 12 − − − − − + + + − −VI➞VI 18 13 − − − − − − − + + −VII➞VII 21 16 − − − − − − − − + +c. Heterotopic graftsIII➞I 8 5 + + - − − − − − − −III➞II 10 6 − + + − − − − − − −III➞IV 11 8 − − − − + + + − − −III➞V 7 5 − − − − − + + + − −I➞III 8 5 − − + + + − − − − −II➞III 9 6 − − + + + − − − − −IV➞III 7 6 − − + + + − − − − −V➞III 8 5 − − + + + − − − − −VI➞III 10 7 − − + + + − − − − −d. Dorsal/Ventral InjectionsIII dorsal 15 11 − − + − + − − − − −III ventral 15 11 − − + + − − − − − −IV dorsal 14 9 − − − − − − + + − −IV ventral 14 9 − − − − − + − − − −

Abbreviations. FNM, frontonasal mass; POM, periocular mesenchyme; MXP, maxillary component of first arch; MDP, mandibular component of the first arch;FM, facial mesenchyme; SA, second arch; CV, cervical mesenchyme; PTM, periotic mesenchyme; TA, third arch; FA, fourth arch.

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mesoderm, a small amount of DiI or DiO solution was expelled andthe labelled sites were visible through the dissecting scope as red (DiI)or orange (DiO) patches of labelled mesoderm (Fig. 2F). For testingdorsoventral regionalisation, different parts of somitomeres III and IVwere labelled with different fluorescent dyes (Fig. 2G). The ventralregion of the somitomere was labelled first, which provided the

reference point for a dorsal label. The two injection sites were alwaysseparated by a zone of unlabelled cells.

Analysis of the results of dye labelling experimentsEmbryos cultured in vitro for 48 hours were viewed without anyfixation as whole mount under a standard fluorescence microscope

P. A. Trainor, S.-S. Tan and P. P. L. Tam

Fig. 2. (A) The bisected halves of a 8.5-day (5-somite) mouse embryo showing the somitomeric boundaries (arrows). Transverse cuts made atthese specific neuromeric landmarks resulted in isolation of single somitomeres. (B) Embryonic fragment containing the Sm-III and itsassociated surface ectoderm and neuroectoderm obtained by cutting the half-head at mid-mesencephalon and at the mesencephalon-metencephalon boundary. (C) The wedge-shaped somitomeric mesoderm (arrowhead) dissected free of the surface ectoderm and neural plate.(D) Grafting somitomeric cells to a 8.5-day early-somite-stage embryo by inserting the injection micropipette through the extraembryonicmembranes into Sm-III. (E) A 8.5-day embryo examined 2 hours after transplantation to reveal the location of the tissue grafts (X-gal-stainedblue cells), which were grafted into the dorsal region of Sm-I and the caudal-ventral region of Sm-IV (arrowheads). (F) A 8.5-day embryoexamined immediately after DiI injection into Sm-I, Sm-III, Sm-IV and Sm-VI (arrows). (G) A 8.5-day embryo with the ventral and dorsalregions of Sm-III labelled by injection of DiO (arrow) and DiI, respectively. Abbreviation: hb, hindbrain; ht, heart; mb, midbrain.Bar, 100 mm.

2401Fate of cranial somitomeres

Fig.

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(Leitz). The DiI and DiO signals were detected using rhodamine andFITC filters respectively. Photographs were taken using Ektachrome400 or 160 films directly with a Wild MPS 46/52 Photoautomat camera.

RESULTS

Cranial somitomeric mesoderm contributes tobranchial arch formationTable 1 summarises the pattern of distribution of transplantedcells and DiI/DiO-labelled cells to various craniofacial struc-tures in the mouse embryo after 48 hours of culture. Embryosto be analysed were selected on the basis that (i) they are mor-phologically similar to the 30- to 36-somite-stage embryo invivo, (ii) the branchial arches were well formed and (iii) boththe forelimb and hindlimb buds had developed.

Results of the transplantation and labelling experimentsshowed that the cranial mesoderm contributes cells to the firstfour branchial arches in a manner strictly in accordance withtheir craniocaudal order in the axis (Table 1a,b). Each of thefirst three branchial arches, were colonised by cells from neigh-bouring regions of the paraxial mesoderm that are defined bytwo consecutive somitomeres: namely Sm-II and III for firstarch, Sm-IV and V for the second arch and Sm-VI and VII forthe third arch (Table 1a,b; Fig. 5). In contrast, only one somit-omere participated in fourth arch development and the fifth andsixth pairs of branchial arches did not receive any contributionfrom the cranial mesoderm.

Both II→II grafts and Sm-II dye injection experimentsrevealed that mesoderm derived from Sm-II extensivelycolonised the proximal maxillary prominence of the firstbranchial arch (Fig. 3A). In 8% (2/25) of cases where Sm-IIwas labelled with either DiO or DiI and in 10% (1/10) of II→IIgrafts, a small number (5-10) of cells were displaced to the

distal region of the first arch which gives rise to the mandibu-lar prominence (Fig. 3B). Mesoderm derived from Sm-IIIcolonised both the mandibular and the maxillary prominencesof the first branchial arch (Fig. 3C). There was a widespreaddistribution of grafted cells in the facial mesenchyme sur-rounding the proximal end of the first arch. Cells derived fromSm-III were also found in the maxillary component of the arch,overlapping with that of Sm-II (Fig. 3D). Histology of the firstarch revealed that the graft-derived cells were localised in thecentral to medial core mesenchyme (Fig. 3E). Sm-II and Sm-III did not participate in morphogenesis of more caudalbranchial arches. The distribution pattern of Sm-III whenlabelled with DiI resembled closely that observed for graftedmesoderm (Fig. 3F). The fluorescent mesoderm againcolonised the central regions of the maxillary and mandibularprominences. The distribution of fluorescent cells, however,seemed broader than that observed for the grafted cells. Thequantitative difference is due to the fact that more cells arelabelled through dye injection than are transplanted. Therewere no instances in which the mandibular epithelia or thesurface ectoderm were colonised with mesoderm derived fromSm-II and Sm-III.

The second branchial (hyoid) arch received contributionfrom the preotic mesoderm associated with the upper hindbrain(Sm-IV and Sm-V). Mesoderm derived from Sm-IV, whichunderlies the metencephalon, contributed primarily to thecentral region of the hyoid arch (Fig. 3G). The distribution ofIV→IV grafted cells was widespread in the rhombencephalicmesenchyme adjacent to the second arch but, within the arch,the mesoderm colonised a central core region (Fig. 3H).Marking somitomere IV with DiI produced similar results toIV→IV grafting experiments, with the labelled cells populat-ing the hyoid arch (Fig. 3I). Sm-V, which underlies the rostralmyelencephalon, also contributed to the hyoid arch. There wasa clear segregation of donor mesoderm derived from the dorsaland ventral regions of Sm-V respectively such that the dorsalmesoderm was found in a more proximal position in the secondarch than the ventral mesoderm (Fig. 3J). The distribution ofSm-V-derived cells overlapped significantly with that of Sm-IV. There was no evidence of Sm-IV and Sm-V contributingto formation of either the first or third branchial arches. In oneinstance only, the investing mesenchyme and epithelium of thefirst pharyngeal pouch were populated with Sm-V-derivedmesoderm (data not shown).

Similar to the first two arches, the third arch received a dualcontribution from two adjacent somitomeres, Sm-VI and Sm-VII of the caudal myelencephalon. VI→VI grafts showed thatthe mesodermal cells were mostly confined to the centralregion of the third arch (Fig. 3G). Sm-VI did not contribute toformation of first, second or fourth branchial arches. Sm-VII,which is the caudal most cranial somitomere, participatesprimarily in formation of the third arch. The distribution ofVII→VII grafted cells in the third arch overlapped with cellsderived from Sm-VI. VII→VII grafts also reveal that Sm-VIIcells were found in the central core region of the fourthbranchial arch.

Prosencephalic and mesencephalic mesodermcontribute to the periocular, frontonasal and facialmesenchymeThe two most rostral somitomeres (Sm-I and Sm-II), con-

P. A. Trainor, S.-S. Tan and P. P. L. Tam

Fig. 3. Sm-II contribution to (A) the maxillary prominence of thefirst arch, caudal to the optic vesicle (arrow) and (B) to the distalmandibular region of the first arch (arrow), the mesenchyme lateralto the diencephalon and the periocular mesenchyme (arrowhead).(C) Sm-III colonisation of the central core mesenchyme (arrowhead)of the mandibular arch and in the maxillary prominence. (D) Sm-IIIdistribution primarily to the maxillary (arrow) and mandibularregions of the first arch. (E) A section through the facialmesenchyme and branchial arch region showing III→III graft-derived cells restricting to the mesenchyme core of the first arch butare dispersed in the facial mesenchyme. (F) DiI-labelled cells in thecore of the maxillary and mandibular portions of the first arch. (G)Non-overlapping distribution of Sm-IV and VI mesoderm cells to thesecond and third branchial arches, respectively, after a doubleorthotopic grafting (IV→IV and VI→VI). Sm-IV colonised the coreof the second arch, the rostral periotic mesenchyme and themesenchyme at the base of the first arch. Some cells are founddispersed rostrally to the facial mesenchyme. Sm-VI colonises thecore of the third arch and the ventral periotic mesenchyme (arrow).(H) A section through the hyoid arch showing the distribution ofIV→IV graft-derived cells in the core region of the hyoid arch and inthe adjacent paraxial mesenchyme. (I) DiI-labelled cells derivedfrom Sm-IV in the second arch. (J) V→V graft-derived cellspopulating the hyoid arch (arrow) and the rostral and ventral perioticmesenchyme (arrowhead). Abbreviations: fb, forebrain; fm, facialmesenchyme; ha, hyoid arch; ht, heart; ma, mandibular arch; mb,midbrain; md, mandibular prominence; mx, maxillary prominence;np, nasal process; op, otic placode; ov, optic vesicle; ta, third arch.Bar, 100 mm for A-D,G and J. Bar, 300 µm for E,F,H,I.

2403Fate of cranial somitomeres

tributed extensively to the periocular mesenchyme. I→I andII→II grafts showed that the rostral and ventral portions of theperiocular mesenchyme were derived from Sm-I, whereas thedorsal and caudal periocular mesenchyme came primarily fromSm-II (Fig. 4A,B). There was, however, some overlap in thedistribution of Sm-I and Sm-II cells. In 9% (1/11) of I→I trans-plantations, the dorsal and caudal periocular mesenchyme werecolonised by graft-derived cells. In 10% (1/10) of II→II grafts,Sm-II-derived cells contributed to the rostral periocular mes-enchyme and also the ventral periocular mesenchyme in thevicinity of the primitive oral cavity. Similar but broader dis-tribution patterns were observed for Sm-I and Sm-II, respec-tively, when they were directly injected with DiO (Fig. 4C). Inaddition to the periocular mesenchyme, Sm-I and Sm-II con-tributed to the endothelium of blood vessels surrounding theoptic epithelium (data not shown). These two somitomeres alsocontributed extensively to the facial mesenchyme lateral to thetelencephalon and diencephalon. The frontal area of theembryo associated with the lateral and medial nasal processeswas colonised by Sm-I only (Fig. 4A).

Sm-III colonised the facial mesenchyme and some vascularand endothelial tissue lateral to the caudal midbrain and meten-cephalon (Fig. 4E). The distribution of Sm-III cells overlappedwith Sm-II cells in the facial mesenchyme lateral to themidbrain and with Sm-IV cells in the region lateral to therostral hindbrain, adjacent to the mandibular and hyoid arches.III→III grafts showed that Sm-III colonised the branchial archartery, the connective tissue surrounding the truncus arteriosusand the endothelium of the primary head vein (anterior cardinalvein).

Rhombencephalic mesoderm contributes to theperiotic and cervical mesenchymeThe otic placode develops superficially to Sm-VI and VII. Theperiotic mesenchyme was derived from Sm-IV, V, VI and VII.IV→IV and V→V grafts revealed that Sm-IV and Sm-V con-tributed significantly to both the rostral and rostroventralperiotic mesenchyme, respectively, and to the facial mes-enchyme associated with the rostral hindbrain (Figs 3J, 4F).VI→VI and VII→VII grafts showed that the ventral and thecaudal periotic mesenchyme were colonised by mesodermderived from Sm-VI and Sm-VII, respectively (Figs 3G, 4G).Sm-VI and Sm-VII also contributed to the cervical mes-enchyme associated with caudal segments of the hindbrain andto the dorsal periotic mesenchyme, which is bounded by theotic placode and neuroectoderm. In one instance, Sm-VI-derived mesoderm transplanted into Sm-VI (VI→VI graft)colonised the otic epithelium. This was probably the result ofgrafting cells too superficially to the surface ectoderm. Asummary of the distribution of somitomeric mesoderm to cran-iofacial structures is presented in Table 2 and Fig. 5.

Evidence for dorsoventral constraintsThe relative positioning of cells within cranial somitomereswas maintained during their deployment to craniofacial struc-tures, such that the dorsal and ventral regions of a somitomerehave different fates with respect to craniofacial morphogene-sis. Table 1d summarises the results of experiments in whichthe ventral and dorsal mesoderm of Sm-III and Sm-IV werelabelled with DiO and DiI, respectively. There was a strongtendency for the ventral mesoderm of Sm-III to colonise the

distal mandibular and maxillary regions of the first branchialarch. Progressively more dorsal mesoderm derived from Sm-III contributed to the maxillary and more proximal regions ofthe first arch, and to the facial mesenchyme (Fig. 4H).Similarly, the ventral Sm-IV mesoderm was confined ulti-mately to the hyoid arch proper and the dorsal mesoderm con-tributed to the cervical mesenchyme and to the proximal regionof the hyoid arch (data not shown). Although some cell mixingoccurred at the interface between the DiO- and DiI-labelledpopulations, mesoderm derived from the dorsal region of thesomitomere seldom colonised the distal region of the branchialarch. Conversely ventrally derived mesoderm never con-tributed to the facial mesenchyme adjacent to the neural tube.These results indicate that regionalisation of cell fate may existin the cranial mesoderm. The dorsoventral partition withinsomitomeres is transposed into a proximal-distal disposition ofcells in the branchial arches and adjacent paraxial mes-enchyme.

The fate of the cranial paraxial mesoderm is notpositionally restricted Heterotopic transplantations showed that mesoderm derivedfrom any axial level, when grafted to a heterotopic site woulddevelop according to its new position and not its site of origin.Table 1c summarises the results of several types of heterotopictransplantation. Sm-III due to its central location in the head,was chosen as the source of donor cells for most of the grafts(45%; 24/53). Sm-III mesoderm was grafted into somitomeresboth rostral and caudal to its origins (III→I, III→II, III→IV,III→V). The final distribution of graft-derived cells wasalways characteristic of the site of transplantation. Forexample, in III→V transplantation experiments, the mesodermcells contributed to the hyoid arch mesenchyme and to therostral periotic mesenchyme (Fig. 4I). This was almostidentical to the distribution pattern observed from V→V grafts(Fig. 3J). Similarly in III→II transplantations, the pattern ofdistribution resembled very closely II→II grafts: the mesodermcontributed to the caudal and ventral periocular mesenchyme,to the mesenchyme lateral to the diencephalon and uppermidbrain, and also to the maxillary component of the firstbranchial arch (Table 1c). As expected there was no colonisa-tion of the mandibular component of the first arch, previouslyshown to be derived from Sm-III (Fig. 3C and 3D). Hetero-topic transplantations were also performed for Sm-I. The dis-tribution of cells observed after I→III grafts showed that theectopic mesoderm cells colonised the maxillary prominenceand facial mesenchyme (Fig. 4J). This pattern of colonisationwas characteristic of III→III grafts. The grafted mesoderm didnot contribute to the mesenchyme of the frontal area, nor to therostral and ventral periocular mesenchyme, which were deriv-atives of I→I grafts (Fig. 4A). Mesoderm cells derived fromSm-IV (IV→III), V (V→III) and VI (VI→III) were also het-erotopically grafted into Sm-III. The grafted mesoderm exten-sively colonised the facial mesenchyme, the maxillary andmandibular prominences and the perivascular tissues of thetruncus arteriosus (Fig. 4K,L). In each case where cranialmesoderm was derived from different craniocaudal levels andgrafted into Sm-III, it always developed in a manner charac-teristic of III→III grafts. These results suggest that the normalfate of the cranial paraxial mesoderm is not positionally orsomitomerically restricted at least at 8.5 days p.c. when the het-

2404 P. A. Trainor, S.-S. Tan and P. P. L. Tam

2405Fate of cranial somitomeres

erotopic grafting was performed. In 11% (6/54) of heterotopictransplantations, the grafted mesoderm failed to disperse. Thecells stayed together as a clump of tissue at the injection site.

DISCUSSION

The impact of regionalisation of cell fate in thecranial paraxial mesoderm on craniofacialpatterningResults of cell transplantation and fluorescent dye markingexperiments reveal that the cranial paraxial mesoderm partici-pates extensively in craniofacial morphogenesis and in partic-ular impacts upon the development of the branchial arches(Table 2). There is an orderly craniocaudal distribution of cellsfrom different regions of the cranial mesoderm to the cranio-facial tissues in the mouse embryo (Table 1a,b). A previousmorphological study has revealed that the murine cranialmesoderm is segmented into clusters of cells known as somit-omeres (Meier and Tam, 1982). Each of the seven somitomeresare related topographically to a particular segment of the brain(Jacobson, 1993; Tam and Trainor, 1994). Our discovery of aregionalisation in cell fate may argue for the presence of asegmental prepattern in the cranial mesoderm although thefindings neither require the existence of somitomeres nor givesupport for their existence. For clarity of discussion, however,we shall refer to the different fragments of cranial mesodermby their somitomeric address.

By following the distribution of cells from individual somit-

Fig. 4. (A) Sm-I contribution to the rostral-ventral periocularmesenchyme and lateral nasal process (arrow). (B) Orthotopic graft(II→II) resulting in the distribution of Sm-II-derived cellspredominantly in the dorsal and caudal periocular mesenchyme (arrow)and in the facial mesenchyme lateral to the diencephalon (arrowhead).(C) DiO-labelled cells derived from Sm-II contributes to thedorsocaudal periocular mesenchyme. (D) III→III graft-derived cellspopulating the facial mesenchyme (arrow) lateral to the midbrainextending from the maxillary prominence to the dorsal neural tube.(E) A section through the midbrain region showing the contribution ofIII→III graft-derived cells to the endothelium of the primary head vein.(F) IV→V graft resulting in colonisation of the rostral perioticmesenchyme (arrowhead). (G) VII→VII graft-derived cells contributeto the caudal periotic mesenchyme (arrowhead). (H) The proximodistalseparation of cells labelled by DiI (yellow fluorescence) in themaxillary and mandibular regions of the first arch and DiO (greenfluorescence) in the facial mesenchyme with considerable mixing ofthe two labelled cell populations in the maxillary prominence. (I) II→Vgraft-derived cells in the hyoid arch (arrowhead) and the mesenchymerostral to the otic vesicle (arrow). (J) V→III graft-derived cells coloniseextensively the mesenchyme associated with the lower mesencephalon(arrow) and maxillary prominence (arrowhead). (K) IV→III graftedcells contributing to the maxillary (arrowhead) and mandibular regionsof the first arch. (L) I→III graft-derived cells in the core of themandibular arch (arrowhead) and the perivascular tissues of the truncusarteriosus (arrow). Abbreviations: fb, forebrain; fm, facialmesenchyme; hb, hindbrain; ht, heart; ma, mandibular arch; mb,midbrain; md, mandibular prominence; mx, maxillary prominence; ne,neural epithelium; np, nasal process; oe, optic epithelium; op, oticplacode; ov, optic vesicle; ta, third arch; ts, truncus arteriosus. Bar, 100mm for A,B,E-H,J-L. Bar, 300 µm for C,D,F and I.

Table 2. The developmental fate of cranial mesoderm during craniofacial morphogenesis. A comparison of thedevelopmental fate of the cranial somitomeres in the chick (Noden, 1983b; Couly et al., 1992) and the mouse (this study)

Tissue distribution: chick Tissue distribution : mouse

Prechordal mesoderm muscles: rectus ventralis unknownrectus medialisobliquus ventralis

SomitomeresI II & III muscles: rectus dorsalis I lateral and medial nasal process

obliquus dorsalis periocular mesenchymerectus lateralisintermandibularis dorsalisintermandibularis ventralis II dorsal and caudal periocular mesenchyme

skeleton: corpus sphenoidalis maxiliary component of branchial arch 1orbitosphenoid

III maxilliary componenet of 1st branchial archmandibular component of 1st branchial archmandibular arch arterytruncus arteriosus

IV & V muscles: pterygoideus medialis IV 2nd (hyoid) archpterygoideus lateralis hyoid arch arteryadductor mandibulae externis rostral periotic mesenchymeadductor mandibulae caudalis cervical mesenchymepseudotemporalis superficialispseudotemporalis profundus V 2nd (hyoid) archprotractor pterygoidei hyoid arch arteryprotractor quadrati ventral and rostral peri-otic mesenchymeadductor mandibulae caudalis

skeleton: orbitosphenoidcorpus sphenoidalisdorso lateral mesencephalic meninges

VI & VII muscles: depressor mandibulae, VI 3rd branchial archskeleton: otic capsule ventral periotic mesenchyme

posterior corpus sphenoidalisventral mesencephalic meningesventral metencephalic meninges VII 3rd branchial arch

4th branchial archventral and caudal periotic mesenchyme

2406

omeres to the branchial arches, it was found quite unexpect-edly that each branchial arch is colonised by cells from a setof two consecutive somitomeres. The mandibular arch isderived form somitomeres II and III. Somitomeres IV and Vcontribute to hyoid arch development and somitomeres VI andVII form part of the visceral arch mesenchyme. The fourth archreceives a cranial mesoderm contribution from somitomere VIIonly. However, an additional contribution by the most rostralcervical somites cannot be ruled out. The fifth and sixth pairsof branchial arches were not colonised by any somitomericmesoderm cells. It is interesting to note that this pairwiserestriction of cell distribution only occurs in the branchialarches. Outside the arches, cells from adjacent somitomeresmixed extensively in the periocular, facial and cervical mes-enchyme. Cell mixing, however, is minimal between alternatesomitomeres.

The contribution by dual somitomeres to each branchial archis reminiscent of the so-called ‘two segment periodicity’ asso-ciated with the derivation of the branchial arch motor neuronsfrom pairs of rhombomeres (Lumsden and Keynes, 1989). Themotor nerves innervating the three arches (trigeminal, facialand glossopharyngeal) develop from nuclei found in consecu-tive pairs of rhombomeres (r2 and r3 for trigeminal; r4 and r5for facial; r6 and r7 for glossopharyngeal; Lumsden, 1990;Lumsden et al., 1991; Hunt et al., 1991a). The segmentallyrepeating patterns of rhombomeres and motor neurons areassociated with the differential expression of genes such asKrox-20, Hoxb-1 and Sek (Wilkinson et al., 1989; Nieto et al.,1991). Differential expression of a similar set of genes is alsofound in the migrating neural crest derived from specific rhom-bomeres (Hunt et al., 1991a). All the known genes forembryonic mesoderm however, such as Mox1, Mox2 (Candiaet al., 1992), M-twist (Wolf et al., 1991), Sek (Nieto et al.,1992) and S8 (de Jong and Meijlink, 1993) do not display anyspatially restricted patterns of expression that may reflect theinitial somitomeric organisation or the distribution of the mes-

enchymal derivatives from these somitomeres (Tam andTrainor, 1994).

Cranial mesoderm does not display any positionalidentity in developmental fateThe orderly and predictable distribution of cells from specificsomitomeres to different craniofacial structures may imply thata stringent specification of developmental fate has beenendowed on individual somitomeres. A comparison of the dis-position of cells from within the dorsal and ventral componentsof the paraxial mesoderm to different parts of the branchialarches further reinforces the view that some form of regionalrestriction in potency may be present. Cells in the ventralregion of the cranial mesoderm contribute to the distal mes-enchyme of the mandibular and hyoid arches, respectively.Progressively more dorsal regions contribute to tissues in theproximal regions of the arches and preferentially to facial mes-enchyme. Whether the maintenance of the dorsoventral segre-gation of these two cell populations is the result of diminishedcell mixing or an intrinsic regional difference in tissue’s fatehas not been tested. It has been shown in the chick, however,that regionalisation of cell fate does exist between the medialand lateral cell populations in the cranial mesoderm (Couly etal., 1992).

Cell fate analysis following heterotopic transplantationrevealed that cells taken from any axial level of the cranialmesoderm can contribute to formation of the maxillary andmandibular prominences of the first arch in a manner typicalof the caudal mesencephalic mesoderm to which they are trans-planted. Similarly mesoderm ectopically grafted into Sm-II orSm-IV will migrate in a manner characteristic of tissues at thenative site. These results indicate that in the 8.5-day early-somite-stage mouse embryo, the differentiative pathway thatcould be taken by cranial mesoderm is not positionallyrestricted. Our results agree with previous studies in the chick,which demonstrate remarkable morphogenetic plasticity of theparaxial mesoderm (McLachlan and Wolpert, 1980; Noden1982; Cauwenbergs et al., 1986; Butler et al., 1988).

Co-distribution of cranial mesoderm and the neuralcrest cells The distribution of cranial mesoderm to the frontonasal andbranchial regions (this study) is generally similar to that of theneural crest cells originating from the same axial level(Lumsden and Keynes, 1989; Serbedzija et al., 1992).Mesoderm from the first two somitomeres and the forebrainand upper mesencephalic neural crest cells contribute primarilyto the periocular region in a similar pattern to the somitomericmesoderm. The maxillary region of the first arch is colonisedby mesenchyme derived from Sm-II and also the mesen-cephalic crest (Lumsden et al., 1991; Serbedzija et al., 1992;Osumi-Yamashita et al., 1994). Neural crest cells from thecaudal mesencephalon and the mesoderm of Sm-III bothmigrate into the distal region of the mandibular arch. Thepreotic or metencephalic neural crest cells and mesodermderived from Sm-IV and Sm-V both contribute to hyoid archmorphogenesis. A similar concordance of cell contribution isalso found for the postotic or myelencephalic neural crest cellsand Sm-VI and SM-VII, which collectively colonise the thirdbranchial arch. Therefore cranial neural crest cells and thesomitomeric mesoderm generally share a common segmental

P. A. Trainor, S.-S. Tan and P. P. L. Tam

III

V

VII

I

VI

II

IVMx

Md

HT

OV

OT

A B

Fig. 5. Schematic diagrams showing the spatial distribution of cellsderived from different regions of the cranial paraxial mesoderm, asdefined by somitomeres, to the craniofacial structures of the mouseembryo at about 10. 5 days p.c. Fig. 5A shows those originating fromodd-numbered somitomeres and Fig. 5B for those from even-numbered somitomeres. Abbreviations: HT, heart; Md, mandibularprominence; Mx, maxillary prominence; OT, otic vesicle; OV, opticevagination.

2407Fate of cranial somitomeres

origin and final destination. This raises the question of how thetwo tissue populations are spatially distributed at their finaldestinations. The Dlx-2 gene, which is a vertebrate homologueof the Drosophila Distal-less gene, is uniformly expressedthroughout the arches excluding a core of mesenchymal cells(Bulfone et al., 1993). These mesenchymal arch cores, whichare devoid of Dlx-2 gene expression, are consistent with thelocation of the mesodermally derived muscle plates. Migratingneural crest cells also express Dlx-2. This suggests that, in thebranchial arches, the muscle plates, which are mesodermallyderived become enveloped by neural crest cells. This could beimportant as it has been shown that, in the limb and the faceof the chick embryo, the region-specific organisation of musclegroups depends critically upon the connective tissue pattern setup locally by the neural crest cells (Chevallier, 1979; Cheval-lier and Kieny, 1982; Noden, 1982, 1983a, 1986). The co-dis-tribution of the craniofacial precursors could therefore beimportant for morphogenetic interactions that lead to the estab-lishment of tissue patterns during craniofacial development.The somitomeric organisation of the paraxial mesoderm, if itdoes exist, may be a part of the metameric patterning processthat operates during the establishment of the neuromeres andthe cranial neural crest cells.

A presumptive myogenic fate of cranial mesodermin the branchial archesCells derived from the cranial somitomeres colonise the mes-enchymal core of the branchial arches. Interestingly, mes-enchyme at the core of newly formed branchial arches has beenfound to express muscle-specific genes such as Myf5 (Ott etal., 1991) and Hlx (R. Harvey, per comm) at 9.25 to 10.0 days.This mesenchyme later condenses into a myogenic massknown as the muscle plate of the branchial arch (Bulfone etal., 1993). In the chick, the cranial mesoderm displays a pre-dominantly myogenic fate (Noden, 1983b). More significantly,the precursors of different groups of craniofacial muscles andskeletogenic cells are regionalised craniocaudally in specificsets of somitomeres (Table 2). For example, the myoblasts ofthe extrinsic ocular muscles in the chick were derived fromSm-I and Sm-II and the jaw opening and hyobranchial musclescame from Sm-VI and Sm-VII. Although the mouse culturesystem only permits in vitro development up to about 10.5d.p.c. and therefore does not allow a definitive elucidation ofthe final histogenetic fate of the cranial somitomeres, parallelfindings in the chick and the initial localisation of somitomericcells to the myogenic regions of the branchial arch are stronglysuggestive of a myogenic fate for the cranial somitomeres inthe mouse.

However, other genes not specific for the myogenic lineage,such as the type II collagen gene (Col2a-1, Cheah et al., 1991;Ng et al., 1993) and the murine homologue of the humanHox11 gene (Tlx-1, Raju et al., 1993) are also expressed in theso-called muscle plate. It has been shown recently in the avianembryo that cranial somitomeres also participate, in additionto the differentiation of craniofacial muscle, in the formationof skull bones such as the orbitosphenoid, sphenoidalis and oticcapsule (Couly et al., 1992). The expression of Col2a-1 genein the mesenchyme colonised by the somitomeric cellstherefore suggests that cranial somitomeres may also con-tribute to the formation of at least part of the facial skeletonwhich previously was thought to be entirely derived from

neural crest cells (Le Lievre, 1978; Noden, 1978; Le Douarin,1982). Finally, the murine cranial mesoderm also containsvascular or endothelial tissue precursors just as in the avianembryos (Peault et al., 1983; Pardanaud et al., 1987; Coffinand Poole, 1988; Noden, 1988; Poole and Coffin, 1988).Somitomeres differentiate to form supporting tissues of theprimitive cephalic vein and also contribute frequently to theendothelium of branchial arch arteries. The cranial paraxialmesoderm therefore consists of a multiprogenitor mesodermalcell population.

We thank Peter Rowe and Gabe Quinlan for discussion andcomments on the manuscript. This work was supported by theNational Health and Medical Research Council of Australia and theClive and Vera Ramaciotti Foundation.

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(Accepted 30 May 1994)

P. A. Trainor, S.-S. Tan and P. P. L. Tam