Development 110, 417-425 (1990) 417 Printed in Great Britain © The Company of Biologists Limited 1990

Differential cytokeratin expression reveals early dorsal-ventral regionalization in chick

TIMOTHY S. CHARLEBOIS*, JONATHAN J. HENRYt and ROBERT M. GRAINGER

Department of Biology, University of Virginia, Charlottesville, VA 22901, USA

Present addresses: •Genetics Institute, One Bum Rd, Andover, MA 01810, USA t Department of Biology, Indiana University, Bloomington, IN 47405, USA

Summary

The induction and spatial patterning of early mesoderm even while the mesoderm layer is being formed, and are known to be critical events in the establishment of persists during the extensive cellular movements and the vertebrate body plan. However, it has been difficult tissue remodelling associated with morphogenesis. These to define precisely the steps by which mesoderm is results point to an early step in which two fundamentally initially subdivided into functionally discrete regions. distinct states are established along the presumptive Here we present evidence for a sharply defined dorsal-ventral axis in the mesoderm, and suggest that distinction between presumptive dorsal and presumptive determination in this occurs in a hierarchical ventral regions in early chick mesoderm. Northern blot manner, rather than by direct specification of individual and in situ hybridization analyses reveal that transcripts types of histological differentiation. The differential corresponding to CKsel, a cytokeratin gene expressed expression of CKsel represents the earliest molecular during early development, are present at high levels in index of dorsoventral regionalization detected thus far in the presumptive ventral mesoderm, but are greatly the mesoderm. reduced or undetectable in the future dorsal region of mesoderm, where the formation of axial structures Key words: chick , mesoderm regionalization, occurs later in development. This distinction is present cytokeratin gene expression.

Introduction histological differentiation, and examining their devel- opment in isolation. In the amphibian embryo, isolated The formation of vertebrate mesoderm has recently cultures of prospective dorsal mesoderm differentiate been the subject of intensive scrutiny because this germ primarily into notochord and muscle (the most dorsal layer plays a central role in the establishment of mesodermal derivatives), but very little mesothelium or embryonic pattern (Slack, 1983). Even at very early blood (ventral mesoderm). Isolates removed from stages, the mesoderm appears not to be homogeneous progressively more ventral locations form much more but instead possesses region-specific characteristics mesothelium and blood, with the amount of muscle or along the dorsal-ventral axis of the embryo. This notochord becoming greatly reduced, particularly in the regional specialization of early mesoderm has particu- most ventral isolates (Dale and Slack, 1987). larly important implications for the formation of the While the above data argue that regional specializ- basic vertebrate body plan, as recognized by Spemann ation occurs along the dorsal-ventral axis in the early and Mangold (1924), who first demonstrated the unique mesoderm, it has been difficult to define the steps by ability of the dorsal lip mesoderm to direct the which cell diversity is first established in this germ layer. formation of a second set of axial structures when One possibility is that local, possibly graded signals placed in a ventral location. In addition to this striking could establish a series of positional states along the regional difference in inductive ability, the mesoderm dorsal-ventral axis. Individual states could then specify becomes subdivided with respect to the types of the various types of histological differentiation, such as phenotypic differentiation that are specified at different blood, mesothelium, muscle or notochord. Alternately, positions along the dorsoventral axis (Slack and the mesoderm might be subdivided in a progressive, Forman, 1980; Dale and Slack, 1987). Such differences hierarchical manner starting perhaps with fundamen- can be detected by removing portions of the prospective tally distinct dorsal and ventral states. Cells within the mesoderm at very early stages, before any sign of dorsal and ventral regions could then interact to 418 T. S. Charlebois, J. J. Henry and R. M. Grainger

generate various intermediate grades of mesoderm. dehyde gel, transferred to nylon membrane, and UV- This latter idea is supported by data indicating that crosslinked at 1200 ^Wcm~2 for 2 min. Equivalency of gel loading was checked by ethidium bromide staining of rRNA interactions within the mesoderm can influence the final 6 1 1 pheriotypic outcome of cells in this germ layer (Dale bands. Filters were hybridized to 4.5xl0 ctsmin~ ml~ of ^P-labelled antisense RNA transcribed from pCKsel (see and Slack, 1987). However, there is considerable Charlebois et al. 1990), in 50% formamide, 0.05% ficoll, overlap in the types of differentiation obtained in 0.05% polyvinylpyrollidone, 0.1% sodium dodecyl sulfate induction and explant studies, and no direct means of (SDS), 5xSSC (lxSSC is 0.15M NaCl, 0.015M sodium detecting regional specialization has been available for citrate), 100/igml"1 salmon sperm DNA at 62°C. Blots were the period before the activation of cell type-specific washed in 2xSSC, 5% SDS, at room temperature, followed . Consequently, it has not been possible to by O.lxSSC, 1 % SDS at 67 °C. Exposures were carried out establish the existence or location of a boundary using X-Omat AR film at —70°C with intensifying screens. delineating fundamentally distinct regions of dorsal and Autoradiograms were scanned using a Biolmage Visage 2000 ventral mesoderm. computerized image analysis system. Here we present evidence for an early and sharply In situ hybridization defined distinction between presumptive dorsal and ventral regions of chick mesoderm. Previously, we were fixed in 4% paraformaldehyde, dehydrated and embedded in Paraplast. 8fim sections were prepared, isolated a chick cDNA clone (CKsel) on the basis of dried on slides, paraffin was removed and sections were differential hybridization within the of the treated with proteinase K and acetic anhydride prior to early embryo (Charlebois et al. 1990). This cDNA clone hybridization. [35S]RNA (antisense) probes were prepared by encodes a putative type II cytokeratin which exhibits transcription of pCKsel using T7 RNA polymerase, and approximately 75 % identity with a number of keratins subjected to partial hydrolysis with 0.2 M NaHCO3/Na2CO3 characteristic of embryonic and simple epithelia, and is (pHIO). Hybridization was carried out for 16 h at 61 °C in most closely related to cytokeratin number 7 of the 50% formamide, 0.3 M NaCl, 20mM Tris-HCl (pH8.0), 5mM human catalog (Moll et al. 1982; Glass et al. 1985). EDTA, 0.02% bovine serum albumin, 0.02% ficoll, 0.02% polyvinylpyrollidone, 10% dextran sulfate, 10 mM dithiothrei- CKsel was found to be expressed at high levels in body 35 ectoderm beginning at neurulation, but transcript levels tol to which [ S]RNA was added to a final concentration of approximately 1.3xl07ctsmin-1 ml"1. Slides were washed in were seen to be greatly reduced in ectoderm of the head 4XSSC, 10 mM dithiothreitol at room temperature and treated and in neural ectoderm (Charlebois et al. 1990). In the with RNase A (20/^gml"1) at 37°C for 30min. Slides were present work we report on our finding that this gene is then washed in 50% formamide, 2xSSC at 50°C for 45min, expressed differentially in the mesoderm as well. The followed by O.lxSSC at 50°C for 45min. Dried slides were pattern of CKsel gene expression in this germ layer dipped in Kodak NTB-2 emulsion and exposed for approxi- appears to delineate two distinct states that are defined mately 3 days at 4°C. Control sections were hybridized under with respect to cellular position along the future identical conditions to a sense probe prepared from the 3' Pstl dorsal-ventral axis. fragment of a chick 6-crystallin cDNA clone (Bhat and Piatigorsky, 1979). No specific hybridization to this control probe was observed; only a very low and uniform level of signal could be seen throughout these sections (Charlebois, Materials and methods 1988). Embryos and tissue dissections Fertile White Leghorn chicken eggs (Truslow Farms, Chester- Results town, MD) were incubated at 38 °C, 60% relative humidity, and staged according to Hamburger and Hamilton (1951). For In the course of carrying out preliminary in situ the isolation of presumptive dorsal and ventral mesoderm, the hybridization studies to determine the regional distri- trunk region (denned as the portion of the body between the first and last somite; see Fig. 1A) of stage 11-13 embryos was bution of CKsel transcripts in early ectoderm, we placed in 2.25% trypsin (Gibco, 1:250), 0.75% pancreatin observed that in the trunk region of stage 10 embryos (Difco) in phosphate-buffered saline (PBS) on ice for (stages are according to Hamburger and Hamilton, 15-30 min, and transferred to 20% fetal bovine serum 1951, for primitive streak and later stage embryos), (Sigma; heat inactivated at 56°C) in PBS for dissection. CKsel was also expressed in the mesoderm and and ectoderm were removed and discarded, and endoderm (Charlebois et al. 1990). However, CKsel mesoderm was separated into presumptive dorsal and ventral transcripts were seen only in the more lateral regions of components by making a longitudinal cut at the ventrolateral mesoderm and did not appear to be detectable in the margin of the somites (see Fig. IB). Tissue fragments were mesoderm immediately flanking the midline. The three rinsed in PBS, and frozen in a dry ice/ethanol bath pending primary germ layers at this stage are still largely RNA isolation, which was performed as described elsewhere (Charlebois et al. 1990). Embryos used for in situ hybridiz- discrete, laying flat on the large yolk mass in the order ation were trimmed at the outer margin of the area pellucida (from the yolk) endoderm, mesoderm, ectoderm (see (stages 4-6) or within 3—4 somite widths of the midline (stage Fig. IB). During the formation of the lateral body 10) prior to fixation. folds, all three germ layers fold downward and inward, undercutting the body of the embryo proper. Thus the Northern blot analysis most medial portion of the embryo constitutes the Approximately 4.5 ^g total RNA from dorsal and ventral future dorsal side; the more lateral regions on either tissues was electrophoresed in a 1.2% agarose, 6% formal- side are destined to become ventrally located (for Dorsal-ventral regionalization in early mesoderm 419 B c » * DORSAL • 0

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Fig. 1. Differential cytokeratin gene expression in chick mesoderm. (A) Diagramatic representation of a stage 11 (38-40 h; Hamburger and Hamilton, 1951) chick embryo, showing the trunk region dissected for mesoderm isolation. (B) Cross- sectional illustration of the trunk region depicted in (A). The shaded region lying between the ectoderm (ect) and endoderm (end) constitutes the mesoderm layer. Broken lines through the mesoderm indicate the position of the cuts made to separate dorsal mesoderm from ventral mesoderm. Abbreviations: nt, neural tube; s, somite; nc, notochord; lpm, ; ect, ectoderm; end, endoderm. (C) Northern blot analysis of cytokeratin gene expression in dorsal and ventral mesoderm regions shown in B. Migration of marker RNAs of known length are indicated in kilobases at left.

review, see Balinsky, 1981). Our initial observation the margins moves ventrally during formation of the therefore suggested that cytokeratin gene expression lateral body folds, and gives rise to a variety of might be regulated along the future dorsal-ventral axis phenotypes characteristic of ventral mesoderm, includ- in the mesoderm. ing mesothelium and blood (Patten, 1951; Romanoff, To pursue this observation further, we performed 1960). Between the lateral plate and the more medial Northern blots to confirm the identity and distribution mesodermal structures (notochord and somites; i.e. of CKsel transcripts in dissected regions of mesoderm. future dorsal mesoderm) lies a thin strip of from the trunk region (illustrated in mesoderm, destined to form nephrotome and venous Fig. 1A) of 40 h embryos (stage 10-12) was isolated and vasculature (Meier, 1980). The pattern of in situ dissected into two regions based on visible morphologi- hybridization of antisense CKsel RNA to this section is cal criteria: a cut was made parallel to the midline at the shown under darkfield illumination in Fig. 2C. As ventrolateral edge of the somites, such that the more expected, CKsel RNA is detected in the trunk medial (presumptive dorsal) fragment containing noto- ectoderm (expression in this tissue was the basis for its chord and somitic mesoderm was separated from the. isolation), and very high levels of hybridization are also (presumptive ventral) mesoderm of the lateral plate seen in the endoderm. Within the mesoderm, consistent (Fig. IB). These fragments were then analyzed for the with the RNA blot result shown above, strong presence of cytokeratin RNA using antisense RNA hybridization is seen in the lateral plate mesoderm. This transcribed from pCKsel as a probe. Hybridization was signal, which is maximal in the most lateral regions of only barely detectable in the medial mesoderm, the mesoderm, disappears abruptly at the boundary whereas a very strong signal was observed in the more between the lateral plate and the intermediate meso- lateral fragment (Fig. 1C). The transcripts detected derm. No hybridization can be discerned in any of the were indistinguishable in size from the major, 2.9 kb more dorsal structures, i.e. the notochord, somites, or band previously detected by the CKsel probe in the intermediate mesoderm. A similar pattern of differen- ectoderm (Charlebois et al. 1990). Densitometric tial cytokeratin gene expression is seen in sections at scanning of autoradiograms indicated that CKsel various levels along the anterior-posterior axis. transcripts are well over 200-fold more abundant in the To investigate the developmental time course of lateral (presumptive ventral) mesoderm. differential cytokeratin gene expression in the meso- In order to obtain more precise spatial and temporal derm, we examined in situ hybridization of the CKsel information concerning the mesodermal distribution of probe to transcripts at earlier stages. At stage 6 CKsel transcripts, in situ hybridization to sectioned (23-25 h; see Fig. 2, D-F), somites have not yet formed embryos was carried out (Fig. 2). Fig. 2A illustrates the and the notochordal rudiment is present only anterior plane of a section cut through the somite region of a to the primitive streak. When sections through the stage 10 (36 h) embryo; this section is shown under primitive streak are examined at this stage, it is evident phase contrast in Fig. 2B. The lateral mesoderm seen at that mesoderm immediately on either side of the 420 T. S. Charlebois, J. J. Henry and R. M. Grainger

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primitive streak (presumptive dorsal region) exhibits morphological organization of this germ layer (e.g. little or no detectable CKsel hybridization. In contrast, formation of somites, notochord, lateral plate). much stronger hybridization is seen at more lateral Sections through the anterior half of the primitive (presumptive ventral) positions in the mesoderm, which streak in stage 4 (18-19 h; see Fig. 2, G-I) embryos lies between and endodermal layers that reveal that a distinct boundary of CKsel gene exhibit very high levels of CKsel hybridization. The expression exists in the mesoderm prior to even the expression pattern in the mesoderm indicates that the most subtle evidence of organization yet detected in this dorsal/ventral difference in CKsel expression pre- germ layer. The primitive streak delineates the portion cedes, and therefore cannot be dependent on, the overt of the embryo undergoing , where prospec- Dorsal-ventral regionalization in early mesoderm 421 Fig. 2. In situ hybridization of pCKsel antisense RNA to Meier, 1982; Packard and Meier, 1983). In the sectioned embryos. For each example shown, the mesoderm at this stage, the level of CKsel RNA illustration at left (A,D,G,J) shows the position and appears to be lower than at later stages, and the orientation of the section photographed under phase hybridization signal is less intense than in the thin layer contrast (B,E,H,K) and darkfield illumination (C,F,I,L). Illustrations are oriented with the anterior of the embryo of endoderm with which it is closely associated (see toward the top; sections are shown with the dorsal surface Fig. 2, H-I). Close inspection of the mesoderm at the top. Arrowheads indicate position where transition demonstrates nonetheless that a well-defined transition in CKsel gene expression is seen between presumptive in CKsel gene expression occurs along the presumptive dorsal and ventral regions of mesoderm. (A-C) Section dorsal-ventral axis: expression is seen in the lateral through the somite region of a stage 10 embryo. mesoderm (presumptive ventral region), but is un- Hybridization is seen throughout the ectoderm and is very detectable in the medial (presumptive dorsal) region of intense in the endoderm. In the mesoderm, hybridization mesoderm (Fig. 21). The region containing the bound- can be seen only in the lateral plate mesoderm and is ary of CKsel gene expression is enlarged in the inset, undetectable in intermediate mesoderm, somites and Fig. 21. The only detectable signal above and to the notochord. (D-F) Section through the primitive streak of a stage 6 embryo. The overall level of CKsel expression in right of the arrowhead (i.e. in the medial region) the mesoderm is somewhat lower than that seen in the corresponds to the heavily labelled endoderm. No endoderm and epiblast. The mesoderm on either side of signal can be seen in the mesoderm in this medial the primitive streak is CKsel-negative, but CKsel RNA is region. In contrast, to the left and below the arrow readily detected in the presumptive ventral mesoderm. (lateral region), in addition to the strong endodermal (G-I) Section through the primitive streak of a stage 4 signal, a band of hybridization is seen over presumptive embryo. In the epiblast, strong hybridization is only seen ventral mesoderm. This signal is not due to autoradio- at the very outer margins of the area pellucida. The graphic grain scattering from the endoderm, as no such endoderm layer exhibits high levels of CKsel RNA grains are observed to the right or beneath the throughout. In the mesoderm (which is adherent to the endoderm layer. The distinct difference in CKsel endoderm in this section), CKsel RNA is seen in the expression along the presumptive dorsal-ventral axis is presumptive ventral region but is not detected in the presumptive dorsal area closest to the midline. The boxed seen in serial sections throughout the anterior half of region in I is shown at higher magnification in inset, to the stage 4 primitive streak (see Fig. 3). facilitate visualization of the boundary of CKsel Although the sharp boundary of CKsel expression hybridization. (J-L) Section through the posterior portion between presumptive dorsal and ventral mesoderm of the primitive streak of a stage 4 embryo. The endoderm extends posterior to any morphological differentiation exhibits very strong hybridization (a portion of this layer is missing at the midline), while CKsel RNA is detectable in the mesoderm of stage 4 embryos, it is interesting to only at the outer margins of the epiblast. In the mesoderm, look at even more posterior regions of these embryos, CKsel expression is low in the presumptive dorsal region, where the overt regionalization of mesoderm (which but becomes elevated in more ventral locations. takes place in an anterior-to-posterior manner) lags Abbreviations: dm, dorsal mesoderm; ect, ectoderm; end, behind. In this part of the embryo, there is still endoderm; epi, epiblast; lp, lateral plate mesoderm; nc, differential CKsel gene expression (Fig. 2, J-L), but notochord; nt, neural tube; ps, primitive streak; s, somite; the boundary between medial and lateral regions is not vm, ventral mesoderm. Scale bars represent 100nm. as sharply defined. In the section shown, no expression is seen in the epiblast layer (except at the very lateral edges), while strong hybridization is again seen in the tive mesoderm and endoderm cells are actively ingress- endoderm (pulled slightly away from the mesoblast in ing from the upper, epiblast layer, leading to the several places, and missing at the midline). In the formation of the three basic germinal layers (Bellairs, mesoderm in the posterior, no distinct boundary of 1982). At stage 4, the 'definitive streak stage', the streak CKsel expression can be discerned. Instead, the level is of maximum length; as development proceeds, the of CKsel hybridization is seen to diminish more streak regresses posteriorly, so that ingression ceases gradually in a lateral-to-medial direction, eventually first in the anterior but continues for many hours, becoming indistinguishable from the background signal restricted to a progressively more posterior location. (at the position of the arrows) in the region flanking the The first subtle evidence of morphological organization primitive streak. Counts of exposed silver grains in the appears to take place in mesoderm just after ingression lateral mesoblast (near the point labelled vm in Fig. 2L) is complete. The condensation of axial mesoderm into are some 2.8-fold higher than in the medial region (near notochord can first be observed just anterior to the the point labelled dm in Fig. 2L). The basic pattern of regressing streak. Flanking the axial mesoderm, tan- CKsel hybridization detected in the stage 4 mesoderm dem circular domains of paraxial mesoderm, known as is summarized in Fig. 3. somitomeres, have been observed in stereo pairs of scanning electron micrographs (Meier, 1979). In Fig. 2, G-I, a section is shown through the mid-anterior Discussion streak, where ingression is still occurring; this is posterior to where even these most subtle measures of The differential detection of cytokeratin gene tran- mesodermal organization have been observed in defini- scripts in the newly forming mesoderm suggests that tive streak stage embryos (Meier, 1981; Triplett and CKsel expression serves as an early molecular index of 422 T. S. Charlebois, J. J. Henry and R. M. Grainger

(Charles de la Brousse and Emerson, 1990). This study also showed that qmfl, but not several contractile protein genes, is expressed in the dorsal medial lip of the dermatome, suggesting that this region of the somite is a site of myogenic lineage determination. In contrast, the differential expression of CKsel is established prior to any morphological evidence of mesodermal organization and seems not to be a reflection of any individual type of histological differen- tiation. experiments (Rosenquist, 1966) have shown that several different mesodermal cell types are derived from the dorsal region (CKsel-negative), and from the ventral region (CKsel-positive). Through- out the period in which the mesoderm changes from a morphologically unorganized to become discretely organized into notochord, somites, inter- mediate mesoderm and lateral plate, the regional distinction in cytokeratin gene expression is main- tained. The formation of the mesoderm layer appears to occur by virtue of perhaps the earliest inductive event during embryogenesis (Dale et al. 1985; Smith et al. 1985; reviewed by Woodland, 1989). The most exten- sive progress toward a mechanistic understanding of this event has been made in amphibian embryos, where recombination experiments have shown that prospec- tive endoderm cells at the vegetal pole can direct animal pole cells (which would otherwise form ) to Fig. 3. Cellular movements of presumptive mesoderm in differentiate into mesodermal derivatives (Nieuwkoop, the definitive streak stage embryo. Anterior of the embryo 1969; Nakamura et al. 1970; Dale et al. 1985; Gurdon et is at top of the figure; the illustration is shown with the epiblast (upper layer) intact at left, but removed at right to al. 1985). There are distinct regional differences in the reveal the underlying mesoderm (and hypoblast/endoblast inductive specificity of the prospective endoderm (Dale at anterior, beyond the limits of mesodermal migration). and Slack, 1987): the most dorsal quadrant induces Arrows are included to show the general properties of 'dorsal-type' mesoderm (notochord and muscle) while mesodermal movement (Rosenquist, 1966; reviewed by the rest is equipotent in inducing 'intermediate-type' Balinsky, 1981). Prospective mesoderm cells initially he (muscle, mesothelium, mesenchyme and blood) or within the upper, epiblast layer. During gastrulation, cells 'ventral-type' mesoderm (mesothelium, mesenchyme that are destined to ingress migrate medially such that they and blood). Recent studies have demonstrated that converge on the center of active ingression, the primitive members of growth factor families are capable of streak. Presumptive mesoderm cells ingress at the primitive inductive effects similar to those obtained with vegetal streak and populate the space between the epiblast and the hypoblast/endoblast. Once beneath the epiblast, these cells cells (Woodland, 1989). Different factors in different spread laterally, moving away from the streak. Shading at combinations and/or concentrations induce different right illustrates the region in which CKsel gene expression types of mesodermal differentiation in culture. There is can be detected. As indicated by the degree of shading, the actually little evidence for a period in which amphibian boundary of CKsel hybridization at the posterior of the 'mesoderm' exists as a truly homogeneous tissue, and embryo is somewhat less sharp than at more anterior such studies point to the possibility that this germ layer positions (see text). possesses regional character even as it is induced (Dale et al. 1985; Smith et al. 1985). dorsal-ventral regionalization within this germ layer. In In the chick embryo, the processes of mesoderm amphibian embryos, the expression of the myogenic induction and regional specialization are not as well gene MyoDl and subsequent accumulation of muscle- understood. The embryonic mesoderm has its cellular specific a--actin transcripts have been found to be early origin in the epiblast and, along with the presumptive markers of mesoderm destined to differentiate into gut endoderm, must ingress through the primitive muscle (Hopwood et al. 1989; Mohun et al. 1984). The streak to "form the three-layered embryo. There is expression of these genes is specific to the forming evidence that some commitment to mesoderm forma- somites and at early stages is probably restricted to their tion occurs as early as the blastula stage, before precursor mesoderm cells (Hopwood et al. 1989). primitive streak formation. that are stripped Similar results have been obtained in avian embryos, of underlying hypoblast prior to the appearance of the where the expression of several contractile protein primitive streak (stage XIII of Eyal-Giladi and Kochav, genes as well as a quail MyoDl homologue (qmfl) was 1976) and cultured either intact (Azar and Eyal-Giladi, seen specifically in the more mature rostral somites 1979) or after dissociation (Mitrani and Eyal-Giladi, Dorsal-ventral regionalization in early mesoderm 423

1982) are able to differentiate into non-axial mesoderm grafted Hensen's node from quail embryos to various such as blood and mesenchyme. Whether all mesoderm dorsoventral positions of definitive streak (stage 4) precursors in the epiblast are capable of this type of chick embryos, and analyzed the origin of somites differentiation is unclear. Recently, the epiblast of stage formed in the resultant secondary axes. Grafts to the XIII (Eyal-Giladi and Kochav, 1976) embryos has been prospective dorsal region (=£200-300fim from the shown to contain two distinct cell populations that primitive streak) lead to the formation of supernumer- differ in their reactivity to the monoclonal antibody ary somites of host origin. In contrast, when grafts were HNK-1 (Stern and Canning, 1990). The cell population placed into prospective ventral locations (500-600//m recognized by HNK-1, which is distributed randomly from the host primitive streak), there were somites throughout the epiblast, is destined to converge to form derived from the transplanted tissue but none from the the primitive streak and give rise to the mesoderm and host. One interpretation of these results (Hornbruch et endoderm (Stern and Canning, 1990). Thus, before the al. 1979) is that the mesoderm in the prospective ventral formation of the primitive streak, HNK-1-positive cells region is not competent to respond to the inductive in the epiblast may already be committed to ingress signals from the graft, whereas more dorsal mesoderm through the streak and become mesoderm and/or can respond and produce supernumerary somites. endoderm. Our results suggest, however, that the Therefore, differential CKsel expression may reflect a mesodermal precursors that are HNK-1-positive in the difference in the ability of dorsal and ventral mesoderm epiblast have not yet undergone the dorsal-ventral to respond to such inductive signals. regionalization reflected by differential CKsel ex- During chick gastrulation, there is extensive mi- pression, since CKsel RNA is not seen in the gration of presumptive mesoderm cells (depicted in presumptive embryonic cells of the epiblast until the Fig. 3), and this must be considered in any model trying time of neurulation (Henry et al. submitted; see also to explain how differential expression of CKsel is set up Fig. 2). It is possible that early regional properties of in this germ layer. These cells migrate from initial chick mesoderm, such as the decision to express CKsel positions in the upper, epiblast layer, by first moving or to form axial structures, are only established during medially in the epiblast and ingressing through the or after gastrulation. primitive streak, and then by spreading out in a The differential distribution of CKsel transcripts ventrolateral direction to occupy the layer beneath the provides the first direct evidence that, even as the epiblast (Rosenquist, 1966). This migration process is mesoderm layer is being formed during gastrulation, it actively occurring at the definitive streak stage (stage 4) is subdivided into sharply defined dorsal and ventral and continues for several hours afterward. Most elements. Previously, Dale and Slack (1987) proposed significantly, many cells that are within the more on the basis of explant and induction studies in Xenopus medial, CKsel-negative region at stage 4 will continue that the first specialization within the prospective to migrate to more lateral positions at subsequent mesoderm involves the establishment of only two stages. Throughout this period, however, the distinc- states: a dorsal type and a ventral type. If simple states tion in CKsel gene expression between presumptive of dorsal and ventral specification do indeed exist, one dorsal and ventral regions is maintained. This obser- might predict that these two states are sharply vation suggests that the developmental state of migrat- distinguished by some feature(s) which is common ing mesoderm cells is determined by their position among cells within each (dorsal or ventral) subdivision. along the future dorsal-ventral axis: as cells move This would be in spite of the fact that several different laterally and cross a morphologically indistinguishable terminal differentiation pathways will ultimately arise boundary, a marked increase in CKsel gene expression from each subdivision. Our current findings are takes place. consistent with such a simple, two-state model in which The position-dependent expression of CKsel in the dorsal and ventral states of mesoderm are distinguish- mesoderm suggests that early regionalization might able with respect to cytokeratin gene expression. specify positional identities which are fundamentally What kind of biological difference(s) might be distinct in the prospective dorsal and ventral regions. specified in the fundamental subdivision of the chick Interactions between these two regions could then be mesoderm into two distinct states? Many studies have responsible for generating finer grades of specialization demonstrated that the properties established for the during the subsequent development of the vertebrate Spemann organizer in amphibians are applicable to mesoderm, as suggested by experiments with amphib- avian embryos (reviewed by Hara, 1978). Thus, ian embryos (Dale and Slack, 1987). This type of 'organizer' activity in the chick mesoderm appears to be progressive specification has been elegantly shown to localized to the prospective dorsal region in the vicinity occur in Drosophila, where the body plan is laid out in a of Hensen's node and the anterior portions of the hierarchical manner. Initially, maternally encoded primitive streak. It is therefore likely that one aspect of determinants specify in broad outline the anterior- early dorsoventral specialization in the mesoderm posterior and dorsal-ventral axes. These maternal gene involves the establishment of region-specific inductive products then act to generate spatially restricted properties necessary for the formation of the body axis. patterns of zygotic gene expression, thereby initiating A second biological difference between dorsal and pathways of genetic instructions to successively sub- ventral components may relate to their capacity to divide the embryo into smaller and smaller domains respond to inductive stimuli. Hornbruch et al. (1979) (reviewed by Ingham, 1988). Currently, we have little 424 T. S. Charlebois, J. J. Henry and R. M. Grainger

information about the steps involved in the regional (EY05542 and EY06675) and the Thomas F. and Kate Miller subdivision of the vertebrate embryo. Our results Jeffress Memorial Trust (J-24) to RMG. T.S.C. was sup- support the idea (Dale et al. 1985) that dorsal-ventral ported in part by a NIH training grant (GM07082). J.J.H. was regionalization of the mesoderm could involve a supported by a postdoctoral award (EY05938) from the NIH. fundamental positional coding which is established early in a hierarchy of decisions in this germ layer. From this perspective, it may not be surprising that the References dorsal/ventral boundary of CKsel gene expression is not as distinct in the posterior region of the mesoderm. AZAR, Y. AND EYAL-GILADI, H. (1979). Marginal zone cells - the Coordinate with primitive streak regression, the mor- primitive streak-inducing component of the primary hypoblast in the chick. J. Embryol. exp. Morph. 52, 79-88. phological organization of mesoderm takes place in an BALJNSKY, B. I. (1981). An Introduction to . Fifth anterior-to-posterior fashion. Therefore, more pos- edition. Philadelphia: Saunders College Publishing. terior mesoderm undergoes dorsal/ventral specializ- BELLAIRS, R. (1982). Gastrulation processes in the chick embryo. ation later in development. In Drosophila, progressive In Cell Behaviour (ed. R. Bellairs, A. Curtis, G. Dunn). sharpening of expression boundaries is seen for a Cambridge: Cambridge Univ. Press, pp. 395-427. BHAT, S. P. AND PIATIGORSKY, J. (1979). Molecular cloning and number of genes involved in the hierarchy of controls partial characterization of <5-crystallin sequences in a bacterial leading to the establishment of embryonic pattern (for plasmid. Proc. natn. Acad. Sci. U.S.A. 76, 3299-3303. reviews, see Carroll, 1990; Ingham, 1988). CARROLL, S. B. (1990). Zebra patterns in fly embryos: activation of stripes or repression of interstripes? 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