Expression Of Ngn1 Research Articles

Hedgehog signaling is directly required for the development of zebrafish dorsal root ganglia neurons.

Hedgehog (Hh) signal transduction is directly required in zebrafish DRG precursors for proper development of DRG neurons. Zebrafish mutations in the Hh signaling pathway result in the absence of DRG neurons and the loss of expression of neurogenin1 (ngn1), a gene required for determination of DRG precursors. Cell transplantation experiments demonstrate that Hh acts directly on DRG neuron precursors. Blocking Hh pathway activation at later stages of embryogenesis with the steroidal alkaloid, cyclopamine, further reveals that the requirement for a Hh signal response in DRG precursors correlates with the onset of ngn1 expression. These results suggest that Hh signaling may normally promote DRG development by regulating expression of ngn1 in DRG precursors.

Ash1a and Neurogenin1 function downstream of Floating head to regulate epiphysial neurogenesis.

The homeodomain transcription factor Floating head (Flh) is required for the generation of neurones in the zebrafish epiphysis. It regulates expression of two basic helix loop helix (bHLH) transcription factor encoding genes, ash1a (achaete/scute homologue 1a) and neurogenin1 (ngn1), in epiphysial neural progenitors. We show that ash1a and ngn1 function in parallel redundant pathways to regulate neurogenesis downstream of flh. Comparison of the epiphysial phenotypes of flh mutant and of ash1a/ngn1 double morphants reveals that reduced expression of ash1a and ngn1 can account for most of the neurogenesis defects in the flh-mutant epiphysis but also shows that Flh has additional activities. Furthermore, different cell populations show different requirements for ash1a and ngn1 within the epiphysis. These populations do not simply correspond to the two described epiphysial cell types: photoreceptors and projection neurones. These results suggest that the genetic pathways that involve ash1a and ngn1 are common to both neuronal types.

Anatomy of neurogenesis in the early zebrafish brain

The detailed architecture of postembryonic (i.e. secondary, as opposed to primary) neurogenesis in the zebrafish brain at 2 days postfertilization was investigated by studying expression domains of various proneural basic helix–loop–helix genes (i.e. neuroD= nrd, neurogenin1= ngn1, Zash-1b) and neurogenic genes (i.e. Notch-1a, deltaA) on the level of in situ-hybridized sectioned material and compared with brain sections of the same age immunostained for PCNA (a proliferation marker) or for Hu-proteins (marker for early neuronal differentiation). Whereas both Notch-1a and deltaA domains are present in all proliferative zones of the brain, only the more diffuse and scattered deltaA expression appears to extend additionally into the adjacent postmitotic gray matter. Zash-1b is the first achaete–scute orthologue shown here to be expressed exclusively in all proliferative central nervous zones (except for the eye). The ngn1 and nrd genes are typically both expressed in overlapping fashion in many—but not all—brain regions, with ngn1 being more restricted towards the ventricular proliferative zones and nrd extending more laterally. This fact as well as comparisons with PCNA- and Hu-immunostains indicate that a great proportion of ngn1-positive cells are mitotic, but some appear to extend into the postmitotic gray matter where the nrd-domains lie. A comparison of the relative extents of PCNA- (proliferative), nrd- (freshly determined) and Hu-positive (differentiating) cell populations allows to determine the relative maturation state of a given brain part. Similar to findings in late embryonic amniote brains, expression of nrd is absent (from 2 to 5 days) in the zebrafish subpallium, ventral preoptic region, ventral thalamus and hypothalamus. These four regions are also free of ngn1 expression (with the exception of an unusual peripheral ngn1 domain in the preoptic region), indicating that a neurogenetic network not involving nrd and ngn1 is at work there. Our characterization of locally distinct patterns and dynamics of secondary neurogenesis in the entire early (2 dpf) postembryonic zebrafish brain delivers the blueprint for more specialized functional studies.

Overexpression of neurogenin1 induces neurite outgrowth in F11 neuroblastoma cells.

Neurogenin1 (Ngn1) is a basic helix-loop-helix (bHLH) transcription factor expressed in neuronal precursors in the developing nervous system. The function of Ngn1 in neurogenesis has been shown in various aspects. In this study, we investigated the neurogenic potential of Ngn1 using neuroblastoma cell line, F11, which could be induced to differentiate into neurons in the presence of cAMP. To investigate the expression of Ngn1, expression vectors for the full-length and the C- terminal deletion mutant of Ngn1 were constructed and their transactivation potential was verified using reporter gene containing the E-box sequence. Overexpression of the full-length Ngn1 induced neurite outgrowth in F11 cells in the absence of cAMP. A C-terminal deletion mutant, Ngn1(1-197), inhibited neurite outgrowth induced by cAMP in F11 cells. These results indicate that the Ngn1 plays an important role in differentiation of neuroblastoma cells and the C terminus of Ngn1 is essential for the efficient differentiation.

Multiple regulatory elements with spatially and temporally distinct activities control neurogenin1 expression in primary neurons of the zebrafish embryo

The basic Helix-Loop-Helix gene neurogenin1 (ngn1) is expressed in a complex pattern in the neural plate of zebrafish embryos, demarcating the sites of primary neurogenesis. We have dissected the ngn1 locus to identify cis-regulatory regions that control this expression. We have isolated two upstream elements that drive expression in precursors of Rohon-Beard sensory neurons and hindbrain interneurons and in clusters of neuronal precursors in the anterior neural plate, respectively. A third regulatory region mediates later expression. Thus, regulatory sequences with temporally and spatially distinct activities control ngn1 expression in primary neurons of the zebrafish embryo. These regions are highly similar to 5′ sequences in the mouse and human ngn1 gene, suggesting that amniote embryos, despite lacking primary neurons, utilize related mechanism to control ngn1 expression.

Suppressor of cytokine signaling 2 regulates neuronal differentiation by inhibiting growth hormone signaling.

The intracellular mechanisms that determine the response of neural progenitor cells to growth factors and regulate their differentiation into either neurons or astrocytes remain unclear. We found that expression of SOCS2, an intracellular regulator of cytokine signaling, was restricted to mouse progenitor cells and neurons in response to leukemia inhibitory factor (LIF)-like cytokines. Progenitors lacking SOCS2 produced fewer neurons and more astrocytes in vitro, and Socs2(-/-) mice had fewer neurons and neurogenin-1 (Ngn1)-expressing cells in the developing cortex, whereas overexpression of SOCS2 increased neuronal differentiation. We also report that growth hormone inhibited Ngn1 expression and neuronal production, and this action was blocked by SOCS2 overexpression. These findings indicate that SOCS2 promotes neuronal differentiation by blocking growth hormone-mediated downregulation of Ngn1.

Neurogenin1 Defines Zebrafish Cranial Sensory Ganglia Precursors

Cells delaminate from epithelial placodes to form sensory ganglia in the vertebrate head. We describe the formation of cranial neurogenic placodes in the zebrafish, Danio rerio, using bHLH transcription factors as molecular markers. A single neurogenin gene, neurogenin1 ( ngn1), is required for the development of all zebrafish cranial ganglia, which contrasts with other described vertebrates. Expression of ngn1 delineates zebrafish ganglionic placodes, including trigeminal, lateral line, and epibranchial placodes. In addition, ngn1 is expressed in a subset of cells within the otic vesicle that will delaminate to form the octaval (statoacoustic) ganglion. The trigeminal placode is the first to differentiate, and forms just lateral and adjacent to the neural crest. Expression of ngn1 is transient and prefigures expression of a related bHLH transcription factor, neuroD. Interfering with ngn1 function using a specific antisense morpholino oligonucleotide blocks differentiation of all cranial ganglia but not associated glial cells. Lateral line sensory neuromasts develop independently of ngn1 function, suggesting that two derivatives of lateral line placodes, ganglia and migrating primordia, are under separate genetic control.

Interkinetic Nuclear Movement May Provide Spatial Clues to the Regulation of Neurogenesis

During the transition from S phase to mitosis, vertebrate neuroepithelial cells displace their nuclei and subsequently migrate from the basal membrane to the apical surface of the neuroepithelium, a phenomenon termed interkinetic nuclear movement (INM). Here we provide evidence that cycling neuroepithelial cells pass through a neurogenic state in which they are situated apically, as defined by the capacity to express Notch1, Delta1, and Neurogenin2 ( Ngn2). Based on this scenario, we have developed a mathematical model to analyze the influence of INM on neurogenesis. In the absence of INM, the model predicted an increase in the rate of neurogenesis due to the reduction in the influence of inhibitory signals on cells in the neurogenic state. This exacerbation in neurogenesis led to the diminished growth of the neuroepithelium and a reduction in the later production of neurons. Pharmacological perturbation of the stereotypical distribution of precursors along the orthogonal axis of the neuroepithelium resulted in an excess of neurogenesis, as seen by the expression of Ngn2, and of the neuronal marker RA4 in the retina. These findings suggest that INM might be important for the efficient and continued production of neurons in G0, since it is involved in defining a proneural cluster in the ventricular part of the neuroepithelium that contains precursors at stages of the mitotic cycle compatible with neuronal differentiation.

Neurogenin2 elicits the genesis of retinal neurons from cultures of nonneural cells.

neurogenin2 (ngn2) encodes a basic helix-loop-helix transcription factor and plays an important role in neurogenesis from migratory neural crest cells. Its role in retinal development is poorly understood. We observed that in the developing chick retina, ngn2 was expressed in a subpopulation of proliferating progenitor cells. Ectopic expression of ngn2 in nonneural, retinal pigment epithelial cell culture triggered de novo generation of cells that expressed neural-specific markers and exhibited neuronal morphologies. Further molecular and morphological analyses showed that the main products of the induced neurogenesis were cells resembling young photoreceptor cells and cells resembling retinal ganglion cells. The generation of multiple cell types suggests that ngn2 induces various retinal pathways. Thus, unlike in the peripheral nervous system where ngn2 specifies one type of sensory neuron, ngn2 in the retina is likely involved in a common step leading to different cellular pathways. Our finding that ngn2 can instruct nonneural retinal pigment epithelial cells to differentiate toward retinal neurons demonstrates one possible way to induce de novo retinal neurogenesis.

Transcription Factor Expression and Notch-Dependent Regulation of Neural Progenitors in the Adult Rat Spinal Cord

Recent studies have demonstrated that neural stem cells and other progenitors are present in the adult CNS. Details of their properties, however, remain poorly understood. Here we examined the properties and control mechanisms of neural progenitors in the adult rat spinal cord at the molecular level. Adult and embryonic progenitors commonly expressed various homeodomain-type (Pax6, Pax7, Nkx2.2, and Prox1) and basic helix-loop-helix (bHLH)-type (Ngn2, Mash1, NeuroD1, and Olig2) transcriptional regulatory factors in vitro. Unlike their embryonic counterparts, however, adult progenitors could not generate specific neurons that expressed markers appropriate for spinal motoneurons or interneurons, including Islet1, Lim1, Lim3, and HB9. Cells expressing the homeodomain factors Pax6, Pax7, and Nkx2.2 also emerged in vivo in response to injury and were distributed in unique patterns in the lesioned spinal cord. However, neither the expression of the neurogenic bHLH factors including Ngn2, Mash1, and NeuroD1 nor subsequent generation of new neurons could be detected in injured tissue. Our results suggest that signaling through the cell-surface receptor Notch is involved in this restriction. The expression of Notch1 in vivo was enhanced in response to injury. Furthermore, activation of Notch signaling in vitro inhibited differentiation of adult progenitors, whereas attenuation of Notch signals and forced expression of Ngn2 significantly enhanced neurogenesis. These results suggest that both the intrinsic properties of adult progenitors and local environmental signals, including Notch signaling, account for the limited regenerative potential of the adult spinal cord.

Crossinhibitory Activities of Ngn1 and Math1 Allow Specification of Distinct Dorsal Interneurons

Distinct classes of neurons are generated from progenitor cells distributed in characteristic dorsoventral patterns in the developing spinal neural tube. We define restricted neural progenitor populations by the discrete, nonoverlapping expression of Ngn1, Math1, and Mash1. Crossinhibition between these bHLH factors is demonstrated and provides a mechanism for the generation of discrete bHLH expression domains. This precise control of bHLH factor expression is essential for proper neural development since as demonstrated in both loss- and gain-of-function experiments, expression of Math1 or Ngn1 in dorsal progenitor cells determines whether LH2A/B- or dorsal Lim1/2-expressing interneurons will develop. Together, the data suggest that although Math1 and Ngn1 appear to be redundant with respect to neurogenesis, they have distinct functions in specifying neuronal subtype in the dorsal neural tube.

Crossregulation between Neurogenin2 and Pathways Specifying Neuronal Identity in the Spinal Cord

We have examined how genetic pathways that specify neuronal identity and regulate neurogenesis interface in the vertebrate neural tube. Here, we demonstrate that expression of the proneural gene Neurogenin2 ( Ngn2) in the ventral spinal cord results from the modular activity of three enhancers active in distinct progenitor domains, suggesting that Ngn2 expression is controlled by dorsoventral patterning signals. Consistent with this hypothesis, Ngn2 enhancer activity is dependent on the function of Pax6, a homeodomain factor involved in specifying the identity of ventral spinal cord progenitors. Moreover, we show that Ngn2 is required for the correct expression of Pax6 and several homeodomain proteins expressed in defined neuronal populations. Thus, neuronal differentiation involves crossregulatory interactions between a bHLH-driven program of neurogenesis and genetic pathways specifying progenitor and neuronal identity in the spinal cord.

Neurogenin2 Expression in Ventral and Dorsal Spinal Neural Tube Progenitor Cells Is Regulated by Distinct Enhancers

The basic helix-loop-helix transcription factor Neurogenin2 (NGN2) is expressed in distinct populations of neural progenitor cells within the developing central and peripheral nervous systems. Transgenic mice containing ngn2/lacZ reporter constructs were used to study the regulation of ngn2 in the developing spinal cord. ngn2/lacZ transgenic embryos containing sequence found 5′ or 3′ to the ngn2 coding region express lacZ in domains that reflect the spatial and temporal expression profile of endogenous ngn2. A 4.4-kb fragment 5′ of ngn2 was sufficient to drive lacZ expression in the ventral neural tube, whereas a 1.0-kb fragment located 3′ of ngn2 directed expression to both dorsal and ventral domains. Persistent β-gal activity revealed that the NGN2 progenitor cells in the dorsal domain give rise to a subset of interneurons that send their axons to the floor plate, and the NGN2 progenitors in the ventral domain give rise to a subset of motor neurons. We identified a discrete element that is required for the activity of the ngn2 enhancer specifically in the ventral neural tube. Thus, separable regulatory elements that direct ngn2 expression to distinct neural progenitor populations have been defined.

Hes genes regulate sequential stages of neurogenesis in the olfactory epithelium.

We have characterised the functions of the bHLH transcriptional repressors HES1 and HES5 in neurogenesis, using the development of the olfactory placodes in mouse embryos as a model. Hes1 and Hes5 are expressed with distinct patterns in the olfactory placodes and are subject to different regulatory mechanisms. Hes1 is expressed in a broad placodal domain, which is maintained in absence of the neural determination gene Mash1. In contrast, expression of Hes5 is restricted to clusters of neural progenitor cells and requires Mash1 function. Mutations in Hes1 and Hes5 also have distinct consequences on olfactory placode neurogenesis. Loss of Hes1 function leads both to expression of Mash1 outside of the normal domain of neurogenesis and to increased density of MASH1-positive progenitors within this domain, and results in an excess of neurons after a delay. A mutation in Hes5 does not produce any apparent defect. However, olfactory placodes that are double mutant for Hes1 and Hes5 upregulate Ngn1, a neural bHLH gene activated downstream of Mash1, and show a strong and rapid increase in neuronal density. Together, our results suggest that Hes1 regulates Mash1 transcription in the olfactory placode in two different contexts, initially as a prepattern gene defining the placodal domain undergoing neurogenesis and, subsequently, as a neurogenic gene controlling the density of neural progenitors in this domain. Hes5 synergizes with Hes1 and regulates neurogenesis at the level of Ngn1 expression. Therefore, the olfactory sensory neuron lineage is regulated at several steps by negative signals acting through different Hes genes and targeting the expression of different proneural gene homologs.

Expression of zebrafish bHLH genes ngn1 and nrd defines distinct stages of neural differentiation.

Two zebrafish bHLH genes, neurogenin-related gene I (ngn1) and neuroD (nrd), have been isolated. ngn1 expression is initiated at the end of gastrulation in the neural plate and defines broad domains of cells that probably possess an ability to develop as neurons. This finding suggests that ngn1 may play a role during determination of cell fate in neuroblasts. ngn1 and pax-b are expressed in a mutually exclusive manner. nrd expression follows that of ngn1 in restricted populations of cells selected from ngn1-positive clusters of cells. The earliest nrd-positive cells in the brain and the trunk are a subset of the primary neurons. ngn1 is not expressed in the eye. Here, nrd transcription is activated at 25 hours postfertilization in the ventral retina. Expression of islet-1 occurs in nrd-positive cells after expression of nrd, and the expression of the two genes partially overlaps in time. These observations suggest that during eye development nrd expression may follow expression of some other neurodetermination gene(s). This supports the idea that expression of nrd is a necessary step leading toward overt neuronal differentiation.

Neurogenin1 Is Essential for the Determination of Neuronal Precursors for Proximal Cranial Sensory Ganglia

The NEUROGENINS (NGNs) are neural-specific basic helix–loop–helix (bHLH) transcription factors. Mouse embryos lacking ngn1 fail to generate the proximal subset of cranial sensory neurons. ngn1 is required for the activation of a cascade of downstream bHLH factors, including NeuroD, MATH3, and NSCL1. ngn1 is expressed by placodal ectodermal cells and acts prior to neuroblast delamination. Moreover, NGN1 positively regulates the Delta homolog DLL1 and can be negatively regulated by Notch signaling. Thus, ngn1 functions similarly to the proneural genes in Drosophila. However, the initial pattern of ngn1 expression appears to be Notch independent. Taken together with the fact that ectopic ngn1 expression can convert ectodermal cells to neurons in Xenopus (Ma et al. 1996), these data and those of Fode et al. 1998) identify ngns as vertebrate neuronal determination genes, analogous to myoD and myf5 in myogenesis.