The regulation of intramuscular nerve branching during normal development and following activity blockade

In vertebrates, approximately 50% of the lumbosacral motoneurons die during a short period of development that coincides with synaptogenesis in the limb. Although it has been postulated that these motoneurons die because they fail to obtain adequate trophic support from the muscles, it is not clear how this factor is supplied. The mechanism by which activity blockade prevents motoneurons cell death is also unknown. In order to begin to understand the nature of these proposed trophic interactions, we have examined the temporal sequence of axonal invasion and ramification within two muscles of the chick hindlimb, the predominantly slow iliofibularis and the fast posterior iliotibialis, during the cell death period. We found striking differences in intramuscular nerve ingrowth and branching between fast and slow muscle. We also observed differences in the molecular composition of fast and slow myotubes that may contribute to the nerve pattern differences. In addition, we observed a progressive increase in the degree of intramuscular nerve fasciculation as well as a precise temporal sequence of nerve branching. The earliest detectable response to chronic curarization was a dramatic decrease in the degree of intramuscular nerve fasciculation. Activity blockade also greatly enhanced nerve branching within the muscles from the time that nerve branches normally formed, and, additionally, interfered with the normal cessation of axon growth. Our results support the idea that nerve endings are the sites of trophic uptake. Furthermore, although our results do not allow us to exclude other activity-dependent influences on motoneuron survival, they suggest the following testable hypotheses: (1) the normal regulation of motoneuron survival may result from the precise control of intramuscular nerve branching, (2) activity blockade may increase motoneuron survival by enhancing intramuscular nerve branching, and (3) anything which affects this complex process of nerve branching may also alter motoneuron survival.     

The relationship of intramuscular nerve branching and synaptogenesis to motoneuron survival

The target has been considered for some time to play a major role in allowing neurons to survive the period of naturally occurring cell death. For the motoneurons that innervate the chick limb, evidence is presented that suggests access to target-derived trophic factor via intramuscular nerve branches and synapses may be important in regulating neuronal survival. Alterations in branching and synapse formation produced by activity blockade as well as by alteration of adhesion molecule function are shown to result in changes in motoneuron survival consistent with the proposed hypothesis. The relevance of these observations to the numerical-matching hypothesis of vertebrate neuronal cell death is also considered. © 1992 John Wiley & Sons, Inc.     

The relationship of intramuscular nerve branching and synaptogenesis to motoneuron survival.

The target has been considered for some time to play a major role in allowing neurons to survive the period of naturally occurring cell death. For the motoneurons that innervate the chick limb, evidence is presented that suggests access to target-derived trophic factor via intramuscular nerve branches and synapses may be important in regulating neuronal survival. Alterations in branching and synapse formation produced by activity blockade as well as by alteration of adhesion molecule function are shown to result in changes in motoneuron survival consistent with the proposed hypothesis. The relevance of these observations to the numerical-matching hypothesis of vertebrate neuronal cell death is also considered.     

Purines as potential morphogens during embryonic development

Components of purinergic signalling are expressed in the early embryo raising the possibility that ATP, ADP and adenosine may contribute to the mechanisms of embryonic development. We summarize the available data from four developmental models-mouse, chick, Xenopus and zebrafish. While there are some notable examples where purinergic signalling is indeed important during development, e.g. development of the eye in the frog, it is puzzling that deletion of single components of purinergic signalling often results in rather minor developmental phenotypes. We suggest that a key step in further analysis is to perform combinatorial alterations of expression of purinergic signalling components to uncover their roles in development. We introduce the concept that purinergic signalling could create novel morphogenetic fields to encode spatial location via the concentration of ATP, ADP and adenosine. We show that using minimal assumptions and the known properties of the ectonucleotidases, complex spatial patterns of ATP and adenosine can be set up. These patterns may provide a new way to assess the potential of purinergic signalling in developmental processes.     

Changes in fatty acid compositions of mitochondria during embryonic development

1. A general trend among biomembranes of hepatocytes in the developing avian embryo is to display increasing percentages of unsaturated fatty acids, especially oleic acid (C18:1). 2. However, once increasing amounts of thyroxine appear in the plasma, mitochondria begin to exhibit increasing percentages of saturated fatty acids, primarily stearic acid (C18:0). 3. Increasing saturation of mitochondrial membrane lipids can be inhibited by exposure of embryonated eggs to 500 R of X-irradiation. 4. Injection of embryonated eggs with estrone increases the proportion of oleic acid (C18:1) in mitochondrial membranes but a balancing increase in palmitic acid (C16:0) enables their lipids to remain more saturated than unsaturated.     

Retinoic acid as a regulator of planarian morphogenesis

The effect of retinoic acid on regeneration of two species of asexual planarian races, Girardia tigrina and Schmidtea mediterranea, was studied. It was established that retinoic acids at physiological concentrations (10⁻⁷–10⁻¹⁰ M) inhibit the regeneration of the head part of planarians but have no effect on tail blastema growth. It is shown that regeneration of the head part is inhibited as a result of arrest of the cell cycle of neoblasts, proliferating stem cells, during the transition from the G ₁/G ₀ to the S phase. Thus, the morphogenetic role of retinoic acids in planarians, primitive bilaterally symmetrical animals, has been demonstrated.     

Sialic acid at the surface of myocardial cells during embryonic development

We tested the hypothesis that the reduction of automaticity during the embryonic development of chick ventricular myocytes is correlated with the number of sialic acid residues at the cell surface. The major findings were twofold. First, the sialic acid content of ventricular tissue fragments declined during the period between 4 and 17 days of development; however, when a 26% reduction of cell surface area was taken into account, the surface density of sialic acid at 7 and 17 days was not significantly different. Second, the sialic acid content of ventricular cell aggregates (after 3 days in gyratory culture) increased during the same two-week period. On the surface of these cells, the density was significantly greater at 17 days than at 7 days, even after a 17% increase in cell surface area had been taken into account. When the developmental increase in sialic acid content was compared with a concomitant decline in aggregate beat rates, we calculated a correlation coefficient of 0.85. Thus, while there could be some relationship between aggregate automaticity and sialic acid content, there appears to be no such correlation for fragments of chick ventricle.     

Fliih, a gelsolin-related cytoskeletal regulator essential for early mammalian embryonic development

The Drosophila melanogaster flightless I gene is required for normal cellularization of the syncytial blastoderm. Highly conserved homologues of flightless I are present in Caenorhabditis elegans, mouse, and human. We have disrupted the mouse homologue Fliih by homologous recombination in embryonic stem cells. Heterozygous Fliih mutant mice develop normally, although the level of Fliih protein is reduced. Cultured homozygous Fliih mutant blastocysts hatch, attach, and form an outgrowing trophoblast cell layer, but egg cylinder formation fails and the embryos degenerate. Similarly, Fliih mutant embryos initiate implantation in vivo but then rapidly degenerate. We have constructed a transgenic mouse carrying the complete human FLII gene and shown that the FLII transgene is capable of rescuing the embryonic lethality of the homozygous targeted Fliih mutation. These results confirm the specific inactivation of the Fliih gene and establish that the human FLII gene and its gene product are functional in the mouse. The Fliih mouse mutant phenotype is much more severe than in the case of the related gelsolin family members gelsolin, villin, and CapG, where the homozygous mutant mice are viable and fertile but display alterations in cytoskeletal actin regulation.     

Expression of mouse Coiled-coil-DIX1 (Ccd1), a positive regulator of Wnt signaling, during embryonic development

Wnt signaling plays an important role in cell growth, differentiation, polarity formation, and neural development. We have recently identified the Coiled-coil-DIX1 (Ccd1) gene encoding a third type of a DIX domain-containing protein. Ccd1 forms homomeric and heteromeric complexes with Dishevelled and Axin, and positively regulates the Wnt/beta-catenin pathway. Here, we examined the spatiotemporal expression pattern of Ccd1 mRNA in mouse embryos from embryonic day 6.5 (E6.5) to E17.5 by in situ hybridization. Ccd1 expression was detected in the node region in gastrula embryos, in the cephalic mesenchyme and tail bud at E8.5, and in the branchial arch and forelimb bud at E9.5. In the central nervous system, Ccd1 expression began and persisted in the regions where the neurons differentiated, so that it was observed throughout the brain and spinal cord at E17.5. Ccd1 expression was also strong in the peripheral nervous system, including sensory cranial ganglia (trigeminal, facial, and vestibulocochlear ganglia), dorsal root ganglia, and autonomic ganglia (sympathetic ganglia, celiac ganglion, and hypogastric plexus). Ccd1 was detected in the sensory organs, such as the inner nuclear layer of the neural retina, saccule and cochlea of the inner ear, and nasal epithelium. Outside the nervous system, Ccd1 mRNA was observed in the cartilage, tongue, lung bud, stomach, and gonad at E12.5-E14.5, and in the tooth bud, bronchial epithelium, and kidney at E17.5. Taken together, these findings demonstrate that Ccd1 expression is observed in all the neurons in the nervous system, closely associated with neural crest-derived tissues, and largely overlapping with the regions where several Wnt genes are reported to play a role.     

Nedd1 expression as a marker of dynamic centrosomal localization during mouse embryonic development

As the primary microtubule-organizing centre of the mammalian cell, the centrosome plays many important roles during cell growth and organization. This is evident across a broad range of cell types and processes, such as the proliferation, differentiation and polarity of neural cells. Additionally, given its localization and function, there are likely to be many more processes that rely on the centrosome that have not yet been characterized. Currently, little is known about centrosomal dynamics during mammalian development. In this study, we have analyzed Nedd1 protein expression to characterize the localization of the centrosome during some aspects of mouse embryogenesis. Using a Nedd1 antibody we have demonstrated the colocalization of Nedd1 with centrosomal markers. We found strong expression of Nedd1, and therefore the centrosome, in highly proliferating cells during neural development. Additionally, Nedd1 was found to have high expression in the cytoplasm of a subset of cells in the dorsal root ganglia. We have also shown a distinct, polarized centrosomal localization of Nedd1 in the developing lens, retina and other polarized tissues. This study reveals the localization of Nedd1 and the centrosome during important processes in mouse embryogenesis and provides a basis for further study into its role in development.     

Quantitative analysis of nerve growth factor in the amniotic fluid during chick embryonic development

Nerve growth factor (NGF) and most neurotrophic factors support the proliferation and survival of particular types of neurons. Besidesthe pivotal role of NGF in the development of neuronal cells, it also has important functions on non-neuronal cells. The amnion surrounds the embryo, providing an aqueous environment for the embryo. A wide range of proteins has been identified in human amniotic fluid (AF). In this study, total protein concentration (TPC) and NGF level in AF samples from chick embryos were measured using a Bio-Rad protein assay, enzyme linked immunosorbent assay (ELISA) and Western blot. TPC increased from days E10 to day E18. There was a rapid increase in AF TPC on day E15 when compared to day E16. No significant changes in NGF levels have been seen from day E10 to day E14. There was a rapid increase in NGF content on days E15 and E16, and thereafter the levels decreased from day E16 to day E18. Since, NGF is important in brain development and changes in AF NGF levels have been seen in some CNS malformations, changes in the TPC and NGF levels in AF during chick embryonic development may be correlated with cerebral cortical development. It is also concluded that NGF is a constant component of the AF during chick embryogenesis.     

Biochemical purification of a mammalian slit protein as a positive regulator of sensory axon elongation and branching

Many neurons in both vertebrates and invertebrates innervate multiple targets by sprouting secondary axon collaterals (or branches) from a primary axon shaft. To begin to identify molecular regulators of axon branch initiation or extension, we studied the growth of single sensory axons in an in vitro collagen assay system and identified an activity in extracts of embryonic spinal cord and of postnatal and adult brain that promotes the elongation and formation of extensive branches by these axons. Biochemical purification of the activity from calf brain extracts led to the identification of an amino-terminal fragment of Slit2 as the main active component and to the discovery of a distinct activity that potentiates its effects. These results indicate that Slit proteins may function as positive regulators of axon collateral formation during the establishment or remodeling of neural circuits.     

Calcium signalling during embryonic development

Consider a hypothetical design specification for an integrated communication-control system within an embryo. It would require short-range (subcellular) and long-range (pan-embryonic) abilities, it would have to be flexible and, at the same time, robust enough to operate in a dynamically changing environment without information being lost or misinterpreted. Although many signalling elements appear, disappear and sometimes reappear during development, it is becoming clear that embryos also depend on a ubiquitous, persistent and highly versatile signalling system that is based around a single messenger, Ca2+.     

The chicken as a model for embryonic development

The traditional strength of chicken embryos for studying development is that they are readily manipulated. This has led to some major discoveries in developmental biology such as the demonstration that the neural crest gives rise to almost the entire peripheral nervous system and the identification of signalling centres that specify the pattern of structures in the central nervous system and limb. More recently with the burgeoning discovery of developmentally important genes, chicken embryos have provided useful models for testing function. Uncovering the molecular basis of development provides direct links with clinical genetics. In addition, since many genes that have crucial roles in development are also expressed in tumours, basic research on chickens has implications for understanding human health and disease. Now that the chicken genome has been sequenced and genomic resources for chicken are becoming increasingly available, this opens up opportunities for combining these new technologies with the manipulability of chicken embryos and also exploiting comparative genomics.     

Smad1 expression and function during mouse embryonic lung branching morphogenesis

Bone morphogenetic protein (BMP) 4 plays very important roles in regulating developmental processes of many organs, including lung. Smad1 is one of the BMP receptor downstream signaling proteins that transduce BMP4 ligand signaling from cell surface to nucleus. The dynamic expression patterns of Smad1 in embryonic mouse lungs were examined using immunohistochemistry. Smad1 protein was predominantly detected in peripheral airway epithelial cells of early embryonic lung tissue [embryonic day 12.5 (E12.5)], whereas Smad1 protein expression in mesenchymal cells increased during mid-late gestation. Many Smad1-positive mesenchymal cells were localized adjacent to large airway epithelial cells and endothelial cells of blood vessels, which colocalized with a molecular marker of smooth muscle cells (alpha-smooth muscle actin). The biological function of Smad1 in early lung branching morphogenesis was then studied in our established E11.5 lung explant culture model. Reduction of endogenous Smad1 expression was achieved by adding a Smad1-specific antisense DNA oligonucleotide, causing approximately 20% reduction of lung epithelial branching. Furthermore, airway epithelial cell proliferation and differentiation were also inhibited when endogenous Smad1 expression was knocked down. Therefore, these data indicate that Smad1, acting as an intracellular BMP signaling pathway component, positively regulates early mouse embryonic lung branching morphogenesis.