Pattern Of Neuronal Differentiation Research Articles

Differentiation signals from glia are fine-tuned to set neuronal numbers during development.

Neural circuit formation and function require that diverse neurons are specified in appropriate numbers. Known strategies for controlling neuronal numbers involve regulating either cell proliferation or survival. We used the Drosophila visual system to probe how neuronal numbers are set. Photoreceptors from the eye-disc induce their target field, the lamina, such that for every unit eye there is a corresponding lamina unit (column). Although each column initially contains ~6 post-mitotic lamina precursors, only 5 differentiate into neurons, called L1-L5; the 'extra' precursor, which is invariantly positioned above the L5 neuron in each column, undergoes apoptosis. Here, we showed that a glial population called the outer chiasm giant glia (xgO), which resides below the lamina, secretes multiple ligands to induce L5 differentiation in response to epidermal growth factor (EGF) from photoreceptors. By forcing neuronal differentiation in the lamina, we uncovered that though fated to die, the 'extra' precursor is specified as an L5. Therefore, two precursors are specified as L5s but only one differentiates during normal development. We found that the row of precursors nearest to xgO differentiate into L5s and, in turn, antagonise differentiation signalling to prevent the 'extra' precursors from differentiating, resulting in their death. Thus, an intricate interplay of glial signals and feedback from differentiating neurons defines an invariant and stereotyped pattern of neuronal differentiation and programmed cell death to ensure that lamina columns each contain exactly one L5 neuron.

Mitochondrial genome mutations and neuronal dysfunction of induced pluripotent stem cells derived from patients with Alzheimer's disease.

ObjectivesPatient‐derived induced pluripotent stem cells (iPSCs) are materials that can be used for autologous stem cell therapy. We screened mtDNA mutations in iPSCs and iPSC‐derived neuronal cells from patients with Alzheimer's disease (AD). Also, we investigated whether the mutations could affect mitochondrial function and deposition of β‐amyloid (Aβ) in differentiated neuronal cells.Materials and MethodsmtDNA mutations were measured and compared among iPSCs and iPSC‐derived neuronal cells. The selected iPSCs carrying mtDNA mutations were subcloned, and then their growth rate and neuronal differentiation pattern were analyzed. The differentiated cells were measured for mitochondrial respiration and membrane potential, as well as deposition of Aβ.ResultsMost iPSCs from subjects with AD harbored ≥1 mtDNA mutations, and the number of mutations was significantly higher than that from umbilical cord blood. About 35% and 40% of mutations in iPSCs were shared with isogenic iPSCs and their differentiated neuronal precursor cells, respectively, with similar or different heteroplasmy. Furthermore, the mutations in clonal iPSCs were stable during extended culture and neuronal differentiation. Finally, mtDNA mutations could induce a growth advantage with higher viability and proliferation, lower mitochondrial respiration and membrane potential, as well as increased Aβ deposition.ConclusionThis study demonstrates that mtDNA mutations in patients with AD could lead to mitochondrial dysfunction and accelerated Aβ deposition. Therefore, early screening for mtDNA mutations in iPSC lines would be essential for developing autologous cell therapy or drug screening for patients with AD.

Basal Protrusions Mediate Spatiotemporal Patterns of Spinal Neuron Differentiation

SummaryDuring early spinal cord development, neurons of particular subtypes differentiate with a sparse periodic pattern while later neurons differentiate in the intervening space to eventually produce continuous columns of similar neurons. The mechanisms that regulate this spatiotemporal pattern are unknown. In vivo imaging in zebrafish reveals that differentiating spinal neurons transiently extend two long protrusions along the basal surface of the spinal cord before axon initiation. These protrusions express Delta protein, consistent with the hypothesis they influence Notch signaling at a distance of several cell diameters. Experimental reduction of Laminin expression leads to smaller protrusions and shorter distances between differentiating neurons. The experimental data and a theoretical model support the proposal that neuronal differentiation pattern is regulated by transient basal protrusions that deliver temporally controlled lateral inhibition mediated at a distance. This work uncovers a stereotyped protrusive activity of newborn neurons that organize long-distance spatiotemporal patterning of differentiation.

Wrapping Glial Morphogenesis and Signaling Control the Timing and Pattern of Neuronal Differentiation in the Drosophila Lamina.

Various regions of the developing brain coordinate their construction so that the correct types and numbers of cells are generated to build a functional network. We previously discovered that wrapping glia in the Drosophila visual system are essential for coordinating retinal and lamina development. We showed that wrapping glia, which ensheath photoreceptor axons, respond to an epidermal growth factor cue from photoreceptors by secreting insulins. Wrapping glial insulins activate the mitogen-activated protein kinase (MAPK) pathway downstream of insulin receptor in lamina precursors to induce neuronal differentiation. The signaling relay via wrapping glia introduces a delay that allows the lamina to assemble the correct stoichiometry and physical alignment of precursors before differentiating and imposes a stereotyped spatiotemporal pattern that is relevant for specifying the individual lamina neuron fates. Here, we further describe how wrapping glia morphogenesis correlates with the timing of lamina neuron differentiation by 2-photon live imaging. We also show that although MAPK activity in lamina precursors drives neuronal differentiation, the upstream receptor driving MAPK activation in lamina precursors and the ligand secreted by wrapping glia to trigger it differentially affect lamina neuron differentiation. These results highlight differences in MAPK signaling properties and confirm that communication between photoreceptors, wrapping glia, and lamina precursors must be precisely controlled to build a complex neural network.

Mouse embryonic stem cell-derived cells reveal niches that support neuronal differentiation in the adult rat brain.

A neurogenic niche can be identified by the proliferation and differentiation of its naturally residing neural stem cells. However, it remains unclear whether "silent" neurogenic niches or regions suitable for neural differentiation, other than the areas of active neurogenesis, exist in the adult brain. Embryoid body (EB) cells derived from embryonic stem cells (ESCs) are endowed with a high potential to respond to specification and neuralization signals of the embryo. Hence, to identify microenvironments in the postnatal and adult rat brain with the capacity to support neuronal differentiation, we transplanted dissociated EB cells to conventional neurogenic and non-neurogenic regions. Our results show a neuronal differentiation pattern of EB cells that was dependent on the host region. Efficient neuronal differentiation of EB cells occurred within an adjacent region to the rostral migratory stream. EB cell differentiation was initially patchy and progressed toward an even distribution along the graft by 15-21 days post-transplantation, giving rise mostly to GABAergic neurons. EB cells in the striatum displayed a lower level of neuronal differentiation and derived into a significant number of astrocytes. Remarkably, when EB cells were transplanted to the striatum of adult rats after a local ischemic stroke, increased number of neuroblasts and neurons were observed. Unexpectedly, we determined that the adult substantia nigra pars compacta, considered a non-neurogenic area, harbors a robust neurogenic environment. Therefore, neurally uncommitted cells derived from ESCs can detect regions that support neuronal differentiation within the adult brain, a fundamental step for the development of stem cell-based replacement therapies.

PLZF Regulates Fibroblast Growth Factor Responsiveness and Maintenance of Neural Progenitors

Author SummaryThe embryonic spinal cord is organized into an array of discrete neural progenitor domains along the dorsoventral axis. Most of these domains undergo two periods of differentiation, first producing specific classes of neurons and then generating distinct populations of glial cells at later times. In addition, each of these progenitors pools exhibit marked differences in their proliferative capacities and propensity to differentiate to produce the appropriate numbers and diversity of neurons and glia needed to form functional neural circuits. The mechanisms behind this regional control of neural progenitor behavior, however, remain unclear. In this study, we identify the transcription factor Promyelocytic Leukemia Zinc Finger (PLZF) as a critical regulator of this process in the chick spinal cord. We show that PLZF is initially expressed by all spinal cord progenitors and then becomes restricted to a central domain, where it helps to limit the rate of neuronal differentiation and to preserve the progenitor pool for subsequent glial production. We also demonstrate that PLZF acts by promoting the expression of Fibroblast Growth Factor (FGF) Receptor 3, thereby enhancing the proliferative response of neural progenitors to FGFs present in developing embryos. Together, these findings reveal a novel developmental strategy for spatially controlling neural progenitor behavior by tuning their responsiveness to broadly distributed growth-promoting signals in the embryonic environment.

Regulation of spinal interneuron development by the Olig-related protein Bhlhb5 and Notch signaling

The neural circuits that control motor activities depend on the spatially and temporally ordered generation of distinct classes of spinal interneurons. Despite the importance of these interneurons, the mechanisms underlying their genesis are poorly understood. Here, we demonstrate that the Olig-related transcription factor Bhlhb5 (recently renamed Bhlhe22) plays two central roles in this process. Our findings suggest that Bhlhb5 repressor activity acts downstream of retinoid signaling and homeodomain proteins to promote the formation of dI6, V1 and V2 interneuron progenitors and their differentiated progeny. In addition, Bhlhb5 is required to organize the spatially restricted expression of the Notch ligands and Fringe proteins that both elicit the formation of the interneuron populations that arise adjacent to Bhlhb5(+) cells and influence the global pattern of neuronal differentiation. Through these actions, Bhlhb5 helps transform the spatial information established by morphogen signaling into local cell-cell interactions associated with Notch signaling that control the progression of neurogenesis and extend neuronal diversity within the developing spinal cord.

Upregulation of SOX2, NOTCH1, and ID1 in supratentorial primitive neuroectodermal tumors: a distinct differentiation pattern from that of medulloblastomas

Supratentorial primitive neuroectodermal tumor (PNET) and medulloblastoma are highly malignant embryonal brain tumors. They share morphological similarities, but differ in their differentiation patterns and global gene expression. The authors compared the expression of specific genes involved in neuroglial differentiation in supratentorial PNETs and medulloblastomas to define the distinct characters of these tumors. The mRNA expression of 8 genes (SOX2, NOTCH1, ID1, ASCL-1, NEUROD1, NEUROG1, NEUROG2, and NRG1) was evaluated in 25 embryonal tumors (12 supratentorial PNETs and 13 medulloblastomas) by quantitative real-time polymerase chain reaction. The expression levels of the transcripts of these genes were compared between the tumor groups. Activation of the JAK/STAT3 pathway was assessed by immunoblotting. Relative expression levels of STAT3 and phosphorylated STAT3 proteins were compared. Supratentorial PNETs expressed significantly higher levels of SOX2, NOTCH1, ID1, and ASCL-1 transcripts, whereas the transcription of proneural basic helix-loop-helix factors, NEUROD1, NEUROG1 (significantly), and NEUROG2 (not significantly) was upregulated in medulloblastomas. The proportion of phosphorylated STAT3alpha relative to STAT3alpha was significantly greater in supratentorial PNETs than in medulloblastomas, indicating activation of the JAK/STAT3 pathway in supratentorial PNETs. These results indicate that supratentorial PNET predominantly has glial features and medulloblastoma largely follows a neuronal differentiation pattern. These divergent differentiation patterns may be related to the location and origin of each tumor.

Development of putative GABAergic neurons in the appendicularian urochordateOikopleura dioica

Studying the developing brain of urochordates can increase our understanding of brain evolution in the chordate lineage. To begin addressing regional patterns of neuronal differentiation in appendicularian urochordates, we examined the development of putative GABAergic neurons in Oikopleura dioica using GABA immunohistochemistry and in situ hybridization for the GABA-synthesizing enzyme GAD. First, we assessed the developmental dynamics of neuron number and organization in the cerebral and caudal ganglia. We then identified and mapped the positions of putative GABAergic neurons using confocal microscopy. We found GAD mRNA-positive and GABA-immunopositive neurons in the first brain nerves and the cerebral and caudal ganglia, but not in the caudal nerve cord. In both ganglia GAD mRNA-positive and GABA-immunopositive neurons are found in the same characteristic intraganglionic locations. The differentiation of these GABAergic markers occurs first in the first brain nerves and the cerebral ganglion and then with a several-hour delay in the caudal ganglion. In all three structures GAD mRNA expression appears 2-3 hours prior to GABA expression. In general, GABA is expressed by the same number of neurons as express GAD. Several discrepancies suggest differential regulation of the GABAergic phenotype in different neurons, however. Our results show that the GABAergic phenotype has a stereotyped pattern of expression along the anteroposterior axis of the CNS. Given recent genome sequencing and developmental patterning gene studies in this species, the GABAergic neurons in O. dioica provide a good model for assessing, at the invertebrate-vertebrate transition, the molecular mechanisms that specify the GABAergic phenotype.

Regionalization of the anterior hypothalamus in the chick embryo

Loss-of-function experiments in mice have shown that the transcription factors Sim1, Otp, Sim2, and Brn2 form a cascade essential for the differentiation of neuroendocrine cells of the anterior hypothalamus that produce vasopressin, oxytocin, somatostatin (SS), thyrotropin-releasing hormone (TRH), and corticotropin-releasing hormone (CRH). Very little is known about how the differentiation of these cell types is regulated in chick. Here, we report the cloning of the chick homolog of Otp. Moreover, we have systematically compared the expression of Sim1, Sim2, Brn2, and Otp with that of the markers of terminal differentiation TRH, SS, CRH, vasotocin, and mesotocin during development of chick embryos. We have found that the cell types studied generally develop in domains expressing these transcriptional regulators but that the pattern of neuronal differentiation and the spatial distribution of some regulators were not the same as in mice. Our results provide a framework useful for the functional analysis of hypothalamus development in chick.

Morphological development of microglia in the postnatal rat brain: A quantitative study

Morphological transformation of lectin-positive microglia/macrophages in the developing rat cerebral hemisphere was analysed using quantitative methods. During the first postnatal month, the cells showed increases in their size and fractal dimension accompanied by a simultaneous decrease in their solidity. Regional variations in dynamics of the process indicated the existence of spatio-temporal developmental gradients within the cerebral hemisphere wall which might correspond with regional patterns of neuronal differentiation. Results of the present study prove that the quantitative methods can be the source of reliable data replacing subjective cell typologies.

Mutations modulating the Argos-regulated signaling pathway in Drosophila eye development.

Argos is a secreted protein that contains an EGF-like domain and acts as an inhibitor of Drosophila EGF receptor activation. To identify genes that function in the Argos-regulated signaling pathway, we performed a genetic screen for enhancers and suppressors of the eye phenotype caused by the overexpression of argos. As a result, new alleles of known genes encoding components of the EGF receptor pathway, such as Star, sprouty, bulge, and clown, were isolated. To study the role of clown in development, we examined the eye and wing phenotypes of the clown mutants in detail. In the eye discs of clown mutants, the pattern of neuronal differentiation was impaired, showing a phenotype similar to those caused by a gain-of-function EGF receptor mutation and overexpression of secreted Spitz, an activating ligand for the EGF receptor. There was also an increased number of pigment cells in the clown eyes. Epistatic analysis placed clown between argos and Ras1. In addition, we found that clown negatively regulated the development of wing veins. These results suggest that the clown gene product is important for the Argos-mediated inhibition of EGF receptor activation during the development of various tissues. In addition to the known genes, we identified six mutations of novel genes. Genetic characterization of these mutants suggested that they have distinct roles in cell differentiation and/or survival regulated by the EGF receptor pathway.

Diamine Oxidase Induces Neurite Outgrowth in Chick Dorsal Root Ganglia by a Nonenzymatic Mechanism

The enzyme diamine oxidase (DAO) catalyzes the oxidative deamination of histamine, diamines, and polyamines. DAO has been localized to several tissues, including thymus, kidney, intestine, seminal vesicles, placenta, and pregnancy plasma. DAO is not constitutively expressed in the mammalian brain, but it becomes detectable following focal injury. Although the physiologic role of DAO remains unknown, the observation that it is present at the interface between rapidly dividing and quiescent cells in several tissues suggests that it might be involved in regulating cell division or differentiation at tissue boundaries. In addition, the observation that DAO is expressed in the brain following injury suggests that the protein might play a role in the CNS response to focal neuronal damage. To test that hypothesis, we assessed the ability of purified DAO to alter the pattern of neuronal differentiation and nerve growth in vitro. In chick dorsal root ganglion explant cultures, purified porcine DAO induced neurite outgrowth in the low nanomolar range. Addition of aminoguanidine, which inhibits DAO enzyme activity, did not inhibit the protein's neurotrophic activity. These findings suggest that DAO can function as a neurotrophic ligand independent of its enzymatic activity.

Patterns of neuronal differentiation in neural tube mutant mice: curly tail and Pax3 splotch-delayed.

A battery of antibodies was used to assess development of the spinal cord and its neurons in mouse embryos with neural tube defects (NTDs). The two mutant strains examined, curly tail (ct) and splotch-delayed (Pax3Sp-d), develop an open neural tube for unrelated reasons, and thus provided for a complementary analysis. Five percent of embryos homozygous for the ct gene and 89% of embryos homozygous for the Pax3Sp-d gene develop spina bifida in the lumbosacral region of the neuraxis. Expression of several neuronal antigens, including Islet-1/2, polysialylated neural cell adhesion molecule (NCAM), neurofilaments, and a neuronal-specific nuclear protein (Neu-N) recognized by monoclonal antibody A60, were used as indicators of the level of differentiation of neuronal tissue. Immunohistochemical labeling suggests that early (embryonic days 12-15) neuronal differentiation in the dorsal and ventral region of the dysraphic neural tube occurs remarkably normally in both of the mutants. Similarly, labeling with antibodies to NCAM and neuroafilaments indicate that axonal development during early neurogenesis is unperturbed. Later stages of neuronal maturation, however, do not occur in the usual manner. Instead, the neuronal tissue begins a prodigious degeneration at embryonic day 17 (E17), so that by E18 only a rudimentary tissue remains. These results suggest that the aberrant morphology of the neural tube does not affect neuronal differentiation. However, the anomalous morphological and chemical environment may contribute to the neuronal degeneration observed at later stages.

The Earliest Patterns of Neuronal Differentiation and Migration in the Mammalian Central Nervous System

With the use of four independent cell markers and Brd-U birthdating we have charted the earliest stages of neuronal differentiation and migration in the developing rat central nervous system, including the cortex, spinal cord, and retina. One of the markers, the monoclonal antibody 2G12, labeled a large subpopulation of differentiating cells that uniformly lined the ventricles throughout these CNS regions at unexpectedly early ages. Immunocytochemistry demonstrated that, in cortex, the 2G12 antigen could appear in cells during mitosis. More mature looking 2G12-positive cell types initially had a primitive radial morphology and were axonless. However, in strict spatio-temporal sequences, the most mature looking 2G12-positive cells had the ability to sprout GAP-43-positive axons before or after the cell body left the ventricular surface and before or after detachment of their pial or ventricular endfoot processes. Double label experiments with 2G12 and Brd-U showed that none of these three 2G12-positive cell types incorporated Brd-U after a short pulse. The primitive neuroepithelial shape of the immature neurons was verified with a polyclonal GAP-43 antibody, a type III beta tubulin antibody, and DiI labeling from a distal portion of the axon. In the cortex and retina, the 2G12 marker persisted in cells that had reached prospective neuronal layers. However, in all CNS regions observed, 2G12 immunoreactivity disappeared from the cell body as the axon extended from the young neuron. Based on the smooth progression of changing 2G12-positive cell shapes, but also because of the transient nature of this label, we can only speculate that the 2G12 epitope may be marking a continuum of neuronal cell states throughout the earliest period of differentiation and migration. Thus, our hypothesis suggests that many of the youngest CNS neurons may have a widespread distribution and may begin their differentiation, and even remain axonless for a time, while retaining a neuroepithelial morphology. Once differentiation resumes, a major mode of transformation into mature neurons during the earliest stages of development could occur via translocation of the cell soma into the pial process. Importantly, these markers have verified at later stages, and especially in cortex, that multiple mechanisms exist for neuronal migration in the CNS depending on the region and stage of development.

Cellular morphology and extracellular space at rhombomere boundaries in the chick embryo hindbrain.

The chick embryo hindbrain is a segmented region of the CNS characterised by repeated blocks of neuroepithelial cells, known as rhombomeres. Individual rhombomeres are polyclonal compartments, defined both by cell lineage restriction and by the restricted expression of development control genes, that later acquire specific patterns of neuronal differentiation and axon outgrowth. The interfaces between adjacent rhombomeres are defined by boundaries across which cells do not move; the boundaries contain specialised cells and are preferentially colonised at early stages of development by extending axons. In this study, routine electron microscopy and high-pressure cryopreservation, a technique that avoids artifacts of chemical fixation, have been used to examine the morphology of rhombomere boundaries through a staged series of chick embryos. We find that the boundary regions contain enlarged extracellular spaces and that these form conduits for axons subsequently extending in the circumferential plane of the hindbrain. Labeling the ventricular surface of the neuroepithelium with DiI crystals in aqueous suspension revealed the morphology of individual cells in the intact neural tube, and demonstrated unusual fan-shaped arrays of cells at the boundaries. These findings contribute further to the evidence that cells at rhombomere boundaries differ from those in rhombomere centres, and leads to hypotheses about both the mechanism of development of the boundaries, and the role they may play in hindbrain patterning.

Early pattern of neuronal differentiation in the Xenopus embryonic brainstem and spinal cord.

Wholemount antibody labeling techniques and horseradish peroxidase backfilling were used to analyze the pattern of neuronal differentiation in the embryonic Xenopus central nervous system between stages 22 and 35/36. In the spinal cord, the first neurons to differentiate are the Rohon-Beard neurons; they are followed by ventral neurons with descending axons (descending interneurons, motoneurons) and lateral interneurons with commissural axons. The somata and axons of these primary neurons form dorsal, ventral, and lateral columns, respectively; the ventral and lateral columns uninterruptedly continue forward into the brainstem. The distribution and projection patterns of spinal neurons were analyzed quantitatively. Rohon-Beard neurons, commissural interneurons, and primary motoneurons vary in number from segment to segment. Thus, these neurons are not distributed in a segmental pattern. In each segment, neurons of a given type project axons whose length varies over a wide range. The numerical distribution of axons formed by a population of neurons of a given type was calculated and expressed as the projection profile of these neurons. For each type of neuron and spinal segment, the projection profile is different. Furthermore, the projection profiles change in a systematic way along the spinal cord. For example, the fraction of Rohon-Beard neurons with long ascending axons steadily increases if one moves towards caudal spinal levels. The findings suggest that suprasegmental cues with a graded distribution along the spinal cord determine the number and projection profile of a particular cell type in a given segment.

Characterization of cytoskeletal and neuronal markers in micromass cultures of rat embryonic midbrain cells.

Micromass cultures of rat embryonic midbrain cells were characterized with regard to the immunolocalization of neuronal and cytoskeletal markers. Cells taken from gestational day-12 embryos and cultured for 5 days in vitro comprise at least two morphologically distinct cells types: fibroblast-like cells and neurons. Antibodies to the following markers yielded preferential staining of neuronal cells: A2B5 (GQ ganglioside), gamma-aminobutyric acid (GABA), microtubule-associated protein 2 (MAP2), MAP5, neuron-specific enolase (NSE), neural cell adhesion molecule (N-CAM), and tau. Antibodies to beta-tubulin, c-neu, MAP1, and neurofilament (NF-H) stained both neuronal and fibroblast-like cells. Antibodies to glial fibrillary acidic protein (GFAP) and vimentin failed to immunoreact with any cells in day-5 CNS cultures. SDS-PAGE and Western analysis were employed to determine the specificity of the antibodies and determine the electrophoretic profiles of the markers. We conclude that the pattern of neuronal differentiation in CNS micromass cultures exhibits certain similarities to that observed in vivo. In addition, certain markers identified in this study may be of potential utility as (1) biomarkers of chemically-induced developmental neurotoxicity, and (2) indicators of differential toxicity toward the diverse cell types that comprise the mammalian central nervous system.

Axonogenesis and morphogenesis in the embryonic zebrafish brain

We have examined early neuronal differentiation and axonogenesis in the fore- and midbrain of zebrafish embryos to address general issues of early vertebrate brain development. AChE expression and HNK-1 antibody immunoreactivity were used as markers for differentiated neurons and axons, respectively. The pattern of neuronal differentiation followed a stereotyped sequence. AChE-positive cells first appeared between 14 and 16 hr in three small, isolated, bilaterally symmetrical clusters on the surface of the brain. The three clusters--the dorsorostral, ventrorostral, and ventrocaudal clusters--proved to be the progenitors of the telencephalon, ventral diencephalon, and mesencephalic tegmentum, respectively. With further development, more cells were added to these three clusters, and new clusters appeared in the anlage of the epiphysis (18 hr) and in the pituitary and dorsal mesencephalon (by 24 hr). Subsequently, as more neurons differentiated, the gaps of unlabeled cells were reduced; by 48 hr, the cluster boundaries were indistinguishable. Axonogenesis also followed a stereotyped sequence. The first HNK-1-labeled processes arose from the first three clusters of AChE-positive cells and connected the clusters. The earliest axonal growth cones appeared at 16 hr, directed caudally from two to three neurons of the ventrocaudal cluster and pioneering the ventral longitudinal tract. By 18 hr, the tract of the postoptic commissure was initiated by growth cones directed caudally from the ventrorostral cluster toward the ventrocaudal cluster. By 20 hr, axons from the dorsorostral cluster projected ventrally to form the supraoptic tract. The other dorsoventral tracts (the dorsoventral diencephalic tract and the tract of the posterior commissure) became evident between 20 and 24 hr. These observations provide a continuous record of the topological distortions involved in the conversion of the tubular embryonic brain into the contorted adult form. The telencephalon, ventral diencephalon, and hypothalamus originate from the same rostrocaudal level of the neural tube. The pattern of differentiation demonstrated that the early development of the rostral neural tube occurs simultaneously in several independent centers, similar to the overtly segmental development of the hindbrain.

The cellular basis of segmentation in the developing hindbrain

One of the most challenging problems in developmental biology is to identify the mechanisms that generate the complex arrangement and interconnections of cells in the vertebrate brain. This review surveys the evidence that the hindbrain region is laid down as a series of similar modules, final complexity being founded on this simple ground plan. Morphological studies, using immunohistochemical and axon-labelling techniques, reveal repeat patterns of neuronal differentiation and organization, whereas cell marking experiments have shown that the segments, or rhombomeres, are lineage-restriction units each constructing a defined piece of the hindbrain.