A Nonneural Epithelial Domain of Embryonic Cranial Neural Folds Gives Rise to Ectomesenchyme

A nonneural epithelial domain of embryonic cranial neural folds gives rise to ectomesenchyme

Marie Anne Breau*†, Thomas Pietri*‡, Marc P. Stemmler§, Jean Paul Thiery*¶, and James A. Weston‡ʈ

*Centre National de la Recherche Scientiﬁque, Unite Mixte de Recherche 144, Institut Curie, 26 Rue d’Ulm, 75248 Paris Cedex 05, France; §Department of Molecular Embryology, Max Planck-Institute of Immunobiology, Stuebeweg 51, D-79108 Freiburg, Germany; and ‡Institute of Neuroscience, University of Oregon, Eugene, OR 97403-1254

Edited by Igor B. Dawid, National Institutes of Health, Bethesda, MD, and approved March 26, 2008 (received for review November 30, 2007) The neural crest is generally believed to be the embryonic source define the general location of an epithelial domain in the murine of skeletogenic mesenchyme (ectomesenchyme) in the vertebrate cranial NFs from which some EM originates. head and other derivatives, including pigment cells and neurons and glia of the peripheral nervous system. Although classical Results transplantation experiments leading to this conclusion assumed Cre-Recombinase Is Expressed in Lateral Neural Fold Epithelium Be- that embryonic neural folds were homogeneous epithelia, we fore EMT in Ht-PA-Cre Transgenic Embryos. In transgenic mouse reported that embryonic cranial neural folds contain spatially and embryos expressing Cre-recombinase (Cre) under the control phenotypically distinct domains, including a lateral nonneural of the human tissue plasminogen activator promoter (Ht-PA- domain with cells that coexpress E-cadherin and PDGFR␣ and a Cre/R26R), cells exhibiting ␤-galactosidase (␤-gal) activity thickened mediodorsal neuroepithelial domain where these pro- appear precociously in BA, frontonasal process, and periocular teins are reduced or absent. We now show that Wnt1-Cre is mesenchyme before any marked cells appear in cranial NF expressed in the lateral nonneural epithelium of rostral neural epithelium (Fig. 1). To reconcile this early appearance of folds and that cells coexpressing Cre-recombinase and PDGFR␣ labeled mesenchyme with its presumed NC origin, it was delaminate precociously from some of this nonneural epithelium. suggested (11) that some crest cells might disperse before the We also show that ectomesenchymal cells exhibit ␤-galactosidase transgenic marker was activated. In early [embryonic days activity in embryos heterozygous for an Ecad-lacZ reporter knock- (E)8.0–E8.5] transgenic embryos, however, we observed Cre in allele. We conclude that a lateral nonneural domain of the neural protein in cell nuclei both in the nonneural epithelium of the fold epithelium, which we call ‘‘metablast,’’ is a source of ecto- cranial NFs and in underlying mesenchyme (Fig. 2). Therefore, mesenchyme distinct from the neural crest. We suggest that closer we conclude that Cre-expression precedes or at least is coin- analysis of the origin of ectomesenchyme might help to under- cident with the delamination of labeled cells from the non- stand (i) the molecular-genetic regulation of development of both neural epithelium. These results are consistent with the neural crest and ectomesenchyme lineages; (ii) the early develop- prediction in ref. 5 that at least some EM originates preco- mental origin of skeletogenic and connective tissue mesenchyme in ciously from a source other than NC. the vertebrate head; and (iii) the presumed origin of head and branchial arch skeletal and connective tissue structures during Wnt1-Cre Is Expressed in NonNeural Epithelium of Embryonic Cranial vertebrate evolution. Neural Folds. Previous cell labeling studies, using Wnt1-Cre transgenic embryos report that both BA mesenchyme and trunk cranial neural crest ͉ EMT ͉ metablast ͉ PDGFR␣ NC derivatives express ␤-gal in E9.5 and older Wnt1-Cre/R26R transgenic embryos (11, 12). Because Wnt1 is generally assumed n vertebrate embryos, dorsal neural epithelium of the neural to be expressed only in dorsal neural tissue (13, 14), it is widely Itube (NT) undergoes an epithelium-to-mesenchyme transition regarded as a definitive marker for NC-derived cells (e.g., ref. (EMT) (1) to produce neural crest (NC) cells. These cells then 15). When we examined Cre expression in the NFs of E8 (4–7 disperse in embryonic interstitial spaces and eventually give rise somite) transgenic embryos carrying Wnt1-Cre, we confirmed to pigment cells and neurons and glia of the peripheral nervous that Cre-immunoreactive (IR) cells are present in the dorsal systems (PNS). At cranial levels of amphibian, avian and mam- neural epithelium of the NFs at and caudal to the Vagal axial malian embryos, the cells derived from neural fold (NF) epi- level [data not shown; see supporting information (SI) Fig. S1]. thelium also give rise to these derivatives. In addition, however, However, in the fore- and midbrain NFs of these embryos, most avian and mammalian cranial NFs, like the NFs at all axial levels Cre-IR was observed in the nonneural (E-cad-IR) epithelium of amphibian embryos, give rise to some connective tissue cells and skeletogenic mesenchyme [‘‘ectomesenchyme’’ (EM)]. It is generally believed that the NC produces EM and that the crest Author contributions: J.P.T. and J.A.W. contributed equally to this work; J.A.W. designed at trunk axial levels of avian and mammalian embryos has lost research; M.A.B., T.P., and M.P.S. performed research; T.P. and M.P.S. contributed new reagents/analytic tools; M.A.B., T.P., M.P.S., J.P.T., and J.A.W. analyzed data; and M.A.B. the ability to do so (ref. 2; see also refs. 3 and 4). and J.A.W. wrote the paper. However, Weston et al. (5) have challenged this idea, noting The authors declare no conﬂict of interest. that mouse cranial NF epithelium is heterogeneous and that a This article is a PNAS Direct Submission. sharp boundary exists between lateral E-cadherinϩ (Ecadϩ) Freely available online through the PNAS open access option. nonneural epithelium and the thickened E-cadϪ neural epithe- † lium (NE) in the dorsomedial ridge (see also refs. 6 and 7). Present address: Division of Developmental Neurobiology, National Institute for Medical ϩ Research, Mill Hill, London NW7 1AA, United Kingdom. Moreover, a subpopulation of E-cad cells in the lateral non- ¶ ␣ To whom correspondence may be addressed at the present address: Institute of Molecular neural epithelium coexpresses PDGFR , which is a well estab- and Cell Biology, Agency for Science, Technology, and Research, 61 Biopolis Drive, Singa- lished marker of mesenchyme and connective tissue in somites pore 138673. E-mail: [email protected]. and in the head and branchial arches (BA) (8–10). They sug- ʈTo whom correspondence may be addressed. E-mail: [email protected]. ϩ ϩ gested that EM arises from this PDGFR␣ /E-cad NF epithe- This article contains supporting information online at www.pnas.org/cgi/content/full/ lium in vivo. Our present results support the idea that the source 0711344105/DCSupplemental. of EM is not restricted to the dorsal NE and have allowed us to © 2008 by The National Academy of Sciences of the USA

7750–7755 ͉ PNAS ͉ June 3, 2008 ͉ vol. 105 ͉ no. 22 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711344105 Downloaded by guest on October 2, 2021 endogenous E-cad expression patterns (17). These embryos reveal that ␤-gal is, or had recently been active in both nonneural epithelium of cranial NFs and in underlying EM cells (see Fig. 5 A–H and Fig. S2). We infer that these mesenchyme cells must have originated in the nonneural epithelium. In addition, however, we occasionally observed a few labeled cells within neural epithelium of E.8.5 Ecad-lacZ knockin embryos (data not shown, but see Discussion), suggesting that these cells also originate from the E-cadϩ (nonneural) epithelium.

Cells Derived from Nonneural Epithelium also Coexpress PDGFR␣. BA mesenchyme persistently expresses PDGFR␣ϩ, and requires its function for normal development of EM derivatives (8–10). Therefore, if EM originated from nonneural epithelium (see refs. 5 and 10), we would expect that (i) some cells in the NF epithelium of Wnt1Ϫ and Ht-PA-Cre transgenic embryos would express both PDGFR␣ and Cre, (ii) some of these PDGFR␣ϩ cells would also coexpress E-cad, and (iii) ␤-gal-expressing cells in Ecad-lacZ knockin embryos would express PDGFR␣. These predictions were verified: Cre/PDGFR␣ double-positive cells were present in both the nonneural epithelium of NFs and in Fig. 1. ␤-galactosidaseϩ mesenchymal cells appear precociously in the underlying mesenchyme of E8 Ht-PA-Cre (Fig. 4 A–D) and branchial arches. (A–C) X-Gal staining of E8-E8.5 Ht-PA-Cre/ROSA26 embryos Wnt1-Cre (Fig. 4 E–H) embryos. Likewise, labeled nonneural at the 4-somite (A), 8-somite (B), and 10-somite (C) stages. (D) High magniﬁ- epithelium and mesenchyme in Ecad-lacZ knockin embryos cation of outlined region in C, showing ␤-galϩ epithelial cells lateral to the coexpressed PDGFR␣ (Fig. 5 G and I). Thus, it seems likely that dorsal ridge of the NF. Labeled cells are present in the BA (white arrowheads), ␣ϩ in the frontonasal process (red arrowhead), and around the optic pit (black skeletogenic (PDGFR ) mesenchymal cells delaminate from arrowheads) before any marked cells are present in the NE. The dorsal ridge this nonneural domain of the NF. of NE, comprising the mediodorsal domain of the NF (the NC), is indicated by dashed lines. Discussion A Lateral Nonneural Domain of Cranial Neural Folds Produces Ecto- mesenchyme. Our results have allowed us to map the general lateral to the dorsal ridges and in underlying mesenchymal cells location of a lateral nonneural epithelial domain in the rostral (Fig. 3). These results contradict the notion that Wnt1-Cre-IR NF of early murine embryos (Fig. 6A, green shading) that cells arise solely from dorsal neural epithelium in these early produces ectomesenchyme. This so-called metablast domain (5) embryos (see refs. 12, 15, and 16) and mitigate the inference that is spatially separate from the locations of neurogenic placodes dorsal neuroepithelium-derived NC cells are the sole source of (Fig. 6A, blue shading; see also ref. 18). The metablast epithe- EM in vivo. lium is also phenotypically and developmentally distinct from the mediodorsal NE, which is believed to give rise to NC-derived Nonneural Epithelial Cells and Underlying Ectomesenchyme Are La- melanogenic and neurogenic cells at all axial levels. We suggest beled in Embryos Where lacZ Is Under the Control of the E-cadherin that the metablast epithelium produces a population of Locus. To confirm that EM cells are derived from E-cad- PDGFR␣ϩmesenchymal cells, presumably skeletogenic EM, expressing epithelium, we examined E8 transgenic embryos in that was previously thought to originate from the NC. which lacZ is expressed under the control of the regulatory We have also observed both Ecad-lacZ activity and sequences of the E-cad locus, which are known to recapitulate PDGFR␣-IR in a few cells within the thickened NE of the NF BIOLOGY DEVELOPMENTAL

Fig. 2. Ht-PA-Cre-recombinase is expressed in the nonneural cranial neural fold epithelium. (A–C and E–G ) Double-staining of Cre and E-cad in cranial NF sections of E8 (4–5 som) Ht-PA-Cre transgenic embryos. Red arrowheads indicate cells of the nonneural epithelium that coexpress Cre and E-cad. White arrowheads mark instances of Cre-IR mesenchyme that also express weak cytoplasmic E-cad staining. (C and G)(Inset) Cells of the nonneural epithelium that coexpress Cre and E-cad cells shown at higher magniﬁcation. (F and G) Blue arrowheads show cells that appear to be down-regulating cell-surface Ecad during EMT, and intermingling with the neural cells. (D–H) Low-magniﬁcation DAPI-stained sections shown for orientation. Planes of section are indicated at right by lines (solid or dashed) on schematic of E8 embryos. Red segments on these lines indicate the location of the double-positive nonneural epithelial cells seen in both left and right NF domains, respectively, lateral to the dorsal ridge.

Breau et al. PNAS ͉ June 3, 2008 ͉ vol. 105 ͉ no. 22 ͉ 7751 Downloaded by guest on October 2, 2021 Fig. 3. Wnt1-Cre-recombinase is expressed in the nonneural cranial neural fold epithelium. (A–C and E–G) Double-staining of Cre and E-cad in cranial NF sections of E8 (4–5 som) Wnt1-Cre transgenic embryos. Red arrowheads indicate instances of the nonneural epithelial cells (C and G) coexpressing Cre and E-cad. White arrowheads indicate Cre-IR mesenchyme with weak cytoplasmic E-cad staining. (C and G)(Inset) Cells of the nonneural epithelium that coexpress Cre and E-cad cells (indicated by asterisks on the arrowheads) shown at higher magniﬁcation. (D and H) Red segments indicate the location of the double-positive nonneural epithelial cells in the plane of section.

(see above). Likewise, weak cytoplasmic E-cad-IR is visible in eventually neuro/gliogenic lineages of the PNS exclusively ex- some of the Cre-IR cells in marginal NE and in underlying EM press Sox1 (19). The metablast and NC domains of the cranial (see Figs. 2 B and F and 3 B and F). Based on these observations, NF epithelia also differ in a number of other ways. Thus, part of we suggest that such E-cadϩ cells within the NE also originate the lateral nonneural epithelial NF has been reported to express from nonneural epithelium, some of which overlaps the NE a proteoglycan link protein (20) and transiently, ␤3-integrin (21), margin, and that these cells transiently intermingle with cells in both of which are characteristic of skeletogenic cells, and neither the NE after down-regulating E-cad from their surfaces during of which is expressed by dorsal NE or trunk NC-derived cells in EMT (see ref. 5). mouse embryos. Therefore, because cells in the metablast domain express phenotypic traits characteristic of cells that will Is the Metablast a Subdomain of the Neural Crest? In much of the produce skeletal/connective tissue, and, because some of these current literature, the cranial NF epithelium and the NC are cells delaminate precociously to produce a population of assumed to be equivalent. Accordingly, what we have called PDGFR␣ϩ EM cells, we consider it likely that some are the metablast might be considered a subdomain of the NC and not precursors of craniofacial skeletogenic and connective tissues. distinct from it. The fact that cells of this NF epithelium share Although we acknowledge that the ultimate fates of the meta- the expression of conventional ‘‘neural crest markers’’ (e.g., Snail blast-derived cells remains to be determined, it is important to gene-family members, Foxd3, and Msx1) is certainly consistent emphasize that different genetic regulatory pathways appear to with this idea. However, because many of these genes are actually operate in the cell populations derived from the two epithelial widely expressed in a variety of cell types before specific domains and therefore that it would probably be appropriate to morphogenetic events like EMT (see ref. 5), their use as specific analyze the mesenchymal cells that emerge from them as pre- markers of NC-derived cells could be misleading. cursors of distinct lineages. Despite these reservations, localized gene expression patterns clearly do distinguish the two epithelial domains in mouse cranial Can Cells Delaminating from Metablast Epithelium Account for All of NFs: In the nonneural epithelial domain, E-cad and PDGFR␣, the Ectomesenchyme That Appears in Developing Branchial Arches? It a marker characteristic of mesodermally derived connective is impossible to estimate the number of BA mesenchyme cells tissues (9, 10), are coexpressed at cell surfaces, whereas NE and that delaminate from the entire metablast domain by sampling

Fig. 4. Nonneural epithelial cells in the cranial neural fold express PDGFR␣. Double-staining of Cre (A, C, E, and G) and PDGFR␣ (B, C, F, and G) in Ht-PA-Cre (A–D) and Wnt1-Cre (E–H) transgenic embryos. PDGFR␣-IR is present as sparse punctate staining at cell surfaces. Red and white arrowheads indicate cells of the nonneural epithelium or underlying mesenchyme, respectively, expressing both Cre and PDGFR␣.(C and G) Dashed lines indicate the boundary of E-cadϪ NE. (D and H) Red segments on the lines show the plane of section and the location of the double-positive nonneural epithelial cells.

7752 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711344105 Breau et al. Downloaded by guest on October 2, 2021 neural folds elevate and fuse during cranial development (see A B ref. 22). Consequently, the cells illustrated in the accompanying figures would represent only a very small fraction of the total number that delaminate during this morphogenetic process. The dramatic growth of the BAs (22) clearly must also involve remarkable rates of cell proliferation, both in the nonneural epithelium and within the mesenchyme itself (ref. 23; see also refs. 24 and 25).

Ectomesenchyme Provides the Conditions Necessary for Neural Crest Cell Dispersal. The first cells to emerge in outgrowths of explanted cranial NFs are reported to produce fibronectin, whereas cells CD that emerge later from the same explants, and NC cells cultured from trunk NT explants do not do so (26). Because NC cells apparently require fibronectin-containing extracellular matrix as a substratum for migration, the precocious emergence of EM in the cranial region could establish the conditions necessary for subsequent dispersal of NC-derived cells in the same region. These ideas are summarized in a schematic diagram (Fig. 6B), showing our inferences that EM emerges precociously by delamination from the lateral nonneural domain of cranial NF, and E F that NC cells subsequently enter the interstitial space where they comingle with EM and disperse in the matrix it produces. In this sense, EM in the head would serve the function of somite- derived sclerotome cells in the trunk of amniote embryos to provide appropriate substrata for NC cell migration.

Assumptions Underlying Classical Grafting Experiments Probably Led to Their Misinterpretation. Pioneering experiments in amphibian embryos suggested that the entire range of ‘‘NC-derived’’ phe- notypes, including EM, arises from grafted tissues (see ref. 3). It G H is important to note, however, that in all these experiments, the entire NF was marked and transplanted. Because NFs contain phenotypically distinct epithelial domains, the results of these grafting experiments cannot exclude the possibility that EM originates from a domain of the NF that is distinct from the NC epithelium. Although not widely acknowledged in the literature, but suggested by E-cad staining patterns (e.g., see Fig. 2), it is impossible to dissect the neural from nonneural epithelia in early I J embryonic NFs. Consequently, the cranial NF grafts in avian embryos would also contain both mediodorsal (neural) and lateral (nonneural) tissues. Accordingly, we suggest an alternative explanation for the widely accepted inference that skeletogenic ability is lost by trunk neural crest cells in avian embryos (2). These authors reported that when cranial NFs were grafted, EM derivatives of donor origin were present in avian host embryos. In contrast, when only NT was transplanted in the trunk, hosts contained donor-derived melanocytes and compo- nents of the PNS, but lacked donor-derived EM. Based on our BIOLOGY Fig. 5. E-cadherin promoter activity is revealed in both nonneural epithe- present results, we suggest that the apparent difference in DEVELOPMENTAL lium of the cranial neural folds and the underlying mesenchyme. (A and B)E8 developmental potential between cranial and trunk grafts is (4–5 som) Ecad-lacZ embryos exhibit ␤-gal activity in domains of the cranial NFs (white arrowheads) that are distinct from the NE. (C and E) In sections of most parsimoniously explained by differences in experimental such embryos, ␤-gal activity (blue cytoplasm or inclusions) is present in non- protocol: NF and NT were grafted at head and trunk axial levels, neural epithelial cells of the head (red arrowheads) and in underlying mes- respectively, and only the NF, which contains a nonneural enchyme (white arrowheads and Insets). (G and I) PDGFR␣ staining (brown) epithelial domain, would include the precursors of donor- performed on X-Gal-stained Ecad-lacZ embryos reveals that ␤-galϩ cells in the derived EM. nonneural epithelium and underlying mesenchyme coexpress PDGFR␣. In- Reports that skeletogenic subpopulations are present in long- stances of such double-positive mesenchymal cells are indicated by white term mouse trunk neural crest cell cultures (27) might appear to arrowheads. (Insets) Magniﬁed cells in insets denoted by asterisks. (D, F, H, and J) Low magniﬁcations and planes of the sections for C, E, G, and I, respectively. be inconsistent with our conclusions about metablast-derived Red segments show the location of ␤-galϩ or ␤-galϩ/PDGFR␣ϩ cells observed mesenchyme in vivo. In this work, however, NFs from early (E8, in the head mesenchyme. 4–7 somite) embryos appear to have been explanted before neural and nonneural epithelial domains had separated during midline fusion of the folds. As mentioned above, we have also individual histological sections of embryos at early developmen- occasionally observed E-cadϩ cells within NE of younger tal stages. It is likely, however, that these mesenchyme cells are Ecad-lacZ knockin embryos. It is possible, therefore, that skel- continuously recruited from the metablast epithelium as the etogenic cells and reported mesenchyme stem cells within

Breau et al. PNAS ͉ June 3, 2008 ͉ vol. 105 ͉ no. 22 ͉ 7753 Downloaded by guest on October 2, 2021 cultured trunk crest cell populations (19) might also originate from nonneural epithelium of the NF. A

It Will Be Productive to Distinguish the Two Neural Fold Domains in Future Experimental Analyses. There are several additional reasons to consider NC and metablast to be developmentally distinct epithelial domains within the NFs: First, if metablast and NC represent spatially and developmentally distinct embryonic tissues, it would be unnecessary to postulate regulatory mecha- nisms at this stage of development to account for the loss of skeletogenic potential by trunk neural crest cell precursors (see ref. 2 and 4), nor would it be necessary to assume the existence of, and therefore to strive to identify, gene regulatory pathways that operate in a common NC ‘‘stem cell’’ population to produce both neurogenic and skeletogenic cells in vivo. Although it has been argued (25, 28) that in vitro cloning studies support the idea that skeletogenic and neurogenic lineages share a common neural crest-cell precursor, we must reiterate that most of these cloning experiments suffer significant technical limitations (29), and that the published evidence from clonal analyses for such B common precursors (e.g., ref. 24) is, at least, susceptible to alternative interpretations (see ref. 5). Consequently, the assumptions underlying these clonal analyses, and their relevance to events in vivo, might beneficially be revisited. Second, if it were acknowledged that two distinct epithelial domains exist in NFs, the need to define NC markers more precisely would be compelling. As previously mentioned, for example, the expression of genes like Wnt1 (and also Snail family members; e.g., ref. 30–32) is unlikely to indicate NC origin because their gene products are present in both mediodorsal and lateral NF epithelia. Thus, these expression patterns appear to be spatially and temporally too general to be used to infer embryonic origins. Third, it is now widely accepted that the ‘‘invention’’ of NC played an important role in the evolution of the vertebrate head (see ref. 33). However, as we suggest, connective tissue and skeletogenic mesenchyme in the vertebrate head could have a distinct developmental origin from that of peripheral neurons, glia, and pigment cells. If this were acknowledged, even tenta- tively, it would provide an incentive to consider heuristic alter- natives to the proposed role of the NC in vertebrate evolution. The discovery of ‘‘NC-like’’ migratory cells in ascidians (34), recently understood to be the vertebrate sister group (35), adds support to our argument. Some of these ascidian cells produce pigment cells, but not other derivatives usually attributed to the neural crest, notably skeletogenic cells. As these authors point Fig. 6. Schematic representation of cranial neural fold complexity during the out, other migratory cell types may have joined with NC-like cells morphogenetic events leading to neural tube formation and EMT. (A) Based to account for the appearance of such new vertebrate derivatives. on combined staining of all transgenic embryos, the green shaded area on the Finally, in light of recent reports of the remarkably expanded diagram suggests the general location of the inferred metablast domain and convergent dermal and chondral skeletogenic abilities for within the NF epithelium. Note that this domain is distinct, at this develop- both mesodermal and putative NC-derived mesenchyme (15), it mental stage, from the locations of cranial placodes (blue shaded areas) that now seems appropriate to explore the possibility that they have represent later sources of dispersing cells in the head. n, nasal placode; t, a common developmental origin, distinct from the NC, and to trigeminal placode; o, otic placode; e, epibranchial placodes. (B) Schematic consider where and when the so-called metablast epithelium summary of EMT events in metablast and neural crest epithelium. As inferred from immunostaining results, the NF epithelium has distinct domains with might originate. Based on earlier intriguing reports (36, 37), we sharply deﬁned boundaries: The E-cadϩ, nonneural cells of NF epithelium are suggest a previously unsuspected contribution to cranial mes- represented by red circles. PDGFR␣-IR cells within, and delaminating from, the enchyme from ectopic mesoderm-like cells: Results reported by E-cadϩ NF epithelium are represented by green circles. NC cells, which arise in these authors indicate that such cells could (i) originate in the and begin to delaminate from the mediodorsal NE domain, are represented by early epiblast before the formation of the primitive streak, (ii) blue circles. In these diagrams, PDGFR␣ϩ cells in the nonneural epithelium initially evade incorporation into the nascent primitive streak overlying and lateral to the NE (a) delaminate precociously into underlying and subsequently remain as a distinct population within ecto- cell-free interstitial spaces (b). This nascent EM, like somite-derived sclero- dermal epithelium during gastrulation, and (iii) eventually un- tomal mesenchyme, then establishes the interstitial environment into which neural crest cells delaminate from the dorsal NE (c). As NFs fuse in the midline dergo delayed involution in NF epithelia through transient NF to form the NT (d), nonneural (E-Cadϩ) epithelium (red) separates from the structures whose morphology resembles the primitive streak (see dorsal NE of the nascent NT (blue), EM (green) occupies the interstitial spaces ref. 38). Likewise, as noted in ref. 5, EM derivatives were absent between neural and nonneural epithelia, and NC cells (blue) intermingle with when NC was experimentally induced in nonneural epithelium and disperse ventrolaterally among the EM cells, similar to trunk crest cell by interaction with NE (39, 40). This also provides support for dispersal among sclerotomal mesenchyme.

7754 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711344105 Breau et al. Downloaded by guest on October 2, 2021 the suggestion that metablast and NC epithelia have different erozygous Ecad-lacZ mice (Ecad-In2flox) (17) were crossed to C57BL/6 females developmental origins. to obtain E8 (4–5 somite) embryos (n ϭ 24). Embryos were fixed in 1% PFA and, after whole-mount staining, dehydrated in an alcohol series, embedded in Ultimately, it will be important to follow the fate of epithelial ␮ ϭ cells in the putative metablast domain of the rostral NF. Precise wax, and sectioned at 7 m. Sections of embryos stained for X-gal only (n 13) were counterstained with FastRed (Vector). marking of individual cells in the metablast epithelium would definitively test the prediction that they are the source of Staining Procedures. ␤-galactosidase activity was revealed in whole-mount skeletogenic and connective tissue lineages in the head and face preparations as described in refs. 17 and 42. Antibodies used for immuno- of vertebrate embryos. Such experiments are feasible in avian, staining included rat anti-mouse PDGFR␣ (APA5) (5 ␮g/ml) (10), rat anti-mouse amphibian, and zebrafish embryos. E-cad (ECCD-2) (10 ␮g/ml) (43) and rabbit anti-Cre recombinase (1/1,000) (PRB-106C; Covance; lot no. 135028002). Sections were incubated for2hin Methods blocking solution consisting of 10% FCS, 0.1% Triton X-100, and 0.5ϫ Blocking Transgenic Embryos. Mice carrying Cre-recombinase under the control of a Reagent (Roche) in PBS. The sections were then incubated overnight at 4°C promoter of human tissue plasminogen activator (Ht-PA-Cre) (11) were mated with primary antibodies in blocking solution, rinsed several times in PBS, and with the ␤-gal reporter strain ROSA26 (R26R) (41). E8–E9 transgenic embryos incubated with secondary antibodies (for APA5 and ECCD-2, goat anti-rat Alexa Fluor 594; for anti-Cre, goat anti-rabbit Alexa Fluor 488) for2hatroom used for whole-mount X-gal staining (n ϭ 15) were removed in cold PBS plus temperature in the dark. Whole-mount APA5 staining was performed as 5% FCS and fixed briefly in cold 1% formaldehyde, 0.2% glutaraldehyde, and described in ref. 10. 0.02% Nonidet-P40. For immunostaining, E8 Ht-PA-Cre embryos (11) (n ϭ 7) and embryos from mice carrying Wnt1-Cre (14) [founders kindly provided by A. McMahon (Harvard University, Cambridge, MA)] (n ϭ 7), were removed in ACKNOWLEDGMENTS. We thank Sylvie Dufour for helpful discussions about this work and Rolf Kemler for his generous cooperation. J.A.W. thanks Charles cold PBS, and fixed in cold 4% PFA for 1.5 h. After washing two times in PBS, Kimmel for his constructive skepticism and trenchant advice. This work was fixed embryos were incubated successively in 12% (1.5 h), 15% (2 h) and 18% supported by an Association pour la Recherche contre le Cancer fellowship (to (overnight) sucrose solutions, then embedded in Tissue-Tek matrix, frozen, M.A.B.). This project was initiated in 2002 when J.A.W. was a visitor at the and sectioned at 7 ␮m on a Leica cryostat microtome before staining. Het- Institut Curie under the auspices of a Rothchild/Mayent Fellowship.

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