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G C Schoenwolf

Publications and source records attributed to G C Schoenwolf.

At least 19 recordsLinked to original sources
PubMed2001

New insights into critical events of avian gastrulation.

The formation and progression of the primitive streak are key events of avian gastrulation. We examine these processes in detail, using various morphological approaches. We show that formation of the primitive streak occurs locally at the caudal midline of the area pellucida, as cells in the caudal midline undergo an epithelial-to-mesenchymal transformation, and that extensive migration of delaminated cells arising from more rostral or peripheral areas of the blastoderm is not involved in streak formation. Instead, such delamination occurs earlier and is restricted to the process of hypoblast formation. Moreover, we provide evidence that progression of the primitive streak involves two processes: convergent-extension movements within the streak per se, and progressive delamination of midline epiblast cells in a caudal-to-rostral sequence. We have identified a subpopulation of primitive-streak cells located at its dorsal midline surface that undergoes extensive rostral displacement concomitant with streak progression. The fact that these cells are located only dorsally and do not elongate ventrally as do adjacent ingressing cells, suggests that these cells retain their residency within the primitive streak, at least until regression of the primitive streak occurs. Finally, by following labeled cells over time we establish the timing of movement of epiblast cells toward and into the primitive streak, providing direct evidence that cell-cell intercalation occurs within the primitive streak during its progression. Collectively, our results provide new insight into complex and central events of avian gastrulation.

Animals↗
PubMed2001

Cellular mechanisms of neural fold formation and morphogenesis in the chick embryo.

The mechanisms underlying neural fold formation and morphogenesis are complex, and how these processes occur is not well understood. Although both intrinsic forces (i.e., generated by the neuroepithelium) and extrinsic forces (i.e., generated by non-neuroepithelial tissues) are known to be important in these processes, the series of events that occur at the neural ectoderm-epidermal ectoderm (NE-EE) transition zone, resulting in the formation of two epithelial layers from one, have not been fully elucidated. Moreover, the region-specific differences that exist in neural fold formation and morphogenesis along the rostrocaudal extent of the neuraxis have not been systematically characterized. In this study, we map the rostrocaudal movements of cells that contribute to the neural folds at three distinct brain and spinal cord levels by following groups of dye-labeled cells over time. In addition, we examine the morphology of the neural folds at the NE-EE transition zone at closely-spaced temporal intervals for comparable populations of neural-fold cells at each of the three levels. Finally, we track the lateral-to-medial displacements that occur in the epidermal ectoderm during neural groove closure. The results demonstrate that neural fold formation and morphogenesis consist of a series of processes comprising convergent-extension movements, as well as epithelial ridging, kinking, delamination, and apposition at the NE-EE transition zone. Regional differences along the length of the neuraxis in the respective roles of these processes are described.

Age Factors↗
PubMed2001

Classification scheme for genes expressed during formation and progression of the avian primitive streak.

We have systematically examined the expression patterns of thirteen genes by in situ hybridization during the formation and progression of the avian primitive streak. Based on common patterns of expression, we classify these genes into three distinct groups. Group 1 genes, subdivided into group 1A (Wnt8c, Slug, Vg1, and Nodal) and group 1B (Fgf8, Brachyury, and Cripto), were expressed first in the epiblast and then, throughout most of the length of the primitive streak. Group 2 genes, namely, cNot1, Sonic hedgehog (Shh), Hnf3 beta and Chordin, were confined to the rostral end of the primitive streak, and then, to Hensen's node. In contrast, Group 3 genes, comprising Goosecoid (GSC) and Crescent, were expressed in the hypoblast. This classification scheme provides a rational basis for categorizing genes expressed during avian gastrulation, and such systematization is likely to provide insight into the relationships among different genes and their potential roles in key events of gastrulation.

Animals↗
PubMed2001

Improved method for chick whole-embryo culture using a filter paper carrier.

We describe a simple method of chick whole-embryo culture, which uses a filter paper carrier to hold the early blastoderm and vitelline membranes under tension while the embryo grows on a substratum of agar-albumen. This is a quick and efficient means of setting up cultures of chick embryos beginning at pre-primitive streak stages to stage 10 (stages X--XIV, Eyal-Giladi and Kochav [1976] Dev Biol 49:321-337; stages 1--10, Hamburger and Hamilton [1951] J Morphol 88:49--92). This is an improvement on the original method of New, which used a glass ring and watch glass (New [1955] Exp Morphol 3:320--331). Our modification of New's method, which we call EC (Early Chick, pronounced EASY) culture, facilitates several manipulations in early chick embryos, including microsurgery, grafting, bead implantation, microinjection, and electroporation. Using the EC method, embryos at stage 8 and older can be readily cultured either dorsal-side up (in contrast to New's method) or ventral-side up, as desired; embryos younger than stage 8 can be culture only ventral-side up (as with New's method). We also discuss some alternative methods for setting up these cultures.

Agar↗
PubMed2001

Towards a cellular and molecular understanding of neurulation.

Neurulation occurs during the early embryogenesis of chordates, and it results in the formation of the neural tube, a dorsal hollow nerve cord that constitutes the rudiment of the entire adult central nervous system. The goal of studies on neurulation is to understand its tissue, cellular and molecular basis, as well as how neurulation is perturbed during the formation of neural tube defects. The tissue basis of neurulation consists of a series of coordinated morphogenetic movements within the primitive streak (e.g., regression of Hensen's node) and nascent primary germ layers formed during gastrulation. Signaling occurs between Hensen's node and the nascent ectoderm, initiating neurulation by inducing the neural plate (i.e., actually, by suppressing development of the epidermal ectoderm). Tissue movements subsequently result in shaping and bending of the neural plate and closure of the neural groove. The cellular basis of the tissue movements of neurulation consists of changes in the behavior of the constituent cells; namely, changes in cell number, position, shape, size and adhesion. Neurulation, like any morphogenetic event, occurs within the milieu of generic biophysical determinants of form present in all living tissues. Such forces govern and to some degree control morphogenesis in a tissue-autonomous manner. The molecular basis of neurulation remains largely unknown, but we suggest that neurulation genes have evolved to work in concert with such determinants, so that appropriate changes occur in the behaviors of the correct populations of cells at the correct time, maximizing the efficiency of neurulation and leading to heritable species- and axial-differences in this process. In this article, we review the tissue and cellular basis of neurulation and provide strategies to determine its molecular basis. We expect that such strategies will lead to the identification in the near future of critical neurulation genes, genes that when mutated perturb neurulation in a highly specific and predictable fashion and cause neurulation defects, thereby contributing to the formation of neural tube defects.

Animals↗
PubMed2001

Cell interactions underlying notochord induction and formation in the chick embryo.

The development of the notochord in the chick is traditionally associated with Hensen's node (the avian equivalent of the organizer). However, recent evidence has shown that two areas outside the node (called the inducer and responder) are capable of interacting after ablation of Hensen's node to form a notochord. It was not clear from these studies what effect (if any) signals from these areas had on normal notochord formation. A third area, the postnodal region, may also contribute to notochord formation, although this has also been questioned. Using transection and grafting experiments, we have evaluated the timing and cellular interactions involved in notochord induction and formation in the chick embryo. Our results indicate that the rostral primitive streak, including the node, is not required for formation of the notochord in rostral blastoderm isolates transected at stages 3a/b. In addition, neither the postnodal region nor the inducer is required for the induction and formation of the most rostral notochordal cells. However, inclusion of the inducer results in considerable elongation of the notochord in this experimental paradigm. Our results also demonstrate that the responder per se is not required for notochord formation, provided that at least the inducer and postnodal region are present, although in the absence of the responder, formation of the notochord occurs far less frequently. We also show that the node is not specified to form notochord until stage 4 and concomitant with this, the inducer loses its ability to induce notochord from the responder. The coincident timing of these changes in the node and inducer suggests that notochord specification and the activity of the inducer are regulated through a negative feedback loop. We propose a model relating our results to the induction of head and trunk organizer activity in the node.

Animals↗
PubMed2001

Cell populations and morphogenetic movements underlying formation of the avian primitive streak and organizer.

The cell populations and morphogenetic movements that contribute to the formation of the avian primitive streak and organizer-Hensen's node-are poorly understood. We labeled selected groups of cells with fluorescent dyes and then followed them over time during formation and progression of the primitive streak and formation of Hensen's node. We show that (1) the primitive streak arises from a localized population of epiblast cells spanning the caudal midline of Koller's sickle, with the mid-dorsal cells of the primitive streak arising from the midline of the epiblast overlying Koller's sickle and the deeper and more lateral primitive streak cells arising more laterally within the epiblast overlying the sickle, from an arch subtending about 30 degrees; (2) convergent extension movements of cells in the epiblast overlying Koller's sickle contribute to formation of the initial primitive streak; and (3) Hensen's node is derived from a mixture of cells originating both from the epiblast just rostral to the incipient (stage 2) primitive streak and later from the epiblast just rostral to the elongating (stage 3a/b) primitive streak, as well as from the rostral tip of the progressing streak itself. Collectively, these results provide new information on the formation of the avian primitive streak and organizer, increasing our understanding of these important events of early development of amniotes.

Animals↗
PubMed2001

Localization of cells of the prospective neural plate, heart and somites within the primitive streak and epiblast of avian embryos at intermediate primitive-streak stages.

By constructing avian transplantation chimeras using fluorescently-labeled grafts and antibodies specific for grafted cells, we have generated a prospective fate map of the primitive streak and epiblast of the avian blastoderm at intermediate primitive-streak stages (stages 3a/3b). This high-resolution map confirms our previous study on the origin of the cardiovascular system from the primitive streak at these stages and provides new information on the epiblast origin of the neural plate, heart and somites. In addition, the origin of the rostral endoderm is now documented in more detail. The map shows that the prospective neural plate arises from the epiblast in close association with the rostral end of the primitive streak and lies within an area extending 250 microm rostral to the streak, 250 microm lateral to the streak and 125 microm caudal to the rostral border of the streak. The future floor plate of the neural tube arises within the midline just rostral to the streak, confirming our earlier study, but unlike at the late-primitive streak stages when both Hensen's node and the midline area rostral to Hensen's node contribute to the floor plate, only the area rostral to the primitive streak contributes to the floor plate at intermediate primitive-streak stages. Instead of contributing to the floor plate of the neural tube, the rostral end of the primitive streak at intermediate primitive-streak stages forms the notochord as well as the rostromedial endoderm, which lies beneath the prechordal plate mesoderm and extends caudolaterally on each side toward the cardiogenic areas. The epiblast lateral to the primitive streak and caudal to the neural plate contributes to the heart and it does so in rostrocaudal sequence (i.e., rostral grafts contribute to rostral levels of the straight heart tube, whereas progressively more caudal grafts contribute to progressively more caudal levels of the straight heart tube), and individual epiblast grafts contribute cells to both the myocardium and endocardium. The prospective somites (i.e., paraxial mesoderm) lie within the epiblast just lateral to the prospective heart mesoderm. Comparing this map with that constructed at late primitive-streak stages reveals that by the late primitive-streak stages, prospective heart mesoderm has moved from the epiblast through the primitive streak and into the mesodermal mantle, and that some of the prospective somitic mesoderm has entered the primitive streak and is undergoing ingression.

Animals↗
PubMed2000

Identification of synergistic signals initiating inner ear development.

Tissue manipulation experiments in amphibians more than 50 years ago showed that induction of the inner ear requires two signals: a mesodermal signal followed by a neural signal. However, the molecules mediating this process have remained elusive. We present evidence for mesodermal initiation of otic development in higher vertebrates and show that the mesoderm can direct terminal differentiation of the inner ear in rostral ectoderm. Furthermore, we demonstrate the synergistic interactions of the extracellular polypeptide ligands FGF-19 and Wnt-8c as mediators of mesodermal and neural signals, respectively, initiating inner ear development.

Animals↗
PubMed2000

Islet-1 marks the early heart rudiments and is asymmetrically expressed during early rotation of the foregut in the chick embryo.

Islet-1 (Isl-1), the LIM domain homeobox gene, is a well-known early marker of neuronal specification. Here, we show its spatial and temporal patterns of expression during early heart and gut development in the chick embryo. Isl-1 transcripts are first detected in the early cardiac progenitors and underlying endoderm at late stage 4. By stages 5-6, it is also expressed in the prechordal plate. From stage 6 onward, transcripts are also detected in the endoderm forming the anterior intestinal portal and floor of the caudal foregut. With progressive rostrocaudal fusion of the paired cardiac rudiments, Isl-1 expression is maintained in the cardiac mesoderm and associated endoderm. By the onset of heart beating, transcripts become restricted to the dorsal mesocardium and more caudal medial splanchnic mesoderm flanking the open gut. Within the foregut, Isl-1 is expressed in the endoderm of the oral membrane, thyroid rudiment, and second pharyngeal pouches, as well as within the second branchial grooves adjacent to the secondary pouches. Interestingly, with the onset of gut rotation, Isl-1 expression is detected unilaterally in the splanchnic mesodermal wall of the future greater curvature of the caudal stomach/rostral duodenum. Thus, Isl-1 is a novel and useful marker of the early cardiac rudiments and of the original left side of the rotating foregut. During early organogenesis, Isl-1 is also expressed in several other discrete domains as reported previously. Additionally, it is expressed at the interface between the hind limbs and trunk.

Animals↗
PubMed2000

Subtractive hybridization identifies chick-cripto, a novel EGF-CFC ortholog expressed during gastrulation, neurulation and early cardiogenesis.

EGF-CFC genes encode a novel class of extracellular, membrane-associated proteins that notably play an important role during vertebrate gastrulation. Whereas the two cysteine-rich domains that characterize these proteins, namely the extracellular EGF-like and the CFC domain, are known to be encoded by two evolutionarily conserved exons, it is generally assumed, based on weak primary sequence identity, that the remaining parts of the protein differ among vertebrates, suggesting that known members of the EGF-CFC family do not represent true orthologs. Here, by characterizing the full cDNA and genomic sequences of a new EGF-CFC gene in chick, and by comparing them with their counterparts in human (CRIPTO), mouse (cripto and cryptic), Xenopus (FRL-1) and zebrafish (one-eyed pinhead), we show that all EGF-CFC genes share an identical genomic organization over the entire coding region. Not only are the central two exons (coding for the EGF-like and CFC motifs) conserved, but also conserved are the total number of exons, their size, their intron phase and their correlation with discrete protein modules, in particular those modules that allow the EGF-CFC motif to become membrane-associated. Therefore, despite apparent divergence between their 5' and 3'-terminal exons, all known CRIPTO-related genes are structurally orthologous. We named this novel ortholog in bird, chick-cripto. We report the mRNA distribution of chick-cripto, which begins in the epiblast of the gastrula, with a pattern similar to EGF-CFC genes of other vertebrates.

Amino Acid Sequence↗
PubMed2000

Ectodermal markers delineate the neural fold interface during avian neurulation.

The formation and morphogenesis of the neural folds are important processes underlying neurulation. We showed previously that these processes comprise four key events in avian embryos: epithelial ridging, kinking, delamination, and apposition. Collectively, these events establish the paired, bilaminar neural folds, which fuse in the dorsal midline during late neurulation to close the neural groove and to establish the neural tube. Here, we use an antisense riboprobe for a new gene called Plato, as well as an antibody for a previously cloned transcription factor, AP-2, as markers to identify critical subpopulations of ectodermal cells during the formation and morphogenesis of the avian neural folds. Plato antisense riboprobe marks the cranial neural ectoderm and premigratory cranial neural crest cells, whereas AP-2 antibody marks the epidermal ectoderm and the early migratory neural crest. We show that subpopulations of ectodermal cells at the forebrain and midbrain levels undergo considerable rearrangement within the neural fold transition zone, which redistributes incipient neural crest cells from the neural ectodermal side of the forming neural fold interface to the epidermal ectodermal side. Additionally, we show that Plato and AP-2 provide useful markers for delineating the incipient neural fold interface.

Animals↗
PubMed2000

Signals from trunk paraxial mesoderm induce pronephros formation in chick intermediate mesoderm.

We used Pax-2 mRNA expression and Lim 1/2 antibody staining as markers for the conversion of chick intermediate mesoderm (IM) to pronephric tissue and Lmx-1 mRNA expression as a marker for mesonephros. Pronephric markers were strongly expressed caudal to the fifth somite by stage 9. To determine whether the pronephros was induced by adjacent tissues and, if so, to identify the inducing tissues and the timing of induction, we microsurgically dissected one side of chick embryos developing in culture and then incubated them for up to 3 days. The undisturbed contralateral side served as a control. Most embryos cut parallel to the rostrocaudal axis between the trunk paraxial mesoderm and IM before stage 8 developed a pronephros on the control side only. Embryos manipulated after stage 9 developed pronephric structures on both sides, but the caudal pronephric extension was attenuated on the cut side. These results suggest that a medial signal is required for pronephric development and show that the signal is propagated in a rostral to caudal sequence. In manipulated embryos cultured for 3 days in ovo, the mesonephros as well as the pronephros failed to develop on the experimental side. In contrast, embryos cut between the notochord and the trunk paraxial mesoderm formed pronephric structures on both sides, regardless of the stage at which the operation was performed, indicating that the signal arises from the paraxial mesoderm (PM) and not from axial mesoderm. This cut also served as a control for cuts between the PM and the IM and showed that signaling itself was blocked in the former experiments, not the migration of pronephric or mesonephric precursor cells from the primitive streak. Additional control experiments ruled out the need for signals from lateral plate mesoderm, ectoderm, or endoderm. To determine whether the trunk paraxial mesoderm caudal to the fifth somite maintains its inductive capacity in the absence of contact with more rostral tissue, embryos were transected. Those transected below the prospective level of the fifth somite expressed Pax-2 in both the rostral and the caudal isolates, whereas embryos transected rostral to this level expressed Pax-2 in the caudal isolate only. Thus, a rostral signal is not required to establish the normal pattern of Pax-2 expression and pronephros formation. To determine whether paraxial mesoderm is sufficient for pronephros induction, stage 7 or earlier chick lateral plate mesoderm was cocultured with caudal stage 8 or 9 quail somites in collagen gels. Pax-2 was expressed in chick tissues in 21 of 25 embryos. Isochronic transplantation of stage 4 or 5 quail node into caudal chick primitive streak resulted in the generation of ectopic somites. These somites induced ectopic pronephroi in lateral plate mesoderm, and the IM that received signals from both native and ectopic somites formed enlarged pronephroi with increased Pax-2 expression. We conclude that signals from a localized region of the trunk paraxial mesoderm are both required and sufficient for the induction of the pronephros from the chick IM. Studies to identify the molecular nature of the induction are in progress.

Animals↗
PubMed2000

Evidence that translation of smooth muscle alpha-actin mRNA is delayed in the chick promyocardium until fusion of the bilateral heart-forming regions.

Heart development in the chick embryo proceeds from bilateral mesodermal primordia established during gastrulation. These primordia migrate to the midline and fuse into a single heart trough. During their migration as a cohesive sheet, the cells of the paired heart fields become epithelial and undergo cardiac differentiation, exhibiting organized myofibrils and rhythmic contractions near the time of their fusion. Between the stages of cardiomyoblast commitment and overt differentiation of cardiomyocytes, a significant time interval exists. Using a new riboprobe (usmaar) for whole-mount in situ hybridization in chick embryos, we report the earliest phases of smooth muscle alpha-actin (smaa) mRNA distribution during the precontractile developmental window. We show that ingressed heart-forming regions express smaa by the head-process stage (Hamburger and Hamilton stage 5). In addition, we used usmaar to study the formation and early morphogenesis of the heart. Consistent with fate mapping studies (Garcia-Martinez and Schoenwolf [1993] Dev. Biol. 159:706-719; Schoenwolf and Garcia-Martinez [1995] Cell Mol. Biol. Res. 41:233-240; Garcia-Martinez et al., in preparation), our results with this probe, combined with detailed histological and SEM analyses of the so-called cardiac crescent, demonstrate unequivocally that the heart arises from separated and paired heart rudiments, rather than from a single crescent-shaped rudiment (that is, prior to fusion of the paired heart rudiments to establish the straight-heart tube, the rostral midline of the cardiac crescent lacks mesodermal cells and consequently fails to label with usmaar). Smaa is also expressed in the splanchnic and somatic mesoderm, marking the earliest step in coelom formation. Consequently, we also used usmaar to describe formation of the pericardium. Finally, we provide evidence of a post-transcriptional level of control of smaa gene expression in the heart fields. Our results suggest that the expression of smaa may mark a primitive mesodermal state from which definitive cell types can be derived through inductive events.

Actins↗
PubMed2000

Characterization of avian frizzled genes in cranial placode development.

To determine the possible role of Wnt signaling in cranial placode development, we have cloned several chick frizzled genes, a family of putative Wnt receptor molecules, and analyzed their expression during chick embryogenesis. Chick frizzled-2 (cFz-2) and frizzled-7 (cFz-7) are expressed broadly in cranial ectoderm, tissue that is competent to express markers of the trigeminal placode (Stark et al., 1997. Development 124, 4287-4295; Baker et al., 1999. Development 126, 147-156). In addition, cFz-2 and cFz-7 are uniquely expressed in other cranial placodes, including the olfactory, lens, and otic placodes. Chick frizzled-1 (cFz-1) is expressed in the lens, otic placode and, along with cFz-7, in epibranchial placodes. Each frizzled gene expressed in the otic placode displays a unique domain of expression: cFz-1 transcripts are detected in the medial wall of the vesicle, cFz-2 in the rostral rim of the vesicle, and cFz-7 in the lateral half of the vesicle. Other chick frizzled family members cloned that do not show striking expression in cranial placodes include frizzled-4 (cFz-4), frizzled-8 (cFz-8), frizzled-9 (cFz-9), and frizzled-10 (cFz-10). A brief summary of their expression is given, along with a brief summary of non-placodal expression of cFz-1, cFz-2, and cFz-7. In all, frizzled genes show dynamic expression at key times during embryonic development, particularly in the cranial placodes.

Animals↗
PubMed2000

Frizzled-4 expression during chick kidney development.

Wnts have been implicated in metanephric kidney development. To determine whether Frizzleds, the genes that encode Wnt receptors, are present at early stages of nephrogenesis, we examined the expression of several recently identified Frizzled genes in the chick by in situ hybridization. Here we report the cloning and characterization of chick Frizzled-4 (cFz-4), which we found to be expressed in the developing chick kidney. cFz-4 was first expressed in the pronephros caudal to the third somite at Hamburger and Hamilton stage 10. Its expression increased with maturation, becoming restricted to the newly induced glomeruli and tubules in the mesonephros and metanephros. Within the metanephros, cFz-4 and Wnt-4 expression patterns were similar, whereas Wnt-11 was expressed solely in the tips of the branching ureteric bud. cFz-4 expression was compared with that of known kidney markers. It preceded that of Lmx-1, but was similarly restricted to developing glomeruli and tubules. In contrast, Pax-2 expression and Lim 1/2 antibody labeling occurred in intermediate mesoderm caudal to the fifth somite in the early pronephros, and each persisted in both the tubules and nephric ducts throughout further development.

Amino Acid Sequence↗