Zebrafish deadly seven functions in neurogenesis.

In a genetic screen, we isolated a mutation that perturbed motor axon outgrowth, neurogenesis, and somitogenesis. Complementation tests revealed that this mutation is an allele of deadly seven (des). By creating genetic mosaics, we demonstrate that the motor axon defect is non-cell autonomous. In addition, we show that the pattern of migration for some neural crest cell populations is aberrant and crest-derived dorsal root ganglion neurons are misplaced. Furthermore, our analysis reveals that des mutant embryos exhibit a neurogenic phenotype. We find an increase in the number of primary motoneurons and in the number of three hindbrain reticulospinal neurons: Mauthner cells, RoL2 cells, and MiD3cm cells. We also find that the number of Rohon-Beard sensory neurons is decreased whereas neural crest-derived dorsal root ganglion neurons are increased in number supporting a previous hypothesis that Rohon-Beard neurons and neural crest form an equivalence group during development. Mutations in genes involved in Notch-Delta signaling result in defects in somitogenesis and neurogenesis. We found that overexpressing an activated form of Notch decreased the number of Mauthner cells in des mutants indicating that des functions via the Notch-Delta signaling pathway to control the production of specific cell types within the central and peripheral nervous systems.     

Disruption of segmental neural crest migration and ephrin expression in delta-1 null mice

Neural crest cells migrate segmentally through the rostral half of each trunk somite due to inhibitory influences of ephrins and other molecules present in the caudal-half of somites. To examine the potential role of Notch/Delta signaling in establishing the segmental distribution of ephrins, we examined neural crest migration and ephrin expression in Delta-1 mutant mice. Using Sox-10 as a marker, we noted that neural crest cells moved through both rostral and caudal halves of the somites in mutants, consistent with the finding that ephrinB2 levels are significantly reduced in the caudal-half somites. Later, mutant embryos had aberrantly fused and/or reduced dorsal root and sympathetic ganglia, with a marked diminution in peripheral glia. These results show that Delta-1 is essential for proper migration and differentiation of neural crest cells. Interestingly, absence of Delta-1 leads to diminution of both neurons and glia in peripheral ganglia, suggesting a general depletion of the ganglion precursor pool in mutant mice.     

Delta signaling mediates segregation of neural crest and spinal sensory neurons from zebrafish lateral neural plate

We examined the role of Delta signaling in specification of two derivatives in zebrafish neural plate: Rohon-Beard spinal sensory neurons and neural crest. deltaA-expressing Rohon-Beard neurons are intermingled with premigratory neural crest cells in the trunk lateral neural plate. Embryos homozygous for a point mutation in deltaA, or with experimentally reduced delta signalling, have supernumerary Rohon-Beard neurons, reduced trunk-level expression of neural crest markers and lack trunk neural crest derivatives. Fin mesenchyme, a putative trunk neural crest derivative, is present in deltaA mutants, suggesting it segregates from other neural crest derivatives as early as the neural plate stage. Cranial neural crest derivatives are also present in deltaA mutants, revealing a genetic difference in regulation of trunk and cranial neural crest development.     

Dorsal and intermediate neuronal cell types of the spinal cord are established by a BMP signaling pathway

We have studied the role of Bmp signaling in patterning neural tissue through the use of mutants in the zebrafish that disrupt three different components of a Bmp signaling pathway: swirl/bmp2b, snailhouse/bmp7 and somitabun/smad5. We demonstrate that Bmp signaling is essential for the establishment of the prospective neural crest and dorsal sensory Rohon-Beard neurons of the spinal cord. Moreover, Bmp signaling is necessary to limit the number of intermediate-positioned lim1+ interneurons of the spinal cord, as observed by the dramatic expansion of these prospective interneurons in many mutant embryos. Our analysis also suggests a positive role for Bmp signaling in the specification of these interneurons, which is independent of Bmp2b/Swirl activity. We found that a presumptive ventral signal, Hh signaling, acts to restrict the amount of dorsal sensory neurons and trunk neural crest. This restriction appears to occur very early in neural tissue development, likely prior to notochord or floor plate formation. A similar early role for Bmp signaling is suggested in the specification of dorsal neural cell types, since the bmp2b/swirl and bmp7/snailhouse genes are only coexpressed during gastrulation and within the tail bud, and are not found in the dorsal neural tube or overlying epidermal ectoderm. Thus, a gastrula Bmp2b/Swirl and Bmp7/Snailhouse-dependent activity gradient may not only act in the specification of the embryonic dorsoventral axis, but may also function in establishing dorsal and intermediate neuronal cell types of the spinal cord.     

Lunatic fringe causes expansion and increased neurogenesis of trunk neural tube and neural crest populations

Both neurons and glia of the PNS are derived from the neural crest. In this study, we have examined the potential function of lunatic fringe in neural tube and trunk neural crest development by gain-of-function analysis during early stages of nervous system formation. Normally lunatic fringe is expressed in three broad bands within the neural tube, and is most prominent in the dorsal neural tube containing neural crest precursors. Using retrovirally-mediated gene transfer, we find that excess lunatic fringe in the neural tube increases the numbers of neural crest cells in the migratory stream via an apparent increase in cell proliferation. In addition, lunatic fringe augments the numbers of neurons and upregulates Delta-1 expression. The results indicate that, by modulating Notch/Delta signaling, lunatic fringe not only increases cell division of neural crest precursors, but also increases the numbers of neurons in the trunk neural crest.     

Zebrafish narrowminded suggests a genetic link between formation of neural crest and primary sensory neurons.

In the developing vertebrate nervous system, both neural crest and sensory neurons form at the boundary between non-neural ectoderm and the neural plate. From an in situ hybridization based expression analysis screen, we have identified a novel zebrafish mutation, narrowminded (nrd), which reduces the number of early neural crest cells and eliminates Rohon-Beard (RB) sensory neurons. Mosaic analysis has shown that the mutation acts cell autonomously suggesting that nrd is involved in either the reception or interpretation of signals at the lateral neural plate boundary. Characterization of the mutant phenotype indicates that nrd is required for a primary wave of neural crest cell formation during which progenitors generate both RB sensory neurons and neural crest cells. Moreover, the early deficit in neural crest cells in nrd homozygotes is compensated later in development. Thus, we propose that a later wave can compensate for the loss of early neural crest cells but, interestingly, not the RB sensory neurons. We discuss the implications of these findings for the possibility that RB sensory neurons and neural crest cells share a common evolutionary origin.     

Expression patterns of HNK-1 carbohydrate and serotonin in sea urchin, amphioxus, and lamprey, with reference to the possible evolutionary origin of the neural crest

We examined deuterostome invertebrates, the sea urchin and amphioxus, and an extant primitive vertebrate, the lamprey, for the presence of structures expressing the HNK-1 carbohydrate and serotonin. In sea urchin embryos and larvae, HNK-1 positive cells were localized in the ciliary bands and in their precursor ectoderm. Serotonergic cells were exclusively observed in the apical organs. In juvenile amphioxus, primary sensory neurons in the dorsal nerve cords were HNK-1 immunoreactive. The juvenile amphioxus nerve cords contained anti-serotonin immunoreactive nerve fibers that seem to be the Rohde axons extending from amphioxus interneurons, the Rohde cells. In lamprey embryos, migrating neural crest cells and primary sensory neurons, including Rohon-Beard cells, expressed the HNK-1 carbohydrate. Lamprey larvae (ammocoetes) contained cell aggregates expressing both the HNK-1 carbohydrate and serotonin in the pronephros and in the regions adjacent to the gut epithelium. Some of these cell aggregates were present in the anti-serotonin positive visceral motor nerve net. Motor neurons and Müller fibers were serotonergic in ammocoetes. Comparison of the expression patterns of HNK-1 carbohydrate among sea urchins, amphioxus and lampreys seem to suggest the possible evolutionary origin of the neural crest, that is, ciliary bands in dipleurula-type ancestors evolved into primary sensory neurons in chordate ancestors, as inferred from Garstang's auricularia hypothesis, and the neural crest originated from the primary sensory neurons.     

Multiple roles of mouse Numb in tuning developmental cell fates.

BACKGROUND: Notch signaling regulates multiple differentiation processes and cell fate decisions during both invertebrate and vertebrate development. Numb encodes an intracellular protein that was shown in Drosophila to antagonize Notch signaling at binary cell fate decisions of certain cell lineages. Although overexpression experiments suggested that Numb might also antagonize some Notch activity in vertebrates, the developmental processes in which Numb is involved remained elusive. RESULTS: We generated mice with a homozygous inactivation of Numb. These mice died before embryonic day E11.5, probably because of defects in angiogenic remodeling and placental dysfunction. Mutant embryos had an open anterior neural tube and impaired neuronal differentiation within the developing cranial central nervous system (CNS). In the developing spinal cord, the number of differentiated motoneurons was reduced. Within the peripheral nervous system (PNS), ganglia of cranial sensory neurons were formed. Trunk neural crest cells migrated and differentiated into sympathetic neurons. In contrast, a selective differentiation anomaly was observed in dorsal root ganglia, where neural crest--derived progenitor cells had migrated normally to form ganglionic structures, but failed to differentiate into sensory neurons. CONCLUSIONS: Mouse Numb is involved in multiple developmental processes and required for cell fate tuning in a variety of lineages. In the nervous system, Numb is required for the generation of a large subset of neuronal lineages. The restricted requirement of Numb during neural development in the mouse suggests that in some neuronal lineages, Notch signaling may be regulated independently of Numb.     

Pax3-expressing trigeminal placode cells can localize to trunk neural crest sites but are committed to a cutaneous sensory neuron fate

The cutaneous sensory neurons of the ophthalmic lobe of the trigeminal ganglion are derived from two embryonic cell populations, the neural crest and the paired ophthalmic trigeminal (opV) placodes. Pax3 is the earliest known marker of opV placode ectoderm in the chick. Pax3 is also expressed transiently by neural crest cells as they emigrate from the neural tube, and it is reexpressed in neural crest cells as they condense to form dorsal root ganglia and certain cranial ganglia, including the trigeminal ganglion. Here, we examined whether Pax3+ opV placode-derived cells behave like Pax3+ neural crest cells when they are grafted into the trunk. Pax3+ quail opV ectoderm cells associate with host neural crest migratory streams and form Pax3+ neurons that populate the dorsal root and sympathetic ganglia and several ectopic sites, including the ventral root. Pax3 expression is subsequently downregulated, and at E8, all opV ectoderm-derived neurons in all locations are large in diameter, and virtually all express TrkB. At least some of these neurons project to the lateral region of the dorsal horn, and peripheral quail neurites are seen in the dermis, suggesting that they are cutaneous sensory neurons. Hence, although they are able to incorporate into neural crest-derived ganglia in the trunk, Pax3+ opV ectoderm cells are committed to forming cutaneous sensory neurons, their normal fate in the trigeminal ganglion. In contrast, Pax3 is not expressed in neural crest-derived neurons in the dorsal root and trigeminal ganglia at any stage, suggesting either that Pax3 is expressed in glial cells or that it is completely downregulated before neuronal differentiation. Since Pax3 is maintained in opV placode-derived neurons for some considerable time after neuronal differentiation, these data suggest that Pax3 may play different roles in opV placode cells and neural crest cells.     

Rohon-Beard sensory neurons are induced by BMP4 expressing non-neural ectoderm in Xenopus laevis

Rohon-Beard mechanosensory neurons (RBs), neural crest cells, and neurogenic placodes arise at the border of the neural- and non-neural ectoderm during anamniote vertebrate development. Neural crest cells require BMP expressing non-neural ectoderm for their induction. To determine if epidermal ectoderm-derived BMP signaling is also involved in the induction of RB sensory neurons, the medial region of the neural plate from donor Xenopus laevis embryos was transplanted into the non-neural ventral ectoderm of host embryos at the same developmental stage. The neural plate border and RBs were induced at the transplant sites, as shown by expression of Xblimp1, and XHox11L2 and XN-tubulin, respectively. Transplantation studies between pigmented donors and albino hosts showed that neurons are induced both in donor neural and host epidermal tissue. Because an intermediate level of BMP4 signaling is required to induce neural plate border fates, we directly tested BMP4′s ability to induce RBs; beads soaked in either 1 or 10 ng/ml were able to induce RBs in cultured neural plate tissue. Conversely, RBs fail to form when neural plate tissue from embryos with decreased BMP activity, either from injection of noggin or a dominant negative BMP receptor, was transplanted into the non-neural ectoderm of un-manipulated hosts. We conclude that contact between neural and non-neural ectoderm is capable of inducing RBs, that BMP4 can induce RB markers, and that BMP activity is required for induction of ectopic RB sensory neurons.     

Differentiation state determines neural effects on microvascular endothelial cells

Growing evidence indicates that nerves and capillaries interact paracrinely in uninjured skin and cutaneous wounds. Although mature neurons are the predominant neural cell in the skin, neural progenitor cells have also been detected in uninjured adult skin. The aim of this study was to characterize differential paracrine effects of neural progenitor cells and mature sensory neurons on dermal microvascular endothelial cells. Our results suggest that neural progenitor cells and mature sensory neurons have unique secretory profiles and distinct effects on dermal microvascular endothelial cell proliferation, migration, and nitric oxide production. Neural progenitor cells and dorsal root ganglion neurons secrete different proteins related to angiogenesis. Specific to neural progenitor cells were dipeptidyl peptidase-4, IGFBP-2, pentraxin-3, serpin f1, TIMP-1, TIMP-4 and VEGF. In contrast, endostatin, FGF-1, MCP-1 and thrombospondin-2 were specific to dorsal root ganglion neurons. Microvascular endothelial cell proliferation was inhibited by dorsal root ganglion neurons but unaffected by neural progenitor cells. In contrast, microvascular endothelial cell migration in a scratch wound assay was inhibited by neural progenitor cells and unaffected by dorsal root ganglion neurons. In addition, nitric oxide production by microvascular endothelial cells was increased by dorsal root ganglion neurons but unaffected by neural progenitor cells.     

Placode and neural crest-derived sensory neurons are responsive at early developmental stages to brain-derived neurotrophic factor

The response of embryonic chick nodose ganglion (neural placode-derived) and dorsal root ganglion (neural crest-derived) sensory neurons to the survival and neurite-promoting activity of brain-derived neurotrophic factor (BDNF) was studied in culture. In dissociated, neuron-enriched cultures established from chick embryos between Day 6 (E6) and Day 12 (E12) of development, both nodose ganglion (NG) and dorsal root ganglion (DRG) neurons were responsive on laminin-coated culture dishes to BDNF. In the case of NG, BDNF elicited neurite outgrowth from 40 to 50% of the neurons plated at three embryonic ages; E6, E9, and E12. At the same ages, nerve growth factor (NGF) alone or in combination with BDNF, had little or no effect upon neurite outgrowth from NG neurons. The response of NG neurons to BDNF was dose dependent and was sustainable for at least 7 days in culture. Surprisingly, in view of a previous study carried out using polyornithine as a substrate for neuronal cell attachment, on laminin-coated dishes BDNF also sustained survival and neurite outgrowth from a high percentage (60-70%) of DRG neurons taken from E6 embryos. In marked contrast to NG neurons, the combined effect of saturating levels of BDNF and NGF activity on DRG neurons was greater than the effect of either agent alone at all embryonic ages studied. Under similar culture conditions, BDNF did not elicit survival and neurite outgrowth from paravertebral chain sympathetic neurons or parasympathetic ciliary ganglion neurons. We propose that primary sensory neurons, regardless of their embryological origin, are responsive to a "central-target" (CNS) derived neurotrophic factor--BDNF, while they are differentially responsive to "peripheral-target"-derived growth factors, such as NGF, depending on whether the neurons are of neural crest or placodal origin.     

Notochord grafts do not suppress formation of neural crest cells or commissural neurons.

Grafting experiments previously have established that the notochord affects dorsoventral polarity of the neural tube by inducing the formation of ventral structures such as motor neurons and the floor plate. Here, we examine if the notochord inhibits formation of dorsal structures by grafting a notochord within or adjacent to the dorsal neural tube prior to or shortly after tube closure. In all cases, neural crest cells emigrated from the neural tube adjacent to the ectopic notochord. When analyzed at stages after ganglion formation, the dorsal root ganglia appeared reduced in size and shifted in position in embryos receiving grafts. Another dorsal cell type, commissural neurons, identified by CRABP and neurofilament immunoreactivity, differentiated in the vicinity of the ectopic notochord. Numerous neuronal cell bodies and axonal processes were observed within the induced, but not endogenous, floor plate 1 to 2 days after implantation but appeared to be cleared with time. These results suggest that dorsally implanted notochords cannot prevent the formation of neural crest cells or commissural neurons, but can alter the size and position of neural crest-derived dorsal root ganglia.