Developing Vertebrate Embryo Research Articles

Quantitative analyses of cell behaviors underlying notochord formation and extension in mouse embryos

Formation and extension of the notochord (i.e., notogenesis) is one of the earliest and most obvious events of axis development in vertebrate embryos. In birds and mammals, prospective notochord cells arise from Hensen's node and come to lie beneath the midline of the neural plate. Throughout the period of neurulation, the notochord retains its close spatial relationship with the developing neural tube and undergoes rapid extension in concert with the overlying neuroepithelium. In the present study, we examined notochord development quantitatively in mouse embryos. C57BL/6 mouse embryos were collected at 8, 8.5, 9, 9.5, and 10 days of gestation. They were then embedded in paraffin and sectioned transversely. Serial sections from 21 embryos were stained with Schiff's reagent according to the Feulgen-Rossenbeck procedure and used for quantitative analyses of notochord extension. Quantitative analyses revealed that extension of the notochord involves cell division within the notochord proper and cell rearrangement within the notochordal plate (the immediate precursor of the notochord). In addition, extension of the notochord involves cell accretion, that is, the addition of cells to the notochord's caudal end, a process that involves considerable cell rearrangement at the notochordal plate-node interface. Extension of the mouse notochord occurs similarly to that described previously for birds (Sausedo and Schoenwolf, 1993 Anat. Rec. 237:58-70). That is, in both birds (i.e., quail and chick) and mouse embryos, notochord extension involves cell division, cell rearrangement, and cell accretion. Thus higher vertebrates utilize similar morphogenetic movements to effect notogenesis.

Retinoids and the alcohol dehydrogenase gene family.

Alcohol dehydrogenase (ADH) is best known as the enzyme which catalyzes the reversible oxidation/reduction of ethanol/acetaldehyde. However, mammalian ADH has also been shown to function in vitro as a retinol dehydrogenase in the conversion of retinol (vitamin A alcohol) to retinoic acid, a hormone which regulates gene expression at the transcriptional level. It is clear that retinol must be converted to more active retinoid forms in order to fulfill its roles in growth, development, and cellular differentiation. An important unsolved issue in retinoid research is the control of retinoic acid synthesis from retinol during differentiation. Several enzymes which participate in the conversion of retinol to retinoic acid in vitro have been isolated, but more information on their relative importance is needed. Human ADH exists as a family of isozymes encoded by seven genes which are differentially expressed in adult and fetal mammalian tissues, being found preferentially in the epithelial cells which are known to synthesize and respond to retinoic acid. Retinoic acid is also known to play a role in neural tube development in vertebrate embryos. Excessive doses of retinoic acid or ethanol are both teratogenic for neural tube development. A relationship may exist between these two types of teratogenesis due to the role of ADH in both retinol and ethanol metabolism and the ability of ethanol to competitively inhibit retinol oxidation. There is a lack of information on the expression patterns of ADH genes in early embryos, but transgenic mouse studies are presented here which show that the human ADH3 gene can be expressed in several mouse embryonic tissues including the neural tube. Thus, ethanol-induced neural tube defects seen in cases of fetal alcohol syndrome may be due to ethanol inhibition of retinol oxidation catalyzed by an embryonic ADH. This could potentially lower retinoic acid levels in the neural tube to the extent that gene expression is not properly regulated, resulting in morphological defects.

Molecular control of neuronal survival in the chick embryo.

Neurotrophins are structurally related proteins which promote the survival and differentiation of specific neuronal populations during the development of vertebrate embryos. Like many growth factors, the neurotrophins mediate their actions by binding to membrane proteins that have a ligand-activated tyrosine kinase activity. The interactions of the neurophins with their neuronal receptors have been mostly studied using chick embryonic neurons. These neurons are also extensively used to characterise biological responses to neurotrophins in physiologically relevant systems. We have recently cloned and expressed the chick homologue of trkB (ctrkB), thought to be a receptor for BDNF, and examined by in situ hybridisation the pattern of expression of the ctrkB gene during development of the chick embryo. We found that whereas the sequence of ctrkB shows a high degree of conservation with the mammalian homologues in the intracellular tyrosine kinase domain, the extracellular binding domain is less well conserved. As in mammals, ctrkB mRNAs appear to exist in differentially spliced forms that result in a full length and a truncated receptor lacking the tyrosine kinase domain. These two forms are differentially expressed in neurons and non-neuronal cells respectively. The binding characteristics of ctrkB expressed in a transfected cell line are similar, but not identical to those of the BDNF binding sites on primary chick neurons, specially with regard to the affinity of BDNF.

Homeotic transformations and limb defects in Hox A11 mutant mice.

Hox A11 is one of the expanded set of vertebrate homeo box (Hox) genes with similarities to the Drosophila homeotic gene, Abdominal-B (Abd-B). These Abd-B-type Hox genes have been shown to be expressed in the most caudal regions of the developing vertebrate embryo and in overlapping domains within the developing limbs, suggesting that these genes play important roles in pattern formation in both appendicular and axial regions of the body. In this report whole-mount in situ hybridization in mouse embryos gave a precise description of Hox A11 gene expression in the developing limbs and in the axial domain of the developing body. In addition, we generated a targeted mutation in Hox A11 and characterized the resulting phenotype to begin to dissect developmental functions of the Abd-B subfamily of Hox genes. Hox A11 mutant mice exhibited double homeotic transformations, with the thirteenth thoracic segment posteriorized to form an additional first lumbar vertebra and with the sacral region anteriorized, generating yet another lumbar segment. Furthermore, skeletal malformations were observed in both forelimbs and hindlimbs. In mutant forelimbs, the ulna and radius were misshapen, the pisiform and triangular carpal bones were fused, and abnormal sesamoid bone development occurred. In mutant hindlimbs the tibia and fibula were joined incorrectly and malformed at their distal ends. Also, an enlarged sesamoid developed ventral to the tibiale bone. Both heterozygous and homozygous mice displayed mutant phenotypes adding an additional level of complexity to the Hox code hypothesis.

Identification of a Drosophila activin receptor.

Activins are cytokines of the transforming growth factor beta superfamily that control various events during vertebrate embryo development and cell differentiation in the adult, and act through transmembrane receptors that contain a cytoplasmic protein-serine/threonine kinase domain. We describe the identification, deduced primary structure, and expression pattern of Atr-II, a receptor serine/threonine kinase found in Drosophila. With the exception of the spacing of 10 cysteine residues, the extracellular domain of Atr-II is very dissimilar from those of vertebrate activin receptors, yet it binds activin with high affinity and specificity. The kinase domain sequence of Atr-II is 60% identical to those of activin receptors from vertebrates, suggesting similarities in their signaling mechanisms. Maternal Atr-II transcript and its product are abundant in the oocyte. During development, the highest levels of Atr-II transcript and protein are observed in the mesoderm and gut. The possible role of an activin signaling system in Drosophila development is discussed.

Cell behaviors underlying notochord formation and extension in avian embryos: quantitative and immunocytochemical studies.

Formation and extension of the notochord is one of the earliest and most obvious events of axis development in vertebrate embryos. In birds, prospective notochord cells arise from Hensen's node and come to lie beneath the midline of the neural plate, where they assist in the process of neurulation and initiate the dorsoventral patterning of the neural tube through sequential inductive interactions. In the present study, we examined notochord development in avian embryos with quantitative and immunological procedures. Extension of the notochord occurs principally through accretion, that is, the addition of cells to its caudal end, a process that involves considerable cell rearrangement at the notochord-Hensen's node interface. In addition, cell division and cell rearrangement within the notochord proper contribute to notochord extension. Thus, extension of the notochord occurs in a manner that is significantly different from that of the adjacent, overlying, midline region of the neural plate (i.e., the median hinge-point region or future floor plate of the neural tube), which as shown in one of the previous studies from our laboratory (Schoenwolf and Alvarez: Development 106:427-439, 1989), extends caudally as its cells undergo two rounds of mediolateral cell-cell intercalation and two-three rounds of cell division.

Identification of a retinoic acid responsive enhancer 3′ of the murine homeobox gene Hox-1.6

The putative vertebrate morphogen retinoic acid (RA) has been shown to induce expression of many mammalian homeobox genes in cell lines, suggesting expression of this gene family in developing vertebrate embryos may be controlled in part by RA. Using the teratocarcinoma cell line F9 as a model system, we have studied the RA-response of the murine homeobox gene Hox-1.6. RA treatment of F9 cells causes the appearance of a DNAse I hypersensitive site 3′ of Hox-1.6, approximately 5 kb downstream of the Hox-1.6 promoter, and this site has been shown to reflect the presence of an RA-responsive enhancer 3′ of the gene. The RA-responsiveness of the enhancer is controlled by a retinoic acid responsive element (RARE) identical to the RARE of the retinoic acid receptor (RAR) β gene; however, other sequences also influence the activity of the enhancer, suggesting the presence of binding sites for novel proteins which regulate Hox-1.6 expression. Experiments with Hox-1.6 minigenes in which lacZ expression is controlled by the Hox-1.6 promoter and enhancer demonstrate that it is the 3′ enhancer which confers RA responsiveness on the endogenous promoter, as constructs which lack the enhancer, or the RARE alone, do not respond to RA. Our results support the idea that RA is an endogenous vertebrate morphogen; identification of the RA-responsive enhancer downstream of Hox-1.6 demonstrates that RA directly controls the transcription of at least one member of a gene family that determines tissue identity in the vertebrate embryo.

Distribution of type II collagen mRNA in Xenopus embryos visualized by whole-mount in situ hybridization.

We have developed a whole-mount histochemical method to monitor the distribution of expressed genes within the intact, developing vertebrate embryo. Background problems that result from alkaline phosphatase- or horseradish peroxidase-based stains have been minimized, enabling both early and late stages of Xenopus embryogenesis to be monitored. The feasibility and utility of this non-isotopic method has been demonstrated by using a specific DNA probe to localize Xenopus laevis Type II collagen mRNA expression to areas surrounding the vacuoles of the notochord in Stage 30 embryos. Expression expands by Stage 41/42 to form a visually striking distribution pattern that includes a variety of chondrogenic tissues such as the vertebrae, otocysts, mandible, and periocular region. Although these experiments focused on expression of a structural gene, the high resolution and sensitivity of the method should allow it also to monitor expression of less abundant mRNA products of non-structural genes such as transcription factors, cytoplasmic regulators, and growth factors. In addition, this approach should be a successful tool to probe expression in normal and perturbed embryos not only of amphibians but also of other vertebrates, including avians and mammals.

Molecular bases of early neural development in Xenopus embryos.

Genes that are differentially expressed in the ectoderm as it diverts along the neural and epidermal pathways of differentiation can be used to study the inducing signals underlying induction as well as how ectoderm responds to these signals by forming neural tissue. Although these genes have provided, and will continue to provide, new information about the induction process, they are not likely to provide the whole story. For example, Notch is a gene required for neurogenesis in Drosophila embryos. When the Notch gene product is absent, the embryo forms far too many neuroblasts at the expense of the hypodermal cell layer (Artavanis-Tsakonis 1988, Campos-Ortega 1988). Even though Notch appears to play a role in deciding the fate of neural/hypodermal cells, the Notch gene product is expressed ubiquitously in early embryos in the neurogenic region (Hartley et al 1987, Kidd et al 1989). Thus, one possibility is that Notch function does not necessarily depend on differential expression of the Notch gene product within cells in the neurogenic regions [although an alternative view has been suggested by Greenspan (1990)]. Thus, some of the molecules controlling early neural development may not be expressed differentially when the ectoderm forms the neural plate. Obviously, other approaches will have to be taken to isolate and characterize these molecules. In this light, it is noteworthy that a molecule has been identified in Xenopus that is remarkably similar to Drosophila Notch in both structure and developmental expression (Coffman et al 1990). One hope is that the analysis of this molecule in combination with the molecules that are differentially expressed during neural induction will eventually lead to a molecular understanding of early neural development in vertebrate embryos.

The contribution made by cells from a single somite to tissues within a body segment and assessment of their integration with similar cells from adjacent segments.

Somites represent the first visual evidence of segmentation in the developing vertebrate embryo and it is becoming clear that this segmental pattern of the somites is used in the initial stages of development of other segmented systems such as the peripheral nervous system. However, it is not known whether the somites continue to contribute to the maintenance of the segmental pattern after the dispersal of the somitic cells. In particular, the extent to which cells from a single somite contribute to all of the tissues of a single body segment and the extent to which they mix with cells from adjacent segments during their migration is not known. In this study, we have replaced single somites in the future cervical region of 2-day-old chick embryos with equivalent, similarly staged quail somites. The chimerae were then allowed to develop for a further 6 days when they were killed. The cervical region was dissected and serially sectioned. The sections were stained with the Feulgen reaction for DNA to differentiate between the chick and quail cells. The results showed that the cells from a single somite remained as a clearly delimited group throughout their migration. Furthermore, the sclerotome, dermatome and myotome portions from the single somites could always be recognised as being separate from similar cells from other somites. The somitic cells formed all of the tissues within a body segment excluding the epidermis, notochord and neural tissue. There was very little mixing of the somitic cells between adjacent segments. The segmental pattern of the somites is therefore maintained during the migration of the somitic cells and this might be fundamental to a mechanism whereby the segmentation of structures, such as the peripheral nervous system, is also maintained during development.

Genetic and Epigenetic Control of Vertebrate Embryonic Development

In this overview, I provide a brief history of preformation (unfolding) and epigenesis (gradual progression) from ARISTOTLE, through HARVEY and WOLFF to the present day. While basic information (DNA) and structure (ribosomes, endoplasmic reticulum) is preformed in the egg, it is the epigenetic activation of fertilization that releases what is otherwise unrealisable potential. Nowadays, epigenesis is the implementation and activation of the genetic instructions found within the zygote; genetics proposes, epigenitics disposes. Epigenetic control of vertebrate development is discussed in the context of hierarchy and increasing complexity. The initial decision in vertebrate development, that between soma or germ line, is used to illustrate how epigenetic and preformed informations interact in decision-making. Differentiation within the soma is then discussed as sequential, epigenetic activation and programming of successive decisions that lay-down the basic plan of the embryo. Epigenetic cascades are used to illustrate the temporal and spatial dependency of tissue and organ development on both prior and adjacent events. After gastrulation, many such cascades involve epithelial-mesenchymal interactions as first-order epigenetic events. These are discussed using the differentiation of cartilage and bone in the developing skeleton as the primary example. Second-order epigenetic control involves interaction with other developing tissues and organs. In sum, these interactions provide the epigenetic control that characterizes development of vertebrate embryos.

An electron-microscope study of cell deletion in the anuran tadpole tail during spontaneous metamorphosis with special reference to apoptosis of striated muscle fibers.

ABSTRACT The mechanism, of cell deletion responsible for involution of the anuran tadpole tail during spontaneous metamorphosis was studied by light and electron microscopy, attention being focused on epidermis and striated muscle. The earliest indication of pending dissolution of epidermal cells was found to be aggregation of condensed chromatin beneath the nuclear envelope. This is followed by breaking up of the nucleus, and cytoplasmic condensation and budding with the production of a number of compact, membrane-bounded cell fragments with relatively well preserved organelles. These are then ingested and degraded by nearby viable cells, the majority by distinctive macrophage-like cells, which are scattered throughout the epidermis, and a few by epithelial cells. The morphological changes observed in the dying epidermal cells are the same as those described both in the ‘programmed cell death’ that plays an important role in the normal development of vertebrate embryos and in the type of cell death that has been shown to be involved in regulating the size of tissues in adult mammals under normal as well as pathological conditions; it has been suggested elsewhere that apoptosis might be a suitable name for the phenomenon. Deletion of striated muscle fibres in the tadpole tail is accomplished by a process that appears to be a modification of classical apoptosis, in which dilatation and confluence of elements of the sarcoplasmic reticulum lead to internal fragmentation, the usual surface budding presumably being precluded by the large volume and specialized structure of these cells. The early and late nuclear changes, and the apparent ultrastructural integrity of organelles in the membrane-bounded muscle fragments are typical of apoptosis, and subsequent degradation within macrophages follows the standard stereotyped pattern. An essentially similar process has been described by others in the muscles of metamorphosing insect larvae, but whether striated muscle cells in adult higher vertebrates can undergo apoptosis is still uncertain.

A high vacuum glass system for thin film study: Anon., Electron. Engng., 35 (425), July 1963, 438

Fibroblast growth factors (FGFs) have been shown to control formation and differentiation of multiple organ systems in the developing vertebrate embryo. The analysis of differential gene expression during embryogenesis is, therefore, a potent tool to identify novel target genes regulated by FGF signalling. Here, we have applied microarray analysis to identify differentially regulated genes in FGF mutant mouse embryos. Surprisingly, transcripts corresponding to vomeronasal receptors (VRs), which so far have been only detected in the vomeronasal organ (VNO), were found to be downregulated in FGF mutant embryos. VR expression was detected in the developing olfactory pit and the anlage of the VNO. Interestingly, several FGFs can be detected in the developing olfactory pit during mouse embryogenesis [Bachler, M., Neubuser, A. 2001. Expression of members of the Fgf family and their receptors during midfacial development. Mech. Dev. 100, 313–316]. FGF signalling may thus control expression of VRs in the olfactory pit and formation of the VNO. Moreover, VR expression was detected in unexpected locations within the developing embryo including retina, dorsal root ganglia and neural tube. The relevance of VR expression in these structures and for formation of the VNO is discussed.

1101. Ultra-high vacuum techniques: R. I. Garrod, Austr. J. Instrum. Techn.,

Fibroblast growth factors (FGFs) have been shown to control formation and differentiation of multiple organ systems in the developing vertebrate embryo. The analysis of differential gene expression during embryogenesis is, therefore, a potent tool to identify novel target genes regulated by FGF signalling. Here, we have applied microarray analysis to identify differentially regulated genes in FGF mutant mouse embryos. Surprisingly, transcripts corresponding to vomeronasal receptors (VRs), which so far have been only detected in the vomeronasal organ (VNO), were found to be downregulated in FGF mutant embryos. VR expression was detected in the developing olfactory pit and the anlage of the VNO. Interestingly, several FGFs can be detected in the developing olfactory pit during mouse embryogenesis [Bachler, M., Neubuser, A. 2001. Expression of members of the Fgf family and their receptors during midfacial development. Mech. Dev. 100, 313–316]. FGF signalling may thus control expression of VRs in the olfactory pit and formation of the VNO. Moreover, VR expression was detected in unexpected locations within the developing embryo including retina, dorsal root ganglia and neural tube. The relevance of VR expression in these structures and for formation of the VNO is discussed.