Development of the enteric nervous system in chick embryonic duodenum in terms of the distribution of tubulin

An immunohistochemical method that uses anti-tubulin was utilized to observe the development of the enteric nervous system in chick embryonic duodenum. Neural crest cells, and enteric neuroblasts, or enteric ganglia, which derive from neural crest cells were clearly shown as sharp immunoreactive regions of tubulin. The distributions of enteric neuroblasts and enteric ganglia in chick duodena were in agreement with results of previous reports in which different techniques were used. The initial stage at which cells of neural crest origin were present in the duodenal walls (4-day-old embryos) was earlier than the initial stage (about 6-day-old embryos) reported earlier. This was verified by transmission electron microscopy. Also, the tubulin that is a component of the enteric nervous system was shown to be stable at a low temperature. This tubulin-immunostaining method provides a useful histochemical technique with which to study the development of the enteric ganglion and the function of tubulin as a component of the enteric nervous system.     

Proliferation pattern of postembryonic neuroblasts in the brain of Drosophila melanogaster.

The spatio-temporal proliferation pattern of postembryonic neuroblasts in the central brain region of the supra-esophageal ganglion of Drosophila melanogaster was studied by labeling DNA replicating cells with 5-bromo-2'-deoxyuridine (BrdU). There are five proliferating neuroblasts per hemisphere in larvae just after hatching: one in the ventro-lateral, and the other four in the postero-dorsal region of the brain. Dividing neuroblasts increase during the late first-late second instar larval stages, reaching a plateau of about 85 neuroblasts per hemisphere. Most neuroblasts cease dividing 20-30 hr after puparium formation (APF), while only four in the postero-dorsal region continue making progenies until 85-90 hr APF. The four distinct neuroblasts proliferating in the early larval and late pupal stages are identical; they lie in the cortex above the calyces of the mushroom bodies (corpora pedunculata), proliferating over a period twice as long as that for the other neuroblasts. Their daughter neurons project into the mushroom body neuropile, and hence are likely to be the Kenyon cells. The cell-cycle period of the four neuroblasts (named mushroom body neuroblasts: MBNbs) is rather constant (1.1-1.5 hr) during the mid larval-early pupal stages and is longer before and after that. The total number of the MBNb progenies made throughout the embryonic and postembryonic development was estimated to be 800-1200 per hemisphere.     

Extrinsic cues orient the cell division axis in Drosophila embryonic neuroblasts

Cell polarity must be integrated with tissue polarity for proper development. The Drosophila embryonic central nervous system (CNS) is a highly polarized tissue; neuroblasts occupy the most apical layer of cells within the CNS, and lie just basal to the neural epithelium. Neuroblasts are the CNS progenitor cells and undergo multiple rounds of asymmetric cell division, ;budding off' smaller daughter cells (GMCs) from the side opposite the epithelium, thereby positioning neuronal/glial progeny towards the embryo interior. It is unknown whether this highly stereotypical orientation of neuroblast divisions is controlled by an intrinsic cue (e.g. cortical mark) or an extrinsic cue (e.g. cell-cell signal). Using live imaging and in vitro culture, we find that neuroblasts in contact with epithelial cells always ;bud off' GMCs in the same direction, opposite from the epithelia-neuroblast contact site, identical to what is observed in vivo. By contrast, isolated neuroblasts 'bud off' GMCs at random positions. Imaging of centrosome/spindle dynamics and cortical polarity shows that in neuroblasts contacting epithelial cells, centrosomes remained anchored and cortical polarity proteins localize at the same epithelia-neuroblast contact site over subsequent cell cycles. In isolated neuroblasts, centrosomes drifted between cell cycles and cortical polarity proteins showed a delay in polarization and random positioning. We conclude that embryonic neuroblasts require an extrinsic signal from the overlying epithelium to anchor the centrosome/centrosome pair at the site of epithelial-neuroblast contact and for proper temporal and spatial localization of cortical Par proteins. This ensures the proper coordination between neuroblast cell polarity and CNS tissue polarity.     

Asymmetric distribution of the apical plasma membrane during neurogenic divisions of mammalian neuroepithelial cells

At the onset of neurogenesis in the mammalian central nervous system, neuroepithelial cells switch from symmetric, proliferative to asymmetric, neurogenic divisions. In analogy to the asymmetric division of Drosophila neuroblasts, this switch of mammalian neuroepithelial cells is thought to involve a change in cleavage plane orientation from perpendicular (vertical cleavage) to parallel (horizontal cleavage) relative to the apical surface of the neuroepithelium. Here, we report, using TIS21-GFP knock-in mouse embryos to identify neurogenic neuroepithelial cells, that at the onset as well as advanced stages of neurogenesis the vast majority of neurogenic divisions, like proliferative divisions, show vertical cleavage planes. Remarkably, however, neurogenic divisions of neuroepithelial cells, but not proliferative ones, involve an asymmetric distribution to the daughter cells of the apical plasma membrane, which constitutes only a minute fraction (1-2%) of the entire neuroepithelial cell plasma membrane. Our results support a novel concept for the cell biological basis of asymmetric, neurogenic divisions of neuroepithelial cells in the mammalian central nervous system.     

Neuronal acquisition of tetanus toxin binding sites: Relationship with the last mitotic cycle

In an earlier study on the developing nervous system, the existence of a temporal correlation between the appearance of tetanus toxin-binding cells and neurogenesis was reported (A. Koulakoff, B. Bizzini, and Y. Berwald-Netter (1982). Using a combined approach of immunocytochemistry and [³H]thymidine autoradiography it is shown that, in the fetal mouse central nervous system, dividing cells do not express membrane binding sites for tetanus toxin. A time-course quantitative autoradiography revealed that the toxin-binding sites become apparent within 7 ± 1 hr, following the last S phase, on cells undergoing the conversion from dividing to postmitotic state. The acquisition of surface binding sites for tetanus toxin may thus be an early property of nascent central neurons, marking the transition from cycling precursor neuroblasts to postmitotic neuronal cells. Parallel studies on in vivo-developing dorsal root ganglia disclosed that at least some peripheral nervous system cells are endowed with tetanus toxin-binding capacity while still capable of DNA synthesis and undergo one or more divisions.     

Segment polarity genes in neuroblast formation and identity specification during Drosophila neurogenesis

The relatively simple central nervous system (CNS) of the Drosophila embryo provides a useful model system for investigating the mechanisms that generate and pattern complex nervous systems. Central to the generation of different types of neurons by precursor neuroblasts is the initial specification of neuroblast identity and the Drosophila segment polarity genes, genes that specify regions within a segment or repeating unit of the Drosophila embryo, have emerged recently as significant players in this process. During neurogenesis the segment polarity genes are expressed in the neuroectodermal cells from which neuroblasts delaminate and they continue to be expressed in neuroblasts and their progeny. Loss-of-function mutations in these genes lead to a failure in the formation of neuroblasts and/or specification of neuroblast identity. Results from several recent studies suggest that regulatory interactions between segment polarity genes during neurogenesis lead to an increase in the number of neuroblasts and specification of different identities to neuroblasts within a population of cells. BioEssays 21:472–485, 1999. © 1999 John Wiley & Sons, Inc.     

Divergent patterns of neural development in larval echinoids and asteroids

The development and organization of the nervous systems of echinoderm larvae are incompletely described. We describe the development and organization of the larval nervous systems of Strongylocentrotus purpuratus and Asterina pectinifera using a novel antibody, 1E11, that appears to be neuron specific. In the early pluteus, the antibody reveals all known neural structures: apical ganglion, oral ganglia, lateral ganglia, and an array of neurons and neurites in the ciliary band, the esophagus, and the intestine. The antibody also reveals several novel features, such as neurites that extend to the posterior end of the larva and additional neurons in the apical ganglion. Similarly, in asteroid larvae the antibody binds to all known neural structures and identifies novel features, including large numbers of neurons in the ciliary bands, a network of neurites under the oral epidermis, cell bodies in the esophagus, and a network of neurites in the intestine. The 1E11 antigen is expressed during gastrulation and can be used to trace the ontogenies of the nervous systems. In S. purpuratus, a small number of neuroblasts arise in the oral ectoderm in late gastrulae. The cells are adjacent to the presumptive ciliary bands, where they project neurites with growth cone-like endings that interconnect the neurons. In A. pectinifera, a large number of neuroblasts appear scattered throughout the ectoderm of gastrulae. The cells aggregate in the developing ciliary bands and then project neurites under the oral epidermis. Although there are several shared features of the larval nervous systems of echinoids and asteroids, the patterns of development reveal fundamental differences in neural ontogeny.     

Spatial and temporal patterns of neurogenesis in the central nervous system of Drosophila melanogaster

Neurogenesis in the ventral CNS of Drosophila was studied using staining with toluidine blue and birth dating of cells monitored by incorporation of bromodeoxyuridine into DNA. The ventral CNS of the larva contains sets of neuronal stem cells (neuroblasts) which are thought to be persistent embryonic neuroblasts. Each thoracic neuromere has at least 47 of these stem cells whereas most abdominal neuromeres possess only 6. They occur in stereotyped locations so that the same neuroblast can be followed from animal to animal. The thoracic neuroblasts begin enlarging at 18-26 hr of larval life, DNA synthesis commences by 31-36 hr, and the first mitoses occur shortly thereafter. Mitotic activity continues through the remainder of larval life with the neuroblasts showing a minimum cell cycle time of less than 55 min during the late third larval instar. By 12 hr after pupariation each neuroblast has produced approximately 100 progeny which are collected with it into a discrete packet. The progeny accumulate in an immature, arrested state and only finish their differentiation into mature neurons with the onset of metamorphosis. Most of the abdominal neuroblasts differ from their thoracic counterparts in their minimum cell cycle time (less than 2 hr) and the duration of proliferation (from about 50 to 90 hr of larval life). Neurons produced during the larval stage account for more than 90% of the cells found in the ventral CNS of the adult.     

Drosophila atypical protein kinase C associates with Bazooka and controls polarity of epithelia and neuroblasts

The establishment and maintenance of polarity is of fundamental importance for the function of epithelial and neuronal cells. In Drosophila, the multi-PDZ domain protein Bazooka (Baz) is required for establishment of apico-basal polarity in epithelia and in neuroblasts, the stem cells of the central nervous system. In the latter, Baz anchors Inscuteable in the apical cytocortex, which is essential for asymmetric localization of cell fate determinants and for proper orientation of the mitotic spindle. Here we show that Baz directly binds to the Drosophila atypical isoform of protein kinase C and that both proteins are mutually dependent on each other for correct apical localization. Loss-of-function mutants of the Drosophila atypical isoform of PKC show loss of apico-basal polarity, multilayering of epithelia, mislocalization of Inscuteable and abnormal spindle orientation in neuroblasts. Together, these data provide strong evidence for the existence of an evolutionary conserved mechanism that controls apico-basal polarity in epithelia and neuronal stem cells. This study is the first functional analysis of an atypical protein kinase C isoform using a loss-of-function allele in a genetically tractable organism.     

Cell division cycle of cultured neural precursor cells from Drosophila

In Drosophila neuroblast cells, which give rise to the embryonic nervous system, undergo a limited number of asymmetric cell divisions. These cell lineages result in the formation of clusters of neurons when neuroblasts are isolated and cultured. A significant proportion of these neural cell clusters (NCC) arise from individual precursor cells. The formation of NCC containing more than two neurons is repressed when DNA synthesis is inhibited. Cell division during NCC development was examined by [3H]thymidine autoradiography. The pattern of DNA synthesis by neural cells was that expected based on observations in situ. The pattern in individual NCC was consistent with single precursor origins for more than 80% of NCC, under our conditions of culture. Based on this, we show that the largest neural precursors at gastrulation undergo the most cell divisions in culture. The neuroblast cell division cycle averages approximately 1.5 hr, and is similar to that of blastoderm cells.     

Commitment of abdominal neuroblasts in Drosophila to a male or female fate is dependent on genes of the sex-determining hierarchy.

Adult specific neurons in the central nervous system of holometabolous insects are generated by the postembryonic divisions of neuronal stem cells (neuroblasts). In the ventral nervous system of Drosophila melanogaster, sex-specific divisions by a set of abdominal neuroblasts occur during larval and early pupal stages. Animals mutant for several sex-determining genes were analyzed to determine the genetic regulation of neuroblast commitment to the male or female pattern of division and the time during development when these decisions are made. We have found that the choice of the sexual pathway taken by sex-specific neuroblasts depends on the expression of one of these genes, doublesex (dsx). In the absence of any functional dxs+ products, the sex-specific neuroblasts fail to undergo any postembryonic divisions in male or female larval nervous systems. From the analysis of intersexes generated by dominant alleles of dsx, it has been concluded that the same neuroblasts provide the sex-specific neuroblasts in both male and female central nervous systems. The time when neuroblasts become committed to generate their sex-specific divisions were identified by shifting tra-2ts flies between the male- and female-specifying temperatures at various times during larval development. Neuroblasts become determined to adopt a male or female state at the end of the first larval instar, a time when abdominal neuroblasts enter their first postembryonic S-phase.     

Polarization of Drosophila Neuroblasts During Asymmetric Division

During Drosophila development, neuroblasts divide to generate progeny with two different fates. One daughter cell self-renews to maintain the neuroblast pool, whereas the other differentiates to populate the central nervous system. The difference in fate arises from the asymmetric distribution of proteins that specify either self-renewal or differentiation, which is brought about by their polarization into separate apical and basal cortical domains during mitosis. Neuroblast symmetry breaking is regulated by numerous proteins, many of which have only recently been discovered. The atypical protein kinase C (aPKC) is a broad regulator of polarity that localizes to the neuroblast apical cortical region and directs the polarization of the basal domain. Recent work suggests that polarity can be explained in large part by the mechanisms that restrict aPKC activity to the apical domain and those that couple asymmetric aPKC activity to the polarization of downstream factors. Polarized aPKC activity is created by a network of regulatory molecules, including Bazooka/Par-3, Cdc42, and the tumor suppressor Lgl, which represses basal recruitment. Direct phosphorylation by aPKC leads to cortical release of basal domain factors, preventing them from occupying the apical domain. In this framework, neuroblast polarity arises from a complex system that orchestrates robust aPKC polarity, which in turn polarizes substrates by coupling phosphorylation to cortical release.Cells use polarity for remarkably diverse functions. In this article, I discuss a polarity that is harnessed to generate daughter cells with different fates. Using polarity to divide asymmetrically addresses several challenges that complex organisms face. The diversification of cell types and tissues that occurs during the development of complex organisms is one such challenge. Drosophila neuroblasts, the subject of this article, undergo repeated symmetry breaking asymmetric cell divisions (ACDs) to populate the central nervous system. In a similar manner in adult organisms, ACDs are important for adult homeostasis, replenishing cells that are turned over during the course of normal physiology (Betschinger and Knoblich 2004).A fundamental aspect of ACD is the production of daughter cells containing distinct fate determinants. To segregate fate determinants, the cell becomes polarized to form mutually exclusive cortical domains, each with a set of fate determinants appropriate for one of the two daughter cells. The cleavage furrow forms at the interface of the two domains, partitioning the fate determinants into the two daughter cells where they function to either self-renew (to keep the progenitor population) or to differentiate (e.g., by changing the pattern of gene expression). One of the unique features of the symmetry breaking that occurs during ACD, at least as implemented by the neuroblast, is that it is remarkably dynamic, developing early in mitosis and depolarizing following the completion of cytokinesis.Since the discovery of the first polarized components, neuroblasts have been an excellent model system for investigating the mechanisms of cell polarization and have been extensively analyzed. Although aspects of neuroblast polarity remain unclear, a core framework for how polarity is created and maintained is emerging. In this article, I focus on neuroblast polarity as centered around the activity of atypical protein kinase C, which has emerged as a key regulator of the process. In this framework, neuroblast polarity can be explained by events that polarize aPKC and those that couple aPKC activity to the polarization of fate determinants.     

Sgt1 acts via an LKB1/AMPK pathway to establish cortical polarity in larval neuroblasts

Drosophila neuroblasts are a model system for studying stem cell self-renewal and the establishment of cortical polarity. Larval neuroblasts generate a large apical self-renewing neuroblast, and a small basal cell that differentiates. We performed a genetic screen to identify regulators of neuroblast self-renewal, and identified a mutation in sgt1 (suppressor-of-G2-allele-of-skp1) that had fewer neuroblasts. We found that sgt1 neuroblasts have two polarity phenotypes: failure to establish apical cortical polarity at prophase, and lack of cortical Scribble localization throughout the cell cycle. Apical cortical polarity was partially restored at metaphase by a microtubule-induced cortical polarity pathway. Double mutants lacking Sgt1 and Pins (a microtubule-induced polarity pathway component) resulted in neuroblasts without detectable cortical polarity and formation of "neuroblast tumors." Mutants in hsp83 (encoding the predicted Sgt1-binding protein Hsp90), LKB1, or AMPKα all show similar prophase apical cortical polarity defects (but no Scribble phenotype), and activated AMPKα rescued the sgt1 mutant phenotype. We propose that an Sgt1/Hsp90-LKB1-AMPK pathway acts redundantly with a microtubule-induced polarity pathway to generate neuroblast cortical polarity, and the absence of neuroblast cortical polarity can produce neuroblast tumors.     

At the nexus between pattern formation and cell-type specification: the generation of individual neuroblast fates in the Drosophila embryonic central nervous system.

The specification of specific and often unique fates to individual cells as a function of their position within a developing organism is a fundamental process during the development of multicellular organisms. The development of the Drosophila embryonic central nervous system serves as an excellent model system in which to clarify the developmental mechanisms that link pattern formation to cell-type specification. The Drosophila embryonic central nervous system develops from a set of neural stem cells termed neuroblasts. Neuroblasts arise from the ectoderm in an invariant pattern, and each neuroblast acquires a unique fate based on its position within this pattern. Two groups of genes recently have been demonstrated to govern the individual fate specification of neuroblasts. One group, the segment polarity genes, enables neuroblasts that develop in different anteroposterior positions to acquire different fates. The second group, referred to as the columnar genes, ensures that neuroblasts that develop in different dorsoventral domains assume different fates. When integrated, the activities of the segment polarity and columnar genes create a Cartesian coordinate system that bestows unique fates to individual neuroblasts as a function of their position of formation within the ectoderm. BioEssays 1999;21:922-931.     

Drosophila neural progenitor polarity and asymmetric division

In the Drosophila embryonic central nervous system, the neural precursor cells called neuroblasts undergo a number of asymmetric divisions along the apical-basal axis to give rise to different daughter cells of distinct fates. This review summarizes recent progress in understanding the mechanisms of these asymmetric cell divisions. We discuss proteins that are localized at distinct domains of cortex in the neuroblasts and their role in generating asymmetry. We also review uniformly cortical localized factors and actin cytoskeleton-associated motor proteins with regard to their potential role to serve as a link between distinct cortical domains in the neuroblasts. In this review, asymmetric divisions of sensory organ precursor and larval neuroblasts are also briefly discussed.     

Development of the nervous system of the pluteus larva of Strongylocentrotus droebachiensis

Summary Development of the nervous system of the pluteus larva of Strongylocentrotus droebachiensis was investigated using indirect immunofluorescence with antibodies against dopamine, GABA, and serotonin, and glyoxylic acid-induced fluorescence of catecholamines. Serotonergic cells first appear in full gastrulae; dopaminergic and GABAergic cells are present in early four-arm plutei. The number of neurons and the complexity of the nervous system increases through development of the pluteus. In the pluteus the dopaminergic component of the nervous system includes a ganglion in the lower lip of the mouth and a pair of ganglia at the base of the post-oral arms which extend axons along the base of the circumoral ciliary band. The distribution of cells visualized by glyoxylic acid-induced fluorescence is similar to that of dopaminergic cells. GABAergic neurons occur in the upper lip and in the wall of the esophagus. Serotonergic neurons are present in the lower lip; the pre-oral hood contains an apical ganglion which extends axons along the base of the epidermis overlying the blastocoel. The dopaminergic and GABAergic components of the nervous system are associated with effectors involved in feeding and swimming. The serotonergic component is not associated with any apparent effectors but may have a role in metamorphosis.     

Cell kinetic studies of chick brain cells in culture: an autoradiographic study with [3H]- and [14C]-thymidine

Labelling index, S-phase duration and cell-cycle time of proliferating brain cells from 6-day-old chick embryos in culture were investigated autoradiographically after labelling with [3H]- and/or [14C]-thymidine. The dissociated cells were cultured in the absence or in the presence of brain extract from 8-day-old chick embryos. Cultures contained essentially two cell types, which could be easily distinguished by the size of their nuclei: small nuclei identified as belonging to precursor cells of neurons and large nuclei corresponding to astroglial cells. The labelling index of astroglial cells (16.4%) was about 2 times higher than that of the neuronal cells (9.9%). Under the influence of brain extract the labelling index of neuroblasts was nearly doubled while that of the astroglial cells remained nearly unchanged. From double-labelling experiments with [3H]- and [14C]-thymidine, the same S-phase duration of about 7 hr was found for both cell types cultured with or without brain extract. A cell-cycle duration of 39 hr for neuronal and of 29 hr for astroglial cells was found. The cycle times remained constant under the influence of brain extract. From the measured data mentioned above, a growth fraction of 50% (neuroblasts) and 68% (astroglial cells) was calculated in control cultures without brain extract. After addition of brain extract, the growth fraction increased for both cell types (neuroblasts: 92%; astroglial cells: 80%). The results demonstrate that more cells proliferate in the presence of brain extract, but the durations of the S-phase and the cell cycle remain unchanged.     

Restriction of developmental potential during divergence of the enteric and sympathetic neuronal lineages.

In the peripheral nervous system, enteric and sympathetic neurons develop from multipotent neural crest cells. While local environmental signals in the gut and in the region of the sympathetic ganglia play a role in the choice of cell fate, little is known about the mechanisms that underlie restriction to specific neuronal phenotypes. We investigated the divergence and restriction of the enteric and sympathetic neuronal lineages using immuno-isolated neural crest-derived cells from the gut and sympathetic ganglia. Analysis of neuronal and lineage-specific mRNAs and proteins indicated that neural crest-derived cells from the gut and sympathetic ganglia had initiated neuronal differentiation and phenotypic divergence by E14.5 in the rat. We investigated the developmental potential of these cells using expression of tyrosine hydroxylase as a marker for a sympathetic phenotype. Tyrosine hydroxylase expression was examined in neurons that developed from sympathetic and enteric neuroblasts under the following culture conditions: culture alone; coculture with gut monolayers to promote enteric differentiation; or coculture with dorsal aorta monolayers to promote noradrenergic differentiation. Both enteric and sympathetic neuroblasts displayed developmental plasticity at E14.5. Sympathetic neuroblasts downregulated tyrosine hydroxylase in response to signals from the gut environment and enteric neuroblasts increased expression of tyrosine hydroxylase when grown on dorsal aorta or in the absence of other cell types. Tracking of individual sympathetic cells displaying a neuronal morphology at the time of plating indicated that neuroblasts retained phenotypic plasticity even after initial neuronal differentiation had occurred. By E19.5 both enteric and sympathetic neuroblasts had undergone a significant loss of their developmental potential, with most neuroblasts retaining their lineage-specific phenotype in all environments tested. Together our data indicate that the developmental potential of enteric and sympathetic neuroblasts becomes restricted over time and that this restriction takes place not as a consequence of initial neuronal differentiation but during the period of neuronal maturation. Further, we have characterized a default pathway of adrenergic differentiation in the enteric nervous system and have defined a transient requirement for gut-derived factors in the maintenance of the enteric neuronal phenotype.