State of Commitment of Prospective Neural Plate And

DEVELOPMENTAL BIOLOGY 181, 102–115 (1997) ARTICLE NO. DB968439 State of Commitment of Prospective Neural Plate View metadata, citation and similar papers at core.ac.uk brought to you by CORE

and Prospective Mesoderm in Late Gastrula/Early provided by Elsevier - Publisher Connector Neurula Stages of Avian Embryos

Virginio Garcia-Martinez,*,1 Diana K. Darnell,* Carmen Lopez-Sanchez,*,1 Drazen Sosic,† Eric N. Olson,† and Gary C. Schoenwolf*,2 *Department of Neurobiology and Anatomy, University of Utah School of Medicine, 50 North Medical Drive, Salt Lake City, Utah 84132; and †Hamon Center for Basic Cancer Research, University of Texas, South West Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, Texas 75235-9148

We examined the ability of epiblast regions of known prospective fate from the late gastrula/early neurula stage of avian embryos to self-differentiate when placed heterotopically, testing their state of commitment. Three sites were examined: paranodal prospective neural plate ectoderm, containing cells fated to form a portion of the lateral wall of the neural tube at essentially all rostrocaudal levels of the neuraxis; prospective mesoderm from the caudolateral epiblast, containing cells fated to ingress through the primitive streak and to form lateral plate mesoderm; and prospective mesoderm from one level of the primitive streak, containing cells fated to continue ingressing and form paraxial mesoderm. Grafts from all sites exhibited plasticity. Grafts from the prospective neural plate ectoderm could readily substitute for regions of prospective mesoderm, when transplanted to either the epiblast or primitive streak, undergoing an epithelial –mesenchymal transition and, where appropriate, expressing paraxis, a gene expressed in paraxial mesoderm. Similarly, grafts containing prospective mesoderm from the epiblast could readily substitute for regions of the prospective neural plate ectoderm, undergoing convergent-extension movements characteristic of neuroectodermal cells and expressing appropriate genes such as En- grailed-2 and Hoxb-1. Grafts containing prospective mesoderm from the primitive streak could also incorporate into the neural plate and undergo convergence-extension movements of neurulation, although their principal contribution was to mesodermal and endodermal structures. Collectively, our results demonstrate that at the late gastrula/early neurula stage, germ layer-speciﬁc properties are not irrevocably ﬁxed for prospective ectodermal and mesodermal regions of the blastoderm. Moreover, the signals responsible for the induction of these two tissue types must still be present and available at these late stages. ᭧ 1997 Academic Press

INTRODUCTION to mesenchymal) and their direction of migration, subsequently moving principally from medial to lateral and ulti- In avian embryos, the late gastrula/early neurula stage mately forming the somitic, intermediate, lateral plate and [i.e., Hamburger and Hamilton’s (1951) late stage 3, early extraembryonic mesoderm (e.g., Nicolet, 1971; Schoenwolf stage 4] is an important transitional period between two et al., 1992; Garcia-Martinez et al., 1993). Concurrently, major phases of vertebrate development. At this stage, pro- cells of the prospective neural plate are initiating coordi- spective mesodermal cells within the epiblast are moving nated lateromedial and rostrocaudal movements, resulting from lateral to medial to ingress through the primitive in shaping of the neural plate (e.g., Schoenwolf and Alvarez, streak, where they change both their morphology (epithelial 1989; Garcia-Martinez et al., 1993). Finally, cells within the prospective epidermal ectoderm are moving centripetally (lateral to medial for most levels; rostral to caudal for those 1 Permanent address: Departamento de Ciencias Morfolo´gicas y forming the head fold of the body; Schoenwolf and Alvarez, Biologı´a Celular y Animal, Facultad de Medicina, Universidad de 1991; Garcia-Martinez et al., 1993; Moury and Schoenwolf, Extremadura, 06071 Badajoz, Spain. 1995). Thus, cells within different areas of the epiblast not 2 To whom reprint requests should be addressed. only have different prospective fates, but also different be-

0012-1606/97 $25.00 Copyright ᭧ 1997 by Academic Press 102 All rights of reproduction in any form reserved.

AID DB 8439 / 6x15$$$101 12-16-96 22:01:29 dba Plasticity of Prospective Neuroectoderm and Mesoderm 103 haviors and patterns of displacement during subsequent de- layer occurs by the beginning of gastrulation. However, velopment. In addition, there are regional morphological these studies utilized only morphological criteria to assess differences within the blastoderm. For example, the pro- cell fate, because relevant markers were not available at the spective neural plate ectoderm is a thickened portion of the time the studies were conducted. epiblast consisting of apicobasally elongated cells, whereas To assess fully the state of commitment of cells, several the prospective mesoderm within the epiblast is a thinner criteria must be met by the experimental system. First, an epithelium consisting of squamous cells; the primitive accurate and detailed fate map must exist so that regions streak is the region of epithelial–mesenchymal transition. representing prospective ectoderm and mesoderm (for ex- Also, there are differences in gene expression at this stage, ample) can be isolated. Second, tissues must be grafted into with prospective mesoderm within the primitive streak ex- a heterotopic location rather than grown in culture (the pressing Brachyury (Kispert et al., 1995), prospective ecto- latter tests prospective potency under the culture condi- derm, including neural plate ectoderm, expressing the FGF tions, not necessarily state of commitment). Third, embryos receptor FREK (Marcelle et al., 1994), and prospective meso- must be staged precisely. Fourth, grafted cells must be dis- derm in the epiblast expressing neither of these genes. An tinguishable from host cells after the culture period. Fifth, obvious question arises regarding these many differences. it must be possible to assess the outcome by several means Are cells in the prospective neural plate ectoderm and pro- (e.g., displacement pattern of grafted cells, their morphol- spective mesoderm fully committed to their particular de- ogy, their behavior, and their expression of specific mark- velopmental fate or even to derivatives of their prospective ers), because no single criterion alone is definitive. Al- germ layer? though a rich classical literature exists on experimental Previous studies have attempted to examine the state of grafting in vertebrates (e.g., reviewed by Detweiler, 1936; commitment of selected populations of prospective ectoder- Spemann, 1938; Weiss, 1939; Ho¨ rstadius, 1950; Le Douarin, mal and mesodermal cells within their respective germ lay- 1982), including classic experiments in chick (Waddington, ers at the late gastrula/early neurula stage of avian embryos. 1932; Abercrombie, 1950), these five criteria have not been Cells of the prospective ectoderm of the epiblast were exam- met previously in one set of experiments. Only recently ined in two studies (Alvarez and Schoenwolf, 1991; Schoen- have extensive fate maps, multiple exogenous and endoge- wolf and Alvarez, 1991). In the first study, it was shown nous cell labels, and region-, tissue-, and cell-specific mark- that prospective lateral neural plate cells could form median ers become available, opening this avenue of inquiry. neural plate cells, and vice versa, but that the preferred In this study, we consider two possibilities: (1) cell fate pathways of cell displacement were already established and/or pattern of displacement of cells is controlled in a (that is, lateral cells when placed into the midline tended manner that is germ layer-specific; that is, prospective ecto- to move back to a lateral position and then to extend down dermal cells and prospective mesodermal cells are not inter- the length of the neuraxis, and median cells when placed changeable; or (2) cell fate and/or pattern of displacement into a lateral position tended to move back into the midline is controlled by the local environment in which a cell re- and then to extend down the length of the neuraxis). In sides, and prospective ectodermal cells and prospective me- the second study, it was shown that prospective epidermal sodermal cells are interchangeable. We have addressed these ectoderm could form neural plate ectoderm, and vice versa. two possibilities by constructing transplantation chimeras Prospective mesodermal cells of the primitive streak were obtained as a result of heterotopic and isochronic grafting. examined in two additional studies (Garcia-Martinez and We have followed the displacement of grafted cells with Schoenwolf, 1992; Selleck and Stern, 1992). In both studies, time-lapse video microscopy and graft-specific labels and the fates of prospective notochordal and somitic cells were have used cell behaviors, morphological features, spatial examined. In the first study, the fate of prospective lateral relationships, and markers to assess the outcome (paraxis plate mesodermal cells was also examined. Cells from all for somitic mesoderm; Engrailed-2, Hoxb-1, and neurofil- examined areas were labile within the mesodermal germ ament-associated protein for neural ectoderm). Our results layer with one exception: prospective notochordal cells con- suggest that cell fate, morphology, and pattern of displace- tained within Hensen’s node always self-differentiated into ment are largely controlled by the local environment in ectopic notochords, demonstrating that they were already which a cell resides and that this plasticity is not limited fully committed to their developmental fate. Thus, the blas- to fates within a given germ layer. toderm at the late gastrula/early neurula stage consists of a mosaic of prospective cell types, some of which have fully committed to a particular developmental fate and others MATERIALS AND METHODS for which fate is still labile, at least within their prospective germ layer. The extent of this lability has not been tested among germ layers, however. Experimental Procedures In Xenopus, the commitment of cells of the blastula and Fertile White Leghorn chick and Japanese quail eggs were incu- gastrula has been investigated using transplantation assays bated to late stage 3, stage 4, or stage 7 (Hamburger and Hamilton, (Heasman et al., 1984; Snape et al., 1987). These experi- 1951; late stage 3 is stage 3d according to the criteria of Schoenwolf ments indicated that the commitment to a specific germ et al., 1992). Chick embryos served as hosts and quail or chick

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from the area adjacent to Hensen’s node consisted of only prospective neural plate and not prospective mesoderm. In one, site b was further fate mapped by transplanting identiﬁable (from quail or from ﬂuorescently labeled chick), epiblast plugs homotopically and

isochronically. In the second, site b1 (250–375 mm caudal to the rostral end of site b; Garcia-Martinez et al., 1993) was fate mapped as just described. This site was also chosen for further mapping to control for the possibility that the position of the rostral end of Hensen’s node may have been occasionally misinterpreted and, consequently, some grafts may have been placed more caudally within the epiblast, near the mesoderm-forming area. Finally, because grafts exhibited lability at the late gastrula–early neurula stage (see Results), we performed two types of heterotopic, heterochronic grafts, using stage 7 quail donors (i.e., midneurula stage) and stage 3d/4 chick hosts to determine whether grafted cells lost their lability by midneurula stages. In the ﬁrst type, the perinodal

FIG. 1. Diagram showing type-1 through type-4 experiments. Do- neural plate was grafted into site c1.5 of the primitive streak. In the nor epiblast plugs were obtained from either the right or left sides second type, the perinodal neural plate was grafted into a pocket of donor blastoderms and transplanted to either the right side (when carved into the ingressed mesoderm underlying site b1 (i.e., into viewed ventrally) or primitive streak of chick hosts. Primitive ingressed somitic mesoderm). streak plugs were also grafted to the right side. (A) Grafts of prospec- Grafts (with the exception of those into the mesodermal pocket) tive neural plate ectoderm to prospective mesoderm. (B) Grafts of were performed essentially as described by Garcia-Martinez et al. prospective mesoderm to prospective neural plate ectoderm. Each (1993; Figs. 2A–2D). Embryos in which further development oc- pair of donor and host embryos was at a comparable stage (i.e., 3d/ curred were virtually identical to unoperated, cultured controls 4). Each figure shows the perimeter of the area pellucida as well as (several hundred collected in the Schoenwolf laboratory during the the primitive streak, including Hensen’s node at its rostral end. past 10 years) when viewed 24 hr later, having largely completed The sites from which the donor graft was isolated are shown in neurulation and attaining stages 9–11 (Fig. 2E). For type-1 through black, and the sites to which the graft was made are shown in type-4 experiments, 243 embryos were successfully grafted [suc- white. Bar Å 125 mm. cessfully defined as grafted embryos developed similarly to unoperated, cultured controls (Fig. 2E) and the graft incorporated into the host, with grafted cells being readily identifiable (Fig. 2F)]. In addition, 65 successful fate-mapping controls and 21 successful embryos as donors. Host embryos were cultured ventral-side up on heterochronic grafts were obtained. The specific number of em- their vitelline membranes according to New (1955); donor embryos bryos in each treatment group can be found in Table 1. were placed ventral-side up on agar/albumen substrates according Typically, each embryo was videotaped at the time of the opera- to Spratt (1947), except when fluorescent dyes were used (see be- tion and at sacrifice to provide a verbatim, permanent record. Nine- low), and they were placed in New culture. Four experiments were teen grafted embryos were examined with time-lapse videomicros- performed at late gastrula/early neurula stages utilizing heterotopic copy. Finally, the morphology of the epiblast at each chosen site and isochronic grafts (Fig. 1). In type-1 experiments, an area of was examined in 5-mm plastic serial transverse sections, collected the donor epiblast designated in previous studies (Schoenwolf and previously for other purposes (Fig. 3; Schoenwolf, 1985 and unpub- Alvarez, 1989; Schoenwolf et al., 1989) as site b (the paranodal site lished data). Locations of graft sites were determined in sections just lateral to Hensen’s node) was grafted to an area of host epiblast as follows: rostrocaudal position was determined by counting trans- designated in a previous study (Garcia-Martinez et al., 1993) as site verse sections (5-mm thick) cut caudal to the rostral end of Hensen’s f3m (the postnodal site 250 to 375 mm lateral to Hensen’s node and node until the appropriate level was reached; mediolateral position 750 to 875 mm caudal to the rostral end of Hensen’s node). Based was determined by morphological landmarks or by measuring dis- on our homotopic, isochronic fate-mapping studies, site b typically tances with an eyepiece micrometer. contributes unilaterally to the lateral wall of the neural tube from the midbrain to the caudal spinal cord and site f3m bilaterally to lateral plate mesoderm. In type-2 experiments, site b of the donor Graft-Specific Labels and Region-, Tissue-, and was grafted to an area of the host primitive streak designated in a Cell-Specific Markers previous study (Schoenwolf et al., 1992) as site c1.5 (a level of the primitive streak 375 –500 mm caudal to the rostral end of Hensen’s To maximize the information available from these operations, node). Site c1.5 typically contributes bilaterally to somitic meso- grafted cells were identified as follows (Table 1): (1) using fluores- derm. Thus, both type-1 and type-2 experiments were designed to cent dyes and fluorescence microscopy, (2) using peroxidase immu- place prospective neural plate ectoderm into sites typically fated nocytochemistry, or (3) using the Feulgen–Rossenbeck procedure. to form mesoderm. In type-3 experiments, prospective mesoderm Fluorescent labeling involved soaking donor embryos for 1 hr in from site f3m of the donor was grafted to site b of the host. In type- Rhodamine 123 (Molecular Probes, Inc., Eugene, OR) and/or CFSE

4 experiments, prospective mesoderm from site c1.5 of the donor [5-(and 6-)carboxyfluorescein diacetate, succinimidyl ester, Molec- was grafted to site b of the host. Thus, both type-3 and type-4 ular Probes, Inc.], as described previously (Yuan et al., 1995). Rhoda- experiments were designed to place prospective mesoderm into mine 123 is a fluorescent mitochondrial label (Chen, 1989), and sites typically fated to form neural plate ectoderm. CFSE is a label that passes through the cell membrane, binds to Additionally, two types of fate-mapping controls were performed protein amines in the cytosol, and becomes fluorescent after reac- to provide greater assurance that donor epiblast grafts obtained tion with nonspecific esterases within the cytoplasm (Haughland,

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1989–1991). Inclusion of Rhodamine 123 allows the grafts to be visualized in living, whole mounts so graft distribution can be doc- umented and embryos can be selected for further processing. Inclu- sion of the fluorescein-containing label CFSE allows for the subsequent use of peroxidase immunocytochemistry using anti-fluorescein. Peroxidase immunocytochemistry was done essentially as described by Patel et al. (1989) with the exception that PBS with 4% paraformaldehyde was substituted as the fixative except when anti-fluorescein was used, and in some cases (usually embryos not also subjected to in situ hybridization) the peroxidase reaction was intensified (changes color from red/brown to black) by adding 25

ml 1% CoCl and 20 ml Ni(NH)2(SO4)2 per milliliter of DAB-PBT. This technique was used on fixed whole mounts, which could be subsequently serially sectioned, and provides whole-mount information on graft distribution and permanent identification of graft cells at single-cell resolution. Two primary antibodies were used: anti-F (anti-fluorescein, Molecular Probes, Inc.; Garton and Schoen- wolf, 1996), which labels whole cells of grafts previously soaked in CFSE; and anti-Q (QCPN, developed by B. M. Carlson and J. A. Carlson, University of Michigan; Inagaki et al., 1993), which labels the nuclei of all quail cells (in this case, in quail–chick chimeras). Due to its nuclear localization, anti-Q is especially useful for double labeling cells in which the cytoplasm is marked by in situ hybridization (see below). To produce histological sections of these labeled embryos, embryos were processed through an ethanol series and Histosol for paraffin embedment, and serial sections were cut at 7 mm. Embryos stained using the Feulgen–Rossenbeck histological stain (Smith and Schoenwolf, 1989) for the naturally occurring quail nucleolar label (Le Douarin, 1973) were fixed with a mixture of glacial acetic acid, 37% formaldehyde, and 100% ethanol (1:2:7, v:v:v) and transversely serially sectioned prior to staining. Due to the excellent histology achieved with this process, this label is especially useful for revealing the morphology of grafted quail cells within the host. Thus, each technique was included to increase the types of information that could be discerned from these grafts: general graft distribution and cell behavior, tissue-specific localization, morphology, and gene expression (via double labeling). Con- clusions about principal graft contributions were consistent regardless of the method used for graft labeling; consequently, results were pooled for general conclusions. In addition to graft-specific labels, a subset of embryos was also examined with region-, tissue-, or cell-specific markers, i.e., ribo- probes and antibodies specific for a particular region, tissue, or cell type within an organ rudiment at the stage during which the results of the experiment were evaluated. The markers used were a riboprobe transcribed from the chick paraxis cDNA (cloned and cur- rently being characterized by D.S. and E.N.O.), which is expressed in the somites and rostral segmental plate mesoderm and is a homo- log of mouse paraxis (Burgess et al., 1995); a riboprobe transcribed from the Hoxb-1 cDNA (provided by R. Krumlauf; Maden et al., 1991), which is expressed in the fourth rhombomere and caudal to the fifth rhombomere in the mylencephalon (Grapin-Botton et al., 1995); a riboprobe transcribed from the Engrailed-2 cDNA (En-2; Darnell, unpublished), which is expressed in the dorsolateral mes-

FIG. 2. Whole mounts of embryos subjected to heterotopic grafting (type-3 experimental paradigm). (A –D) Same blastoderm show- embryo approximately 24 hr after grafting (rostral is toward the top ing reﬂection of the endoderm (A), removal of the host site b (B), of the illustration). Donor-derived cells within the lateral wall of insertion of the donor graft into the host site b (C), and replacement one side of the caudal neural tube at the spinal cord level are shown of the endoderm over the graft (D). (E) Similar embryo approxi- with anti-Q. Arrows, a pair of somites. Bars Å 300 mm (A–E); 130 mately 24 hr after grafting. (F) Enlargement of the trunk of a similar mm (F).

AID DB 8439 / 6x15$$$101 12-16-96 22:01:29 dba 106 Garcia-Martinez et al. encephalon and rostral metencephalon (Bally-Cuif et al., 1992); an grafting (Schoenwolf et al., 1992). In embryos from type- antibody for the En-2 homeodomain (anti-En-2 Å 4D9; Patel et al., 1, -2, and -4 experiments, cells derived from grafts were 1989); and an antibody for a neurofilament-associated protein (anti- consistently dispersed within the mesoderm (e.g., Figs. 4, NAP Å 3A10, developed by T. Jessell and J. Dodd), which is ex- 5B, 7A, and 7B), whereas those within the lateral wall of pressed in differentiating neurons in the hindbrain and spinal cord. the neural tube from type-3 and 4 experiments were usually Neurofilament-associated protein, En-2, and Hoxb-1 are not well expressed in the regions containing experimental grafts prior to tightly clustered (e.g., Figs. 6F, 6I, and 7C). This is in accor- stage 10, which limited the number of embryos available for region- dance with our previous results from fate-mapping studies and cell-specific marking. The specific combinations of graft-spe- (e.g., Garcia-Martinez et al., 1993). cific labels and region-, tissue-, and cell-specific markers used and A fate map published by Bortier and Vakaet (1992) seem- the number of embryos successfully examined with each combina- ingly is at variance with respect to the fate of our site b, tion are given in Table 1. Some specific markers were visualized calling into question the designation of the region lateral using peroxidase immunocytochemistry (anti-NAP and anti-En-2), to Hensen’s node as prospective neuroectoderm. They im- while the remainder were visualized using in situ hybridization ply that the prospective neural plate is found only rostral (paraxis, En-2, and Hoxb-1). Cell type-specific marking by in situ to the node. To address this issue and to provide further hybridization was essentially as described by Wilkinson (1993), fate-mapping control data, we collected 65 additional em- with the exception that proteinase K was reduced (5 mg/ml for 5 min) or eliminated and BM purple (Boehringer-Mannheim, India- bryos, successfully grafted at site b and b1 . The results of napolis, IN) replaced NBT/BCIP in the final reaction. After in situ these control grafts were identical to those of our previous hybridization, embryos were dehydrated and processed for paraffin studies (Schoenwolf and Alvarez, 1989; Schoenwolf et al., embedment with isopropanol rather than Histosol, and serial sec- 1989; Garcia-Martinez et al., 1993). Namely, the vast ma- tions were cut at 15 mm. A subset of embryos (Table 1) was double jority of cells fluently incorporated into the length of the labeled with anti-Q after in situ hybridization and prior to em- neural tube from the midbrain or hindbrain to the caudal bedment. end of the spinal cord (Fig. 2F). Also as reported previously by us, a very small number of cells contributed unilaterally to the mesoderm immediately underlying the graft Photography and Digital Image Processing within the neural tube. We believe these few cells repre- Color photographic slides were taken of both whole-mount and sent a contaminant (e.g., ingressed mesodermal cells sectioned embryos using Kodak Ektachrome 100 plus film. These attached to the epiblast plug at the time of transplanta- slides were scanned into Adobe Photoshop 3.0 (Adobe Systems, tion) and are not a normal derivative of the epiblast per Inc., Mountain View, CA) using a Leaf 45 scanner (Leaf Systems, se. Regardless, they cannot account for the large contribu- Inc., Southborough, MA). Scanned images were cropped, adjusted tion of graft cells to mesoderm seen in heterotopic grafting for brightness, contrast, and color balance, and otherwise photo- experiments (type-1 and type-2). graphically optimized without compromising the integrity of the data. Final files for each figure were printed at 320 dpi on a Fujix Pictrography 3000 printer (Fuji Photo Film Co., Ltd., Tokyo, Japan). Structural Differences among Plugs of Different Sites of Origin RESULTS During the grafting procedure, it was obvious that the The aim of this set of experiments was to determine at structure of the plugs from different sites differed consider- or after the late gastrula/early neurula stage (stage 3d/4) ably. For example, plugs from site c1.5 and site b seemed whether prospective neuroectodermal regions of the blasto- robust, whereas plugs from site f3m seemed flimsy. Serial derm were capable of forming mesoderm and, conversely, transverse plastic sections from stage 4 embryos were exam- whether a prospective mesodermal region was capable of ined to assess critically the morphology of plugs at the three forming neuroectoderm. In type-1, -3, and -4 experiments, sites used in the present study. Plugs from site c1.5 consisted grafting was done on one side of the blastoderm and the of cells undergoing an epithelial –mesenchymal transition unoperated side served as an internal control. No differences (Fig. 3A). Plugs from site b consisted of an epithelium com- were detected in any of the experimental groups in the rate posed of tall cells arranged as a pseudostratified, columnar of neurulation or in morphological features between the epithelium (ca. 40 mm tall; Fig. 3B). In contrast, plugs from grafted and unoperated sides. A few embryos from type-4 site f3m consisted of a simple squamous epithelium (ca. 5– experiments had wide or dysraphic neural tubes in the head 10 mm tall; Fig. 3C). Time-lapse videomicroscopy revealed region. In type-2 experiments, midline grafts gave rise to delayed healing of grafts between site b donors and site f3m cells that were unilaterally or bilaterally distributed, and hosts, and vice versa; such delayed healing was likely owing no differences were detected between either group and un- to the striking morphological differences between these two operated, cultured controls with regard to neurulation or types of plugs. Specifically, histological analysis showed general development, with one exception: in some cases, that cells from such grafted plugs elongated or contracted somites appeared misshapen, duplicated, or large, a result apicobasally, approximating the heights of adjacent host similar to one previously reported for homotopic streak cells.

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tion of bilateral distribution, underwent morphogenetic movements appropriate to their new germ layer and location, as determined with time-lapse videomicroscopy and histological evaluation of their morphology and position. That is, cells from the round, cohesive, and thickened epithelial grafts became freely dispersed within the lateral mesoderm from medial to lateral and from caudal to rostral. The evidence from location and morphology is consistent with the interpretation that these cells have lost the identity of (prospective) neural plate ectoderm and acquired the identity of mesoderm.

Type-2 Experiments In type-2 experiments, prospective neural plate ectoderm was grafted to a site normally forming somitic mesoderm. Grafted plugs contributed cells principally to somitic mesoderm (n Å 45; 71%) and/or to mesoderm in the segmental plate (n Å 28; 44%). In 52 cases (83%), grafts contributed to the region in which paraxis is expressed. Graft cells were visualized by fluorescence and/or by peroxidase immunocytochemistry and results from both methods were similar and were pooled. Graft cells were bilaterally distributed in 43 cases (68%; Figs. 5A–5C) and unilaterally distributed in 20 (32%; not shown). In most cases the graft cells were fluently incorporated into the host mesoderm and were displaced appropriately along with the host mesoderm, from medial to lateral and from caudal to rostral. Thirty embryos previously labeled with fluorescence were marked with the FIG. 3. Transverse sections of 5-mm plastic sections of control mesodermal marker paraxis. In addition, 17 embryos embryos viewed with Hoffman optics. (A) Site c1.5 ; (B) site b; (C) site f3m . ps/sm, primitive streak consisting of prospective somitic mesoderm; e/np, epiblast consisting of prospective neural plate; e/lpm, epiblast consisting of prospective lateral plate mesoderm; m, ingressed mesoderm; en, endoderm. (Note: the endoderm typically wrinkles during fixation and yolk may become adherent; hence, this endoderm is cut obliquely and has attached yolk.) Bar Å 50 mm.

Type-1 Experiments In type-1 experiments, prospective neural plate ectoderm was grafted to the site normally forming predominantly mesoderm. Grafted plugs contributed cells to three types of structures. The principal contribution was to the mesoderm: lateral plate and intermediate (Fig. 4) and somitic. A lesser contribution was also made in a few cases to the lateral wall of the neural tube in the caudal spinal cord and FIG. 4. Type-1 experiments. Epiblast grafts from prospective neu- to the epidermal ectoderm of the caudal trunk. In no case ral plate ectoderm (site b) into prospective lateral plate mesoderm (site f ) yielded graft cells unilaterally distributed in the meso- did grafted plugs contribute cells to the mesoderm bilater- 3m derm. A transverse section of one of these embryos taken approxi- ally, although homotopic grafts to site f do contribute 3m mately 24 hr after grafting and stained according to the Feulgen – bilaterally to lateral plate mesoderm. No speciﬁc markers Rossenbeck procedure shows quail cells (arrows) occupying a por- were used in type-1 experiments because none were avail- tion of the lateral plate mesoderm (lpm) and intermediate meso- able for lateral plate or intermediate mesoderm, the princi- derm or nephrotome, just ventral to the pronephric cord (pc). som, pal yield of these grafts. However, grafted cells were ﬂuently somite; e, ectoderm; en, endoderm. Medial is toward the left of the incorporated into the host mesoderm and, with the excep- illustration. Bar Å 40 mm.

AID DB 8439 / 6x15$$$101 12-16-96 22:01:29 dba 108 Garcia-Martinez et al. grafted as in type-2 experiments (not included in the n of the neural tube, as far rostral as the mesencephalon (n Å 7; 63) were marked with paraxis, but were not labeled for graft 7%) and more commonly beginning in the hindbrain or ros- location. Fourteen of these embryos (83%) can statistically tral spinal cord and extending the length of the cord (n Å 45; be included with the 30 double-labeled embryos (because 45%; Fig. 6A). In some cases the extent of extension of the 83% of the embryos in our initial cohort had grafts that graft was restricted to the midbrain (n Å 24; 24%), the hind- had incorporated into the paraxis-expressing region), thus brain (n Å 17; 17%), or the caudal spinal cord (n Å 8; 8%). increasing our population of paraxis-marked experimental Some graft cells were also identified in the head mesen- embryos to 44. In every case for both control and experimen- chyme, somites, heart, extraembryonic endoderm, and sur- tal embryos, paraxis expression appeared normal in whole- face ectoderm. Graft cells located in the neuroectoderm were mount (Figs. 5D and 5E) and in transverse sections (not fluently incorporated into the host epithelium and were dis- shown). For embryos with unilaterally distributed grafts, placed appropriately along with the host ectoderm (converg- paraxis expression seemed equivalent on the side of the ing and extending) from lateral to medial and from rostral to embryo with and without grafted cells (not shown). These caudal. This is the opposite direction of displacement from results were compelling, but not definitive, so 18 paraxis- the mesoderm, from which the graft is derived. Sixty-three marked embryos were secondarily labeled with anti-Q via of these embryos were labeled successfully with markers peroxidase immunocytochemistry. In 12 of these embryos, specific for neuroectoderm, 48 with antibodies and 15 with cells could be identified in which both the marker and label antibodies and in situ probes (Table 1). In no case could a were expressed, demonstrating at the single cell level that difference in marker intensity or pattern be discerned be- cells of the graft form paraxial mesoderm. One such embryo tween control embryos and experimental embryos or be- is shown in whole mount in Fig. 5F and in transverse section tween the control and experimental sides of grafted embryos revealing bilateral graft distribution in right and left somites for any of these 63 embryos. Of the 48 embryos labeled with (Figs. 5G and 5H). Thus, the evidence from displacement, antibodies, 3 anti-NAP/anti-Q double-labeled embryos and morphology, and the expression of a tissue-specific marker 5 anti-En-2/fluorescein double-labeled embryos apparently is that these cells have lost the identity of (prospective) expressed neural markers in graft cells (the other 40 lacked neural plate ectoderm and acquired the identity of paraxial cells in the correct dorsoventral or rostrocaudal localization mesoderm. within the neural tube to make an assessment with these markers). One of the anti-En-2/fluorescein double-labeled embryos is shown in Figs. 6B–6D. These results were com- Type-3 Experiments pelling, but not definitive, so 15 embryos (7 En-2- and 8 Hoxb-1-marked embryos) were secondarily labeled with anti- In type-3 experiments, prospective lateral plate mesoderm Q via peroxidase immunocytochemistry. In 2 of the En-2- was grafted to the site normally forming neural plate ecto- marked embryos (one shown in Figs. 6E–6G) cells could be derm. Grafted plugs contributed cells to the lateral wall of identified in which both the marker and label were expressed.

FIG. 5. Type-2 experiments. Epiblast grafts from prospective neural plate ectoderm (site b) into prospective somitic mesoderm (site c1.5) yielded graft cells bilaterally distributed in the somites and segmental plate mesoderm as shown here in a whole-mount (A) and transverse section (B) of a embryo immunolabeled with anti-F and in another embryo (C) labeled with a ﬂuorescent dye and shown in whole mount. In the same embryo shown in C, paraxis marking (D) could not be distinguished from paraxis marking in a similar stage control embryo (E). (F) A whole-mount embryo (rostral up) double labeled with a riboprobe for paraxis (purple) and anti-Q (brown). Black lines delineate the extent of obvious anti-Q labeling and paraxis marking, showing extensive overlap. A transverse section through this embryo (white line) reveals opposing somites (G, H) in which labeled, individual quail nuclei (arrows) are surrounded by paraxis-marked cytoplasm (brackets). Bars Å 150 mm (A); 30 mm (B); 300 mm (C); 100 mm (D, E); 50 mm (F); 10 mm (G, H).

FIG. 6. Type-3 experiments. Grafts from prospective lateral plate mesoderm (site f3m) into prospective neural plate ectoderm (site b) resulted in graft cells within the lateral neural tube. (A) A type-3 embryo is shown with its graft ﬂuorescently labeled with Rhodamine 123. The rostrocaudal extent of the graft (hindbrain to caudal spinal cord) is consistent with the normal behavior (convergence extension) of the neuroectoderm at site b. An embryo with ﬂuorescently labeled graft cells in the expanded mesencephalon (B) was also marked subsequently with anti-En-2 (C, D). En-2 protein expression in this embryo was normal for this age embryo, as seen in the whole-mount embryo (C) and in a transverse section taken at the level of the white line in C. [Note: En-2 protein is expressed throughout the dorsoventral extent of the neural tube on both the control (arrowhead) and grafted (arrow) side (D)]. (E) A whole-mount embryo is shown (rostral up) double labeled with a riboprobe for En-2 (purple) and an antibody for quail cells (brown). Black lines delineate the extent of obvious anti- Q and En-2 double labeling, showing considerable overlap. A transverse section through this embryo (white line) reveals the double- marked neural tube (F) including a large lateral cohort of quail cells and a small cluster of more rostral quail cells (arrow; expanded view at lower right). An expanded view of the boxed area (G) shows quail nuclei surrounded by En-2-marked cytoplasm (bracket). (H) A whole- mount embryo (rostral up) double labeled with a riboprobe for Hoxb-1 (purple) and an antibody for quail cells (brown). Black lines delineate the extent of obvious anti-Q labeling and Hoxb-1 marking, showing extensive overlap. A transverse section through this embryo (white line) reveals the double-labeled neural tube (I). An expanded view of the boxed area (J) shows quail nuclei surrounded by Hoxb-1 marked cytoplasm (bracket). Bars Å 300 mm (A–C); 50 mm (D, E, H); 25 mm (F); 10 mm (G, J); 20 mm (I).

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TABLE 1 Experimental Treatments

Type (n) Graft-speciﬁc label (n) Speciﬁc marker (n)

Type-1 (11) PIa (7) Feulgenb (4) Type-2 (63) Fluorescencec (35) paraxisd (12) PI (10) Fluorescence and PI (18) paraxis (18) Ectoderm to mesoderm subtotal: 74

Type-3 (103) Fluorescence (53) Anti-NAP (21), Anti-En-2e (10) PI (19) Anti-NAP f (17) Fluorescence and PI (27) En-2g (7), Hoxb-1h (8) Feulgen (4) Type-4 (66) Fluorescence (34) paraxis (4) Fluorescence and PI (32) paraxis (3) Mesoderm to ectoderm subtotal: 169 Total heterotopic, isochronic grafts: 243

Site b controls (26) PI (26)

Site b1 controls (39) PI (39) Total homotopic, isochronic fate mapping grafts: 65

NP to streak (13) Fluorescence (3) Fluorescence and PI (10) paraxis (10) NP to pocket (8) Fluorescence and PI (8) paraxis (8) Total heterotopic, heterochronic grafts: 21

a Peroxidase immunocytochemistry for graft-specific labels (anti-Q or anti-F) on fixed whole mounts, subsequently sectioned. b Histological stain used to identify quail nucleoli in fixed, serial sectioned embryos. c Fluorescently labeled (Rhodamine 123 and CFSE) graft cells visualized in living, intact embryos. d Riboprobe marker for paraxial mesoderm. e Antibody marker for mesencephalon/metencephalon. f Antibody marker for differentiating neurons, rhombencephalon, and rostral spinal cord. g Riboprobe marker for mesencephalon/metencephalon. h Riboprobe marker for myelencephalon.

In the other 5 cases grafts and En-2 signal did not colocalize bryos labeled with anti-Q showed that grafts unilaterally con- dorsoventrally. In 6 of the Hoxb-1-marked embryos individ- tributed cells principally to head mesoderm (27% of grafts; ual cells were also double labeled. One of these embryos is Fig. 7A) and somites (72%; Fig. 7B), notochord (44%; Fig. 7A), shown in Figs. 6H–6J. It should be pointed out that in cases and endoderm (10%; Figs. 7A and 7C). In addition, some grafts in which the graft extended rostral to the Hoxb-1-expressing (n Å 6, 33%) contributed cells to the lateral wall of the neural area or within the nonexpressing fifth rhombomere, double tube (Figs. 7C and 7D), in two cases bilaterally (Fig. 7D). labeling revealed that Hoxb-1 expression did not occur in Grafted cells were usually fluently incorporated into the host grafted cells at these levels (data not shown). This appropriate and were often displaced along the rostrocaudal axis of the rostrocaudal regulation eliminates the possibility that Hoxb- embryo (Fig. 7E). The bilateral distribution within the neural 1 expression in graft cells within the neural tube is merely tube in two cases (not seen in homotopic grafts to site b) misplaced mesodermal expression of this gene. Collectively, could be due to inefficient healing, which might result in the the evidence from displacement, morphology, and tissue-spe- graft moving into the midline during neurulation. In some cific expression of appropriate markers is that these cells cases then, the graft cells that incorporated into the neural have lost the identity of (prospective) lateral plate mesoderm plate were cleft by other prospective neural plate cells (pro- and have acquired the identity of neural plate ectoderm. spective floor plate cells from site a; Schoenwolf and Alvarez, 1989) moving caudally from the region rostral to Hensen’s node. The mesodermal and endodermal contribution of graft Type-4 Experiments cells was similar to rostral streak homotopic grafts (site c0.5; In type-4 experiments, prospective somitic mesoderm from Schoenwolf et al., 1992) or midstreak to rostral streak hetero- the primitive streak was grafted to the site normally forming topic grafts (type-4 grafts; Garcia-Martinez and Schoenwolf, the lateral neural plate. Data derived from 18 sectioned em- 1992), but extended more rostrally and occurred unilaterally.

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FIG. 7. Type-4 experiments. (A–D) Grafts from prospective somitic mesoderm (site c1.5) into prospective neural plate ectoderm (site b) resulted in graft contributions to head mesenchyme (hm), endoderm (e), notochord (n), somite (s), and neural tube (nt) including bilateral cells in the neural tube in two cases (D). Arrowheads show graft cells labeled with anti-Q. (E) An embryo with a graft in the head (arrowhead) and elongated rostrocaudally near the midline (arrows) is shown. This same embryo labeled with paraxis after further culture (F) shows that graft cells fail to label with paraxis in the head (arrowhead) or near the midline (arrows). Dorsal is up in A–D and rostral is to the right in E and F. Bars Å 50 mm (A –D); 500 mm (E); 400 mm (F).

Because of the small contribution to the neural tube in tions containing graft cells (Fig. 7F), indicating that graft type-4 grafts, speciﬁc neural marking of graft cells with En- cells outside the somites were not expressing a gene appro- 2 or Hoxb-1 was not attempted for these embryos. Seven priate to their original position. Thus, the evidence from additional embryos were labeled with paraxis to determine displacement, morphology, and paraxis expression is that if a gene appropriate to the tissue of origin was expressed in primitive streak cells have frequently lost the identity of the heterotopically grafted cells. This paraxial mesodermal (prospective) somitic mesoderm and have acquired the iden- marker was detected in the somites and segmental plate tity of more rostral streak derivatives. Surprisingly, in some mesoderm, but in no case were paraxis-marked cells de- cases grafted cells acquired the identity of the neuroectoder- tected in the neural plate ectoderm or other ectopic loca- mal site into which they were placed.

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well as the ability to express one of three neuroectodermal markers, En-2, Hoxb-1, or a neurofilament-associated protein. It is generally believed that neural induction in the chick begins very early, perhaps by stage 2 (Gallera, 1971), and is likely a progressive event. Moreover, the conven- tional wisdom is that the neural plate is induced by mesoderm, which in itself is believed to be induced from epiblast (presumably at a somewhat earlier stage than the onset of neural induction) by a factor secreted by the endoderm (pos- sibly activin; Mitrani et al., 1990; Mitrani, 1992; Ziv et al., 1992). In our current study we have found that the prospective neural plate from late gastrula/early neurula aged hosts can form mesoderm (lateral plate, intermediate and somitic). This result implies not only that prospective neural plate cells are competent to respond to mesodermal-inductive signals but also that mesodermal-inductive signals are still present at late gastrula/early neurula stages in chick. This competence seems to be lost by stage 7, an outcome that FIG. 8. (A) A fluorescently labeled neural plate graft (arrow) from may result in, or be confounded by, the inability of the graft a stage 7 donor embryo is shown in the somitic mesoderm after 24 cells to incorporate fluently into the host in the experi- hr of culture. (B) The same embryo labeled with paraxis shows that ments with older donors. Loss of competence is also sup- graft cells fail to label (arrow), whereas somites label well. Bar Å 200 mm. ported by additional experiments using the paradigm in which epiblast grafts from neuroectodermal epiblast were grafted between the epiblast and endodermal layers of the blastoderm at a heterotopic location prior to embryo culture Timing of Commitment (Waddington, 1931). In these experiments, the persistence of the neural phenotype, as shown by the expression of a Epiblast plugs from the neural plate of stage 7 hosts were neuroectodermal marker, was observed in grafts from stages grafted into either of two sites normally forming somite to 5 through 8 but not in grafts from earlier stages (Darnell test the lability of the neural plate at the midneurula stage. and Schoenwolf, in preparation). Graft cells remained coherent, that is, the graft appeared In addition, our past and present results suggest that neu- as a slightly elongated, discrete disk that was not fluently ral-inductive signals must still be operative at the late gas- integrated into the surrounding mesodermal tissue (Figs. 8A trula/early neurula stage in chick, and they further provide and 8B). Regardless of whether the graft was placed into the remarkable finding that a prospective mesodermal re- the streak or into the mesenchyme containing ingressed gion of the epiblast is competent to respond to these signals somitic mesoderm, the coherent graft cells failed to label by forming neural plate ectoderm, as are a subset of cells with paraxis, whereas the neighboring host somitic cells from the primitive streak. This competence also seems to labeled normally (Fig. 8B). Thus, the lability of epiblast cells be lost by the majority of cells as they enter the primitive that has been demonstrated at stage 3d/4 (late gastrula/early streak and by prospective mesoderm within the epiblast by neurula) is no longer observed at stage 7 (midneurula) using stages 5 to 7 (early to midneurula) based on experiments this paradigm. using the Waddington technique mentioned earlier. In these experiments, expression of appropriate neuroectodermal markers was not observed in mesodermal grafts from stages DISCUSSION 5 through 8 when grafted to a neuroectodermal site, whereas they were at earlier stages (Darnell and Schoenwolf, in prep- In this study we demonstrate that at the late gastrula/ aration). Extraembryonic ectoderm exhibits a similar loss early neurula stage, the germ layer identity of an epiblast of plasticity in its ability to be induced by grafted Hensen’s region, that is, mesoderm or ectoderm (neural), is not fixed. node to become neuroectoderm (Dias and Schoenwolf, Prospective neural plate regions have the ability to respond 1990; Storey et al., 1992), as does headfold stage posterior to mesodermal-inductive signals to form mesoderm, as indi- ectoderm (epiblast) to be induced to form neuroectoderm cated by considerable morphological and displacement by rostral mesendoderm in mouse explant cultures (Ang changes as well as by the ability (where appropriate) to ex- and Rossant, 1993). These neurula stage restrictions con- press a mesoderm-specific marker, paraxis. A prospective trast with an earlier, but similar, gradual loss of competence mesodermal region has the ability to respond to neural- that is observed for prospective endoderm (and probably inductive signals and to form neuroectoderm, as indicated ectoderm) at the early gastrula stage in Xenopus (Heasman by considerable morphological and displacement changes as et al., 1985; Wylie et al., 1987).

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Immediate healing of some heterotopic grafts was un- The nature of the regional cues and how their signals are doubtedly inhibited by overt structural differences between interpreted remain as major challenges for future studies. epiblast donor and host sites. In some of our experiments, epiblast cells may have formed mesoderm without actually undergoing ingression, that is, cells grafted to the epiblast ACKNOWLEDGMENTS that subsequently populated the mesoderm unilaterally may have either ingressed through the streak and migrated We gratefully acknowledge the technical assistance of Arlene only on the side grafted, or such cells may have directly Carillo. The Hoxb-1 cDNA was graciously provided by R. Krum- delaminated into the mesoderm. Conversely, in the major- lauf, National Institute for Medical Research, London. The QCPN, ity of cases for type-1 through type-3 experiments, grafted 3A10, and 4D9 antibodies were obtained from the Developmental plugs contributed to the mesoderm bilaterally or to the neu- Studies Hybridoma Bank maintained by the Department of Phar- ral tube unilaterally and must have healed into the epi- macology and Molecular Sciences, the Johns Hopkins University blast—in the former case where grafts contributed bilater- School of Medicine (Baltimore, MD), and the Department of Biol- ally to the mesoderm they must have ingressed or they ogy, University of Iowa (Iowa City, IA), under Contract NO1-HD- could not have crossed the midline to contribute cells to 2-3144 from the NICHD. This research was supported by NIH the side opposite the graft site; in the latter case where grafts Grant NS 18112 to G.C.S.; FIS Grant 94-0431 and DGICYT Grant contributed to the ectoderm, healing must have occurred or PR95-243 of the Spanish Government to V.G.M.; grant of the Junta de Extremadura to C.L.S.; and NIH Grants HL53351, AR40339, and contributions would have been impossible (i.e., the graft AR39849, as well as grants from the Muscular Dystrophy Associa- would have merely been lost). The healing of heterotopic tion and the Human Frontier Science Program, to E.N.O.; D.K.D. grafts into the epiblast suggests that regional cell morphol- was supported by NIH Developmental Biology Training Grant ogy is also a function of position within the epithelial sheet 1T32 HD07491-01. and, like fate and displacement pathways, occurs in a non- cell-autonomous fashion. In addition, although rigorous Note added in proof. While this manuscript was in press, a paper analyses of cell division rates and cell death were not con- appeared showing similarly that cells of ectodermal and germ-line ducted in this study, based on histological evaluation, time- lineages exhibit plasticity in gastrulating mouse embryos (TAM, lapse videomicroscopy, sequential viewing of grafts in cul- P.P.L., and S.X. Zhou (1996). The allocation of epiblast cells to tured embryos, and comparison of experimental embryos to ectodermal and germ-line derivatives is inﬂuenced by the position matched homotopically grafted control embryos, experi- of the cells in the gastrulating mouse embryo. Dev. Biol. 78, 124– mental grafts and control grafts contributed similar num- 132). bers and distributions of cells. 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