Candidate genes for congenital diaphragmatic hernia from animal models: sequencing of FOG2 and PDGFRalpha reveals rare variants in diaphragmatic hernia patients.

Congenital diaphragmatic hernia (CDH) is a common, life threatening birth defect. Although there is strong evidence implicating genetic factors in its pathogenesis, few causative genes have been identified, and in isolated CDH, only one de novo, nonsense mutation has been reported in FOG2 in a female with posterior diaphragmatic eventration. We report here that the homozygous null mouse for the Pdgfralpha gene has posterolateral diaphragmatic defects and thus is a model for human CDH. We hypothesized that mutations in this gene could cause human CDH. We sequenced PDGFRalpha and FOG2 in 96 patients with CDH, of which 53 had isolated CDH (55.2%), 36 had CDH and additional anomalies (37.5%), and 7 had CDH and known chromosome aberrations (7.3%). For FOG2, we identified novel sequence alterations predicting p.M703L and p.T843A in two patients with isolated CDH that were absent in 526 and 564 control chromosomes respectively. These altered amino acids were highly conserved. However, due to the lack of available parental DNA samples we were not able to determine if the sequence alterations were de novo. For PDGFRalpha, we found a single variant predicting p.L967V in a patient with CDH and multiple anomalies that was absent in 768 control chromosomes. This patient also had one cell with trisomy 15 on skin fibroblast culture, a finding of uncertain significance. Although our study identified sequence variants in FOG2 and PDGFRalpha, we have not definitively established the variants as mutations and we found no evidence that CDH commonly results from mutations in these genes.

Improvements in histological quality and signal retention following in situ hybridization in early chick embryos using plastic resin and recolorization.

We describe a novel method that allows reliable detection of in situ hybridization signals in thin sections of plastic embedded embryos. Sections from plastic embedded embryos are thinner and have superior histological quality compared to paraffin, gelatin, agarose embedded sections or cryosections; however, plastic resin traditionally has not been used as an embedding medium following in situ hybridization because of loss of signal. When signal is detected with alkaline phosphatase and NBT/BCIP, the resulting colored precipitate is subject to fading when samples are exposed to organic compounds. The colored precipitate can be redeposited by repeating the NBT/BCIP reaction following plastic sectioning. This recolorization shows no loss of specificity, because signal is detected only where the anti-digoxigenin/alkaline phosphatase conjugated antibody is bound to the riboprobe. Strong signals can be detected without recolorization; however, weaker signals require the recolorization step. This novel method of re-depositing colored precipitate after processing and sectioning allows accurate determination of the location of gene expression and study of this expression in high quality histological sections of early chick embryos.

Principles of developmental biology.

The field of developmental biology has a history that spans the last 500 years. Within the last 10 years, our understanding of developmental mechanisms has grown exponentially by employing modern techniques of genetics and molecular biology, frequently combined with experimental embryology and the use of molecular markers, rather than solely morphology, to identify critical populations of cells and their state of differentiation. Three main principles have emerged. First, mechanisms of development are highly conserved, both among developing rudiments of a variety of organ systems and among diverse organisms. This conservation occurs both at the level of tissue and cellular mechanisms, and at the molecular level. Second, the development of organ rudiments is influenced by surrounding tissues through interactions called inductive interactions. Such interactions are mediated by highly conserved growth factors and signaling systems. Third, development is a life-long process and can be reawakened in events such as wound healing and regeneration, and in certain diseases. Advances in understanding normal development provide hope that diseases in which development runs amuck, such as cancer, may soon be preventable and fully treatable. Supported by NS 18112 and DC 04185 from the NIH.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Molecular genetic control of axis patterning during early embryogenesis of vertebrates.

Formation of the axis and its subsequent patterning to establish the tube-within-a-tube body plan characteristic of vertebrates are initiated during gastrulation. In higher vertebrates (i.e., birds and mammals), gastrulation involves six key events: establishment of the rostrocaudal/mediolateral axis; formation and progression of the primitive streak and organizer; epiboly of the epiblast, ingression of prospective mesodermal and endodermal cells through the primitive streak, and migration of cells away from the primitive streak; regression of the primitive streak; establishment of the right-left axis; and formation of the tail bud. Over 50 years of study of these processes have provided a morphological framework for understanding how these events occur, and recent advances in imaging, microsurgical intervention, and cell tracking are beginning to elucidate the underlying cell behaviors that drive morphogenetic movements. Moreover, homotopic transplantation and dye microinjection studies are being used to generate high-resolution fate maps, and heterotopic transplantation studies are revealing the cell-cell interactions that are sufficient as well as required for mesodermal and ectodermal commitment. Additionally, the roles of the organizer and secondary signaling centers in establishing the body plan are being defined. With the advent of the molecular/genetic age, the molecular basis for axis formation is beginning to become understood. Thus, it is becoming clear that secreted growth factors/signaling molecules produced by localized signaling centers induce and pattern the axis, presumably through downstream activation of signal-transduction proteins and cascades of transcription factors.

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.

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.

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.

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.

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.