Embryonic Nervous System Development Research Articles

Embryonic and paralarval development of the central nervous system of the loliginid squid Sepioteuthis lessoniana

The embryonic development of the central nervous system (CNS) in the oval squid Sepioteuthis lessoniana is described. It has three distinct phases: (1) The ganglionic accumulation phase: Ganglionic cell clusters develop by ingression, migration, and accumulation of neuroblasts. (2) The lobe differentiation phase: Ganglia differentiate into lobes. The phase is identified by the beginning of an axogenesis. During this phase, neuropils are first formed in the suboesophageal mass, then in the basal lobe system, and finally in the inferior frontal lobes and the superior frontal-vertical lobe systems. (3) The neuropil increment phase: After the shape of the lobes reached its typical form, neuropil growth occurs, specifically in the vertical lobe. The paralarval central nervous system (CNS) is characterized by neuronal gigantism of the giant fibers and some suboesophageal commissures and connectives. The neuropil formation in the CNS of S. lessoniana occurs somewhat earlier than in Octopus vulgaris, although the principal developmental plan is quite conservative among the other coleoids investigated. Some phylogenetic aspects are discussed based on the similarities in the morphologic organization of their brains.

Expression of the intermediate filament protein nestin by sustentacular cells in mature olfactory neuroepithelium.

The intermediate filament protein nestin has been widely used as a marker for proliferating neural progenitor cells in the nervous system. The mammalian olfactory neuroepithelium is a region of the nervous system that robustly supports ongoing neurogenesis, yet where nestin has not been reported to mark proliferating progenitors. Using immunohistochemistry, we examined nestin expression in the mature olfactory neuroepithelium and found it to be tightly restricted to the basal compartment where the olfactory neuronal progenitor cell population resides. The pattern of nestin immunoreactivity was consistent with expression by the endfeet and inferior processes of sustentacular cells rather than basal cells. Using a bank of defined antibody markers, we confirmed nestin's pattern of distribution to be different from that of cytokeratin, vimentin, GBC-1, GAP43, and carnosine. It was highly similar to the pattern of SUS-4 immunoreactivity in the basal region of the neuroepithelium. Following surgical bulbectomy, nestin expression was up-regulated and became evident in the cell bodies of sustentacular cells situated more apically in the neuroepithelium. We have shown nestin to be present in the basal region of the adult olfactory neuroepithelium in the zone that supports ongoing neurogenesis in the adult, but its expression is restricted to the inferior parts of sustentacular cells rather than the neuronal progenitor cells. Nestin may play a potential role in the migration of recently proliferated olfactory neurons on the scaffolding of sustentacular cells in a manner analogous to its proposed role in radial glia during embryonic development of the central nervous system.

Regulated nuclear export of the homeodomain transcription factor Prospero.

Subcellular distribution of the Prospero protein is dynamically regulated during Drosophila embryonic nervous system development. Prospero is first detected in neuroblasts where it becomes cortically localized and tethered by the adapter protein, Miranda. After division, Prospero enters the nucleus of daughter ganglion mother cells where it functions as a transcription factor. We have isolated a mutation that removes the C-terminal 30 amino acids from the highly conserved 100 amino acid Prospero domain. Molecular dissection of the homeo- and Prospero domains, and expression of chimeric Prospero proteins in mammalian and insect cultured cells indicates that Prospero contains a nuclear export signal that is masked by the Prospero domain. Nuclear export of Prospero, which is sensitive to the drug leptomycin B, is mediated by Exportin. Mutation of the nuclear export signal-mask in Drosophila embryos prevents Prospero nuclear localization in ganglion mother cells. We propose that a combination of cortical tethering and regulated nuclear export controls Prospero subcellular distribution and function in all higher eukaryotes.

In vivo study of motoneuron death induced by nerve injury in mice deficient in the caspase 1/ interleukin-1β-converting enzyme

The apoptotic cell death program is orchestrated by members of the caspase family. Among these caspases, several in vitro and in vivo reports indicate that the interleukin-1β-converting enzyme (or caspase 1) may be involved in neurodegenerative processes. In view of these findings, and in order to characterize the role of the interleukin-1β-converting enzyme in mediating or modulating cell death processes in vivo, we have investigated the effects of its deletion on motoneuron survival after a facial nerve transection in newborn and adult interleukin-1β-converting enzyme knock-out mice. During the postnatal period of development, when facial motoneurons are highly vulnerable to axotomy, we did not observe any significant effect of the interleukin-1β-converting enzyme-deletion on the percentage of cell death in the lesioned nuclei. In addition, the spontaneous cell death characteristic of the postnatal period was not altered in knock-out mice. In contrast, in adult knock-out mice, a significant reduction (16%) in the number of surviving facial motoneurons was observed six weeks after axotomy. We therefore conclude that the interleukin-1β-converting enzyme does not appear to be critical for cell death during the postnatal period but may favor motoneuron survival during adulthood. Given the key role of caspase 3 in neuronal apoptosis during embryonic development of the central nervous system, we also investigated the role of this caspase in cell death following axotomy. Combined immunofluorescence revealed that, at least during the postnatal period, axotomized motoneurons that have apoptotic nuclear morphologies were immunopositive for the active form of caspase 3. Double-stained cells could be also observed on the unlesioned side. These results strongly suggest that caspase 3 may be involved in both the postnatal spontaneous- and axotomy-induced facial motoneuron death processes. Similar results were obtained in interleukin-1β-converting enzyme-deficient and wild-type mice, indicating that the interleukin-1β-converting enzyme may not be required for caspase 3 activation.

Ontogeny of vesicular monoamine transporter mRNAs VMAT1 and VMAT2: I. The developing rat central nervous system

We used in situ hybridization histochemistry to study the expression of the mRNA of the two vesicular monoamine transporters (VMAT1 and VMAT2) during embryonic and postnatal development of the central nervous system (CNS) in the rat. In the adult rat, VMAT2 mRNA is present exclusively in monoaminergic cell groups of the CNS and VMAT1 mRNA was reported to be present in the adrenal medulla and certain intestinal epithelial cells. In contrast to the above, the expression of VMAT1 mRNA has previously never been detected in the central nervous system. This study shows the first evidence that both transporter molecules are expressed in CNS during ontogenesis. We here demonstrate four main expression patterns detected during development: 1. VMAT2 mRNA expression in monoaminergic neurons of the brainstem beginning as early as embryonic day E13. 2. Expression of VMAT2 mRNA in all major sensory relay nuclei of central nervous system. 3. Co-expression of VMAT1 and VMAT2 mRNA in most limbic structures, basal ganglia, as well as in some hypothalamic nuclei. 4. Exclusive expression of VMAT1 mRNA in the neocortical subventricular zone, in the amygdala at early (E15–18) and late (P1–P28) timepoints, the granular cell layer of cerebellum, and in several brainstem motor nuclei. Based on their distribution during development we suggest that monoamines, released in a controlled fashion, might affect wiring of sensory and also motor circuits. VMAT1 mRNA expression may reflect a specific effect of monoamines in glial differentiation and cerebellar granule cell migration and/or differentiation.

Miranda directs Prospero to a daughter cell during Drosophila asymmetric divisions

Asymmetric cell division is a general process used in many developmental contexts to create two differently fated cells from a single progenitor cell. Intrinsic mechanisms like the asymmetric transmission of cell-fate determinants during cell division, and extrinsic cell-interaction mechanisms, can mediate asymmetric divisions. During embryonic development of the Drosophila central nervous system, neural stem cells called neuroblasts divide asymmetrically to produce another multipotent neuroblast and a ganglion mother cell (GMC) of more restricted developmental potential. Intrinsic mechanisms promote asymmetric division of neuroblasts: for example, the transcription factor Prospero localizes to the basal cell cortex of mitotic neuroblasts and then segregates exclusively into the GMC, which buds off from the basal side of the neuroblast. In the GMC, Prospero translocates to the nucleus, where it establishes differential gene expression between sibling cells. Here we report the identification of a gene, miranda, which encodes a new protein that co-localizes with Prospero in mitotic neuroblasts, tethers Prospero to the basal cortex of mitotic neuroblasts, directing Prospero into the GMC, and releases Prospero from the cell cortex within GMCs. miranda thus creates intrinsic differences between sibling cells by mediating the asymmetric segregation of a transcription factor into only one daughter cell during neural stem-cell division.

CSox21 exhibits a complex and dynamic pattern of transcription during embryonic development of the chick central nervous system

cSox21 is a novel member of the Sox gene family of transcription factors. This gene is a member of the subgroup B, which includes Sox1, Sox2 and Sox3. Although all of these genes are predominantly expressed in the nervous system, only cSox21 expression is positionally restricted within the CNS. Longitudinal stripes are seen in the spinal cord and a more complex pattern is seen in the brain. The timing and position in which cSox21 stripes of expression appear provides further insight into dorsoventral patterning of the CNS. The expression of cSox21, and other genes (such as Delta, Serrate and Pax genes), may play a part in defining the developmental fate of cells along the dorsoventral axis.

Embryonic expression pattern of H218, a G-protein coupled receptor homolog, suggests roles in early mammalian nervous system development

Heterologous expression studies employing mammalian cell tissue culture techniques and in vivo studies of lower eukaryotes suggest that G-protein coupled receptors may play critical roles in regulating early stages of vertebrate nervous system development. Previous work suggests that H218, a rat G-protein coupled receptor homolog, could serve such a role. Most importantly, northern blot data indicate that whole brain H218 mRNA levels are highest during embryogenesis. In the present studies we raised, affinity-purified and characterized several anti-H218, polyclonal antisera and immunohistochemically mapped the expression of H218 during the early stages of rat embryonic nervous system development. The resulting data indicate that H218 is preferentially expressed in young, differentiating neuronal cell bodies and axons. Moreover, the expression is temporally regulated such that highest H218 levels are found in neuronal cell bodies during their early stages of differentiation and in axons during their outgrowth. Therefore, we propose that H218 signal transduction may widely participate in the regulation of some of the first steps in neuronal differentiation including axon outgrowth.

Protease nexin-1 is expressed at the mouse met-/mesencephalic junction and FGF signaling regulates its promoter activity in primary met-/mesencephalic cells.

The expression pattern of the serine protease inhibitor gene Protease nexin-1 (PN-1) has been analyzed by in situ hybridization during embryonic nervous system development. PN-1 was found to be specifically expressed at the junction between the mes- and metencephalon (mid- and hindbrain). Transgenic embryos expressing the bacterial lacZ gene under the control of different fragments of the PN-1 upstream regulatory region were used to demarcate an enhancer sufficient for expression at this putative segmental border. Primary cell cultures derived from the embryonic neural tube at the level of the met-/mesencephalic junction were used to demonstrate a specific effect on transcriptional activity by basic fibroblast growth factor and Engrailed transcription factors, providing evidence that PN-1 is a target gene of these factors. The results of this study place PN-1 into the signaling cascade(s) considered to be important for the development of this junction and suggest a role for PN-1 in the establishment or maintenance of the differences between these adjacent neuromeres.

Embryonic development of the enteric nervous system of the grasshopper Schistocerca americana.

The enteric nervous system (ENS) of the grasshopper Schistocerca americana is organized into four ganglia located in the foregut (the dorsal unpaired frontal and hypocerebral ganglia, and the paired ingluvial ganglia), and two plexuses that innervate the foregut and midgut. A dorsomedial recurrent nerve and two lateral esophageal nerves connect the ganglia. The midgut plexus is arranged in four nerves running along the midgut surface. In this study, we have focused on the embryonic development of the grasshopper ENS; we have studied the proliferation pattern, morphogenesis, and some aspects of neuronal differentiation by using a number of specific molecular markers. The grasshopper ENS develops early in embryogenesis (25-30%) from three neurogenic zones (NZs) located on the roof of the stomodeum. These NZs slightly invaginate from an epithelial placode. The expression pattern of specific cell surface proteins and the analysis of the mitotic activity showed that NZs cells delaminate from the epithelium, become neuronal precursors, divide symmetrically, and then actively migrate to their final position in the enteric ganglia or plexuses. The grasshopper enteric ganglia are composed of mixed populations of cells from different NZs. The foregut and midgut plexuses are formed by the dispersal of cells from the developing hypocerebral and ingluvial ganglia. The main ENS nerves are pioneered by axons extending anteriorly from hypocerebral and ingluvial neurons. The insect ENS exhibits an enormous variation in design. Several features of the grasshopper program of neurogenesis and pattern of cell migration are compared to other insects, and some evolutionary implications are discussed.

Insulin Acts as an Embryonic Growth Factor forDrosophilaNeural Cells

The isolation of theDrosophilainsulin receptor gene and the recent analysis of loss of function mutants have clearly implicated insulin signalling in embryonic nervous system development. Here we study the presence of insulin in the embryo and we characterize the cellular processes affected by insulin in embryonic neural cells. We find that 7.5% of the cells in the 15-18 hDrosophilaembryo contain insulin immunoreactivity by flow cytometry. In the embryonic-derived cell line Schneider 1, we show that human insulin is capable of stimulating proliferation and neural differentiation. Thus, the action of insulin on the developingDrosophilanervous system appears to be as pleiotropic as in vertebrates.

The zebrafish brain: a neuroanatomical comparison with the goldfish

The zebrafish Danio rerio is an important model system for genetic and developmental studies of the vertebrate central nervous system. Considerable knowledge concerning the embryonic development of the central nervous system of the zebrafish has accumulated in recent years. However, there is an apparent lack of information on the organization of the adult zebrafish brain. We have therefore recently studied in detail the neuroanatomy of the adult zebrafish. Here we compare the brains of the zebrafish and of the closely related and neurobiologically well-investigated goldfish, Carassius auratus. Two sensory systems, the visual and the gustatory systems, were identified as differing on the gross morphological and histological levels in the two species. The goldfish shows the simple (evolutionarily reduced) pattern of pretectal organization, and its gustatory system is massively enlarged. The pretectum of the zebrafish conforms to this simplified visual pretectal pattern, although the retention of some ancestral pretectal characters indicates a lesser degree of reduction of the visual system compared to the goldfish. The gustatory system shows many similarities with the evolutionarily derived and functionally specialized gustatory system of the goldfish. However, some peripheral and central gustatory characters are missing in the zebrafish, indicating a less specialized gustatory system.

Embryonic expression of vasoactive intestinal peptide (VIP) and VIP receptor genes.

Vasoactive intestinal peptide (VIP) exhibits pronounced effects on the growth rate of cultured mouse embryonic day (E) 9.5 embryos and acts in tissue culture as a potent glial mitogen and neuron survival factor. However, previous studies using immunohistochemistry or in situ hybridization in the rat have not revealed the presence and location of VIP or VIP mRNA in the early developing embryo CNS. Using a sensitive in situ hybridization assay with a 33P-labeled riboprobe, we show here that the VIP gene is expressed at least as early as E11 in the mouse hindbrain. Northern blot analysis on RNA from brain dissected from mouse embryos beginning at E14 confirmed that a correct-size mRNA for VIP was present by E14 and at later time points. Expression of the VIP2 receptor gene was also detected by northern analysis in E14 mouse brains. These studies support the hypothesis that VIP produced by the embryo exerts important effects on embryonic nervous system development.

P-element mutations affecting embryonic peripheral nervous system development in Drosophila melanogaster.

The Drosophila embryonic peripheral nervous system (PNS) is an excellent model system to study the molecular mechanisms governing neural development. To identify genes controlling PNS development, we screened 2000 lethal P-element insertion strains. The PNS of mutant embryos was examined using the neural specific marker MAb 22C10, and 92 mutant strains were retained for further analysis. Genetic and cytological analysis of these strains shows that 42 mutations affect previously isolated genes that are known to be required for PNS development: longitudinals lacking (19), mastermind (15), numb (4), big brain (2), and spitz (2). The remaining 50 mutations were classified into 29 complementation groups and the P-element insertions were cytologically mapped. The mutants were classified in five major classes on the basis of their phenotype: gain of neurons, loss of neurons, organizational defects, pathfinding defects and morphological defects. Herein we report the preliminary phenotypic characterization of each of these complementation groups as well as the embryonic lacZ expression pattern of each P-element strain. Our analysis indicates that in most of the P-element insertion strains, the lacZ reporter gene is not expressed in the developing PNS.

Phyllopod functions in the fate determination of a subset of photoreceptors in drosophila

phyllopod ( phyl) encodes a novel protein required for fate determination of photoreceptors R1, R6, and R7, the last three photoreceptors to be recruited into the ommatidia of the developing Drosophila eye. Genetic data suggests that phyl acts downstream of Ras1, raf, and yan to promote neuronal differentiation in this subset of photoreceptors. Ectopic expression of phyl in the cone cell precursors mimics the effect of ectopic activation of Ras1, suggesting that phyl expression is regulated by Ras1. phyl is also required for embryonic nervous system and sensory bristle development.

Reconstruction of neural tube-like structures in vitro from primary neural precursor cells.

Vertebrate central nervous system develops from a neural tube derived from the embryonic ectoderm. In mouse, the neural tube around embryonic day 10 primarily consists of neural precursor cells (NPCs). During the development of embryonic central nervous system, NPCs proliferate and migrate outward; thus later stages show NPCs toward the lumen of the neural tube and neurofilament-positive differentiated cells toward the periphery. In conventional liquid culture, NPCs isolated from mouse on embryonic day 10 proliferate and differentiate into neurofilament-positive neurons. In the present communication, we show that fragments of neural tubes and aggregates of NPCs, when placed into collagen gel matrix, form three-dimensional structures which resemble the neural tube formed in vivo in the developing embryos. Even dissociated NPCs form the three-dimensional structures in the collagen gel matrix. Our results indicate that individual NPCs or fragments of neural tubes carry morphogenetic information which allows them to reconstruct neural tube-like structures in vitro.

Neovascularization and the appearance of morphological characteristics of the blood-brain barrier in the embryonic mouse central nervous system

Vascularization and expression of blood-brain barrier (bbb)-associated morphological characteristics during the embryonic development of the mouse cnetral nervous system (CNS) was studied by ultrastructural analysis. At embryonic day 9 (E9) capillaries were only found in the perineural mesenchymal tissue. These capillaries showed fenestrations, and pericyte like cells (PC) were found joined to the vessel walls. Around E10 endothelial cells (EC) together with PC started invading the intraneural section. At this stage, the immigrating endothelial cells lost their fenestrations and exhibited numerous, partly extended junctional complexes, which appeared ‘tight’ in some places. A first intraneural anastomotic plexus was observed at E10, as evidenced by the presence of blood cells in all capillary lumens. While the number of junctional complexes remained constant in intraneural capillaries, the frequency of pinocytotic vesicles decreased significantly from E10 to E17. These findings indicate that from the first day of intraneural vascularization onwards, the morphological properties of the bbb are present in the early embryonic mouse cerebral cortex.

Retinoic acid receptors and cellular retinoid binding proteins. III. Their differential transcript distribution during mouse nervous system development.

We have studied the transcript distribution of the retinoic acid receptors (RARs) and the cytoplasmic retinoid binding proteins during embryonic development of the mouse nervous system. Of the three retinoic acid receptors, only RAR-gamma was not expressed in developing neural structures. RAR-beta and RAR-alpha both showed rostral limits of expression in the medulla oblongata equivalent to their patterns of expression in the neuroepithelium of the early hindbrain neural tube. Within their expression domains in the spinal cord and brain, RAR-alpha was ubiquitously expressed, whereas RAR-beta transcripts showed very specific patterns of expression, suggesting that this receptor is involved in mediating retinoic acid-induced gene expression in relation to the development of specific neural structures or pathways. The cytoplasmic binding proteins, cellular retinoic acid binding proteins type I and II (CRABP I and CRABP II) and cellular retinol binding protein type I (CRBP I), were widely distributed in developing neural structures. Their differential spatiotemporal patterns of expression suggest that fine regional control of availability of retinoic acid (RA) to the nuclear receptors plays an important role in organization and differentiation of the nervous system. For instance, expression of CRABP I in the migrating cells that give rise to the olivary and pontine nuclei, which develop abnormally in conditions of retinoid excess, is consistent with observations from a variety of other systems indicating that CRABP I limits the access of RA to the nuclear receptors in normal physiological conditions. Similarly, expression of CRBP I in the choroid plexuses, which develop abnormally in conditions of vitamin A deficiency, is consistent with observations indicating that this binding protein mediates the synthesis of RA in tissues requiring high levels of RA for their normal developmental programme. RAR-beta and CRABP II, which are both RA-inducible, were coexpressed with CRBP I in the choroid plexus and in many other sites, perhaps reflecting the fact that all three genes are RA-inducible. The function of CRABP II is not well understood; its domains of expression showed overlaps with both CRABP I and CRBP I.

Expression of DRG during murine embryonic development

We had previously characterised a cDNA which encodes a novel GTP-binding protein DRG. The expression of drg gene is down-regulated during the embryonic development of murine central nervous system. Further analysis of drg mRNA and protein in adult mouse tissues and various cell lines of different origins indicated that it is expressed widely, albeit at low and variable levels. In situ hybridisation analysis of mRNA expression in sections of mouse embryos indicated that drg is expressed strongly in various embryonic tissues. The expression of drg mRNA is greatly reduced in newborn animals. At cellular level, DRG protein can be detected in the cytoplasm. These observations suggest that DRG may play multiple roles in development and normal cell metabolism.

Identification of a set of genes with developmentally down-regulated expression in the mouse brain

Using a subtraction cloning approach, we have isolated a set of cDNA clones from mouse neural precursor cells whose respective mRNA levels are down-regulated during the development of mouse brain. Single stranded DNA prepared from neuronal precursor cell cDNA library in lambda Zap vector was subtracted with poly (A) + RNA prepared from postnatal and adult mouse brain to obtain several clones which show developmental down-regulation of expression. Their patterns of expression indicate that these genes may play important roles during the embryonic development and differentiation of central nervous system.