Neuroblast Identity Research Articles

Control of successive unequal cell divisions by neural cell fate regulators determines embryonic neuroblast cell size.

Asymmetric cell divisions often generate daughter cells of unequal size in addition to different fates. In some contexts, daughter cell size asymmetry is thought to be a key input to specific binary cell fate decisions. An alternative possibility is that unequal division is a mechanism by which a variety of cells of different sizes are generated during embryonic development. We show here that two unequal cell divisions precede neuroblast formation in the C lineage of Caenorhabditis elegans. The equalisation of these divisions in a pig-1/MELK mutant background has little effect on neuroblast specification. Instead, we demonstrate that let-19/MDT13 is a regulator of the proneural basic helix-loop-helix transcription factor hlh-14/ASCL1 and find that both are required to concomitantly regulate the acquisition of neuroblast identity and neuroblast cell size. Thus, embryonic neuroblast cell size in this lineage is progressively regulated in parallel with identity by key neural cell fate regulators. We propose that key cell fate determinants have a previously unappreciated function in regulating unequal cleavage, and therefore cell size, of the progenitor cells whose daughter cell fates they then go on to specify.

Nuclear localization of Goosecoid is positively associated with neuroblast identity during the early development of the chicken inner ear

Nuclear localization of Goosecoid is positively associated with neuroblast identity during the early development of the chicken inner ear

ASC proneural factors are necessary for chromatin remodeling during neuroectodermal to neuroblast fate transition to ensure the timely initiation of the neural stem cell program

BackgroundIn both Drosophila and mammals, the achaete-scute (ASC/ASCL) proneural bHLH transcription factors are expressed in the developing central and peripheral nervous systems, where they function during specification and maintenance of the neural stem cells in opposition to Notch signaling. In addition to their role in nervous system development, ASC transcription factors are oncogenic and exhibit chromatin reprogramming activity; however, the impact of ASC on chromatin dynamics during neural stem cell generation remains elusive. Here, we investigate the chromatin changes accompanying neural commitment using an integrative genetics and genomics methodology.ResultsWe found that ASC factors bind equally strongly to two distinct classes of cis-regulatory elements: open regions remodeled earlier during maternal to zygotic transition by Zelda and less accessible, Zelda-independent regions. Both classes of cis-elements exhibit enhanced chromatin accessibility during neural specification and correlate with transcriptional regulation of genes involved in a variety of biological processes necessary for neuroblast function/homeostasis. We identified an ASC-Notch regulated TF network that includes likely prime regulators of neuroblast function. Using a cohort of ASC target genes, we report that ASC null neuroblasts are defectively specified, remaining initially stalled, unable to divide, and lacking expression of many proneural targets. When mutant neuroblasts eventually start proliferating, they produce compromised progeny. Reporter lines driven by proneural-bound enhancers display ASC dependency, suggesting that the partial neuroblast identity seen in the absence of ASC genes is likely driven by other, proneural-independent, cis-elements. Neuroblast impairment and the late differentiation defects of ASC mutants are corrected by ectodermal induction of individual ASC genes but not by individual members of the TF network downstream of ASC. However, in wild-type embryos, the induction of individual members of this network induces CNS hyperplasia, suggesting that they synergize with the activating function of ASC to consolidate the chromatin dynamics that promote neural specification.ConclusionsWe demonstrate that ASC proneural transcription factors are indispensable for the timely initiation of the neural stem cell program at the chromatin level by regulating a large number of enhancers in the vicinity of neural genes. This early chromatin remodeling is crucial for both neuroblast homeostasis as well as future progeny fidelity.

Dephosphorylation of the Proneural Transcription Factor ASCL1 Re-Engages a Latent Post-Mitotic Differentiation Program in Neuroblastoma.

Pediatric cancers often resemble trapped developmental intermediate states that fail to engage the normal differentiation program, typified by high-risk neuroblastoma arising from the developing sympathetic nervous system. Neuroblastoma cells resemble arrested neuroblasts trapped by a stable but aberrant epigenetic program controlled by sustained expression of a core transcriptional circuit of developmental regulators in conjunction with elevated MYCN or MYC (MYC). The transcription factor ASCL1 is a key master regulator in neuroblastoma and has oncogenic and tumor-suppressive activities in several other tumor types. Using functional mutational approaches, we find that preventing CDK-dependent phosphorylation of ASCL1 in neuroblastoma cells drives coordinated suppression of the MYC-driven core circuit supporting neuroblast identity and proliferation, while simultaneously activating an enduring gene program driving mitotic exit and neuronal differentiation. IMPLICATIONS: These findings indicate that targeting phosphorylation of ASCL1 may offer a new approach to development of differentiation therapies in neuroblastoma. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/12/1759/F1.large.jpg.

Prox1 identifies proliferating neuroblasts and nascent neurons during neurogenesis in sympathetic ganglia

Neurogenesis in embryonic sympathetic ganglia involves neuroblasts that resume proliferation following neuronal differentiation. As cell cycle exit is not associated with neuronal differentiation, the identity of proliferating neuroblasts is incompletely understood. Here, we use sympathetic ganglia of chick embryos to define the timing of neurogenesis and neuroblast identity focusing on the expression and function of the transcription factor Prox1. We show that a large fraction of neuroblasts has initially withdrawn from the cell cycle at embryonic day 3 (E3), which is reflected by a high proportion of p27(+)/Islet1(+) neuroblasts (63%) and low numbers of EdU(+)/Islet1(+) cells (12%). The proportion of proliferating Islet1(+) neuroblasts, identified by EdU pulse labeling and by the absence of the postmitotic marker p27 increases to reach maximal levels at E5, when virtually all neuroblasts are in the cell cycle (95%). Subsequently, the proportion of EdU-labeled and p27(-) neuroblasts is reduced to reach low levels at E11. Interestingly, the expression of the transcription factor Prox1 is restricted to the neuronal lineage, that is, Sox10(+)/Phox2b(+) neuron progenitors, proliferating p27(-)/Islet1(+) neuroblasts and nascent neurons but is rapidly lost in postmitotic neurons. In vitro and in vivo knockdown and overexpression experiments demonstrate effects of Prox1 in the support of neuroblast proliferation and survival. Taken together, these results define the neurogenesis period in the chick paravertebral sympathetic ganglia including an initial cell cycle withdrawal and identify Prox1 as a marker and regulator of proliferating sympathetic neuroblasts.

Drosophila neuroblasts as a new model for the study of stem cell self-renewal and tumour formation.

Drosophila larval brain stem cells (neuroblasts) have emerged as an important model for the study of stem cell asymmetric division and the mechanisms underlying the transformation of neural stem cells into tumour-forming cancer stem cells. Each Drosophila neuroblast divides asymmetrically to produce a larger daughter cell that retains neuroblast identity, and a smaller daughter cell that is committed to undergo differentiation. Neuroblast self-renewal and differentiation are tightly controlled by a set of intrinsic factors that regulate ACD (asymmetric cell division). Any disruption of these two processes may deleteriously affect the delicate balance between neuroblast self-renewal and progenitor cell fate specification and differentiation, causing neuroblast overgrowth and ultimately lead to tumour formation in the fly. In this review, we discuss the mechanisms underlying Drosophila neural stem cell self-renewal and differentiation. Furthermore, we highlight emerging evidence in support of the notion that defects in ACD in mammalian systems, which may play significant roles in the series of pathogenic events leading to the development of brain cancers.

The role of Dichaete in transcriptional regulation during Drosophila embryonic development

BackgroundGroup B Sox domain transcription factors play conserved roles in the specification and development of the nervous system in higher metazoans. However, we know comparatively little about how these transcription factors regulate gene expression, and the analysis of Sox gene function in vertebrates is confounded by functional compensation between three closely related family members. In Drosophila, only two group B Sox genes, Dichaete and SoxN, have been shown to function during embryonic CNS development, providing a simpler system for understanding the functions of this important class of regulators.ResultsUsing a combination of transcriptional profiling and genome-wide binding analysis we conservatively identify over 1000 high confidence direct Dichaete target genes in the Drosophila genome. We show that Dichaete plays key roles in CNS development, regulating aspects of the temporal transcription factor sequence that confer neuroblast identity. Dichaete also shows a complex interaction with Prospero in the pathway controlling the switch from stem cell self-renewal to neural differentiation. Dichaete potentially regulates many more genes in the Drosophila genome and was found to be associated with over 2000 mapped regulatory elements.ConclusionsOur analysis suggests that Dichaete acts as a transcriptional hub, controlling multiple regulatory pathways during CNS development. These include a set of core CNS expressed genes that are also bound by the related Sox2 gene during mammalian CNS development. Furthermore, we identify Dichaete as one of the transcription factors involved in the neural stem cell transcriptional network, with evidence supporting the view that Dichaete is involved in controlling the temporal series of divisions regulating neuroblast identity.

Generation of striatal projection neurons extends into the neonatal period in the rat brain

Substantial advances have been made in the last decade on our understanding of the basic physiology underlying neurogenesis in the postnatal mammalian brain. The bulk of the work in this area has been based on analysis of the adult brain. Relatively less is known about the capacity for neurogenesis in specific structures within the neonatal brain. Here we report that the production of medium spiny striatal projection neurons extends into the early neonatal period under normal physiological conditions in the rat brain. Birth-dating of newborn cells with bromodeoxyuridine at postnatal days 0, 2 and 5 showed a peak production close to birth, which sharply declined at the later time-points. Additionally, there was a low-level but stable contribution of neurons with interneuron identity over the same time-period. Importantly, retroviral labelling of new striatal projection neurons with green fluorescent protein showed long-term survival and terminal differentiation with characteristic morphology, including highly elaborated spiny dendrites, and appropriate axonal targeting of the globus pallidus and midbrain. This latent period of striatal neurogenesis in the early neonatal brain represents an interesting target for regenerative approaches aimed at restoring striatal circuitry in perinatal pathologies, such as hypoxic and ischaemic damage associated with cerebral palsy.

Changes in Notch signaling coordinates maintenance and differentiation of the Drosophila larval optic lobe neuroepithelia

A dynamic balance between stem cell maintenance and differentiation paces generation of post-mitotic progeny during normal development and maintenance of homeostasis. Recent studies show that Notch plays a key role in regulating the identity of neuroepithelial stem cells, which generate terminally differentiated neurons that populate the adult optic lobe via the intermediate progenitor cell type called neuroblast. Thus, understanding how Notch controls neuroepithelial cell maintenance and neuroblast formation will provide critical insight into the intricate regulation of stem cell function during tissue morphogenesis. Here, we showed that a low level of Notch signaling functions to maintain the neuroepithelial cell identity by suppressing the expression of pointedP1 gene through the transcriptional repressor Anterior open. Increased Notch signaling, which coincides with transient cell cycle arrest but precedes the expression of PointedP1 in cells near the medial edge of neuroepithelia, defines transitioning neuroepithelial cells that are in the process of acquiring the neuroblast identity. Transient up-regulation of Notch signaling in transitioning neuroepithelial cells decreases their sensitivity to PointedP1 and prevents them from becoming converted into neuroblasts prematurely. Down-regulation of Notch signaling combined with a high level of PointedP1 trigger a synchronous conversion from transitioning neuroepithelial cells to immature neuroblasts at the medial edge of neuroepithelia. Thus, changes in Notch signaling orchestrate a dynamic balance between maintenance and conversion of neuroepithelial cells during optic lobe neurogenesis.

Drosophilatype II neuroblast lineages keep Prospero levels low to generate large clones that contribute to the adult brain central complex

Tissue homeostasis depends on the ability of stem cells to properly regulate self-renewal versus differentiation. Drosophila neural stem cells (neuroblasts) are a model system to study self-renewal and differentiation. Recent work has identified two types of larval neuroblasts that have different self-renewal/differentiation properties. Type I neuroblasts bud off a series of small basal daughter cells (ganglion mother cells) that each generate two neurons. Type II neuroblasts bud off small basal daughter cells called intermediate progenitors (INPs), with each INP generating 6 to 12 neurons. Type I neuroblasts and INPs have nuclear Asense and cytoplasmic Prospero, whereas type II neuroblasts lack both these transcription factors. Here we test whether Prospero distinguishes type I/II neuroblast identity or proliferation profile, using several newly characterized Gal4 lines. We misexpress prospero using the 19H09-Gal4 line (expressed in type II neuroblasts but no adjacent type I neuroblasts) or 9D11-Gal4 line (expressed in INPs but not type II neuroblasts). We find that differential prospero expression does not distinguish type I and type II neuroblast identities, but Prospero regulates proliferation in both type I and type II neuroblast lineages. In addition, we use 9D11 lineage tracing to show that type II lineages generate both small-field and large-field neurons within the adult central complex, a brain region required for locomotion, flight, and visual pattern memory.

SOX9 induces and maintains neural stem cells

Neural stem cells (NSCs) are uncommitted cells of the CNS defined by their multipotentiality and ability to self renew. We found these cells to not be present in substantial numbers in the CNS until after embryonic day (E) 10.5 in mouse and E5 in chick. This coincides with the induction of SOX9 in neural cells. Gain- and loss-of-function studies indicated that SOX9 was essential for multipotent NSC formation. Moreover, Sonic Hedgehog was able to stimulate precocious generation of NSCs by inducing Sox9 expression. SOX9 was also necessary for the maintenance of multipotent NSCs, as shown by in vivo fate mapping experiments in the adult subependymal zone and olfactory bulbs. In addition, loss of SOX9 led ependymal cells to adopt a neuroblast identity. These data identify a functional link between extrinsic and intrinsic mechanisms of NSCs specification and maintenance, and establish a central role for SOX9 in the process.

Apical/Basal Spindle Orientation Is Required for Neuroblast Homeostasis and Neuronal Differentiation in Drosophila

Precise regulation of stem cell self-renewal/differentiation is essential for embryogenesis and tumor suppression. Drosophila neural progenitors (neuroblasts) align their spindle along an apical/basal polarity axis to generate a self-renewed apical neuroblast and a differentiating basal cell. Here, we genetically disrupt spindle orientation without altering cell polarity to test the role of spindle orientation in self-renewal/differentiation. We perform correlative live imaging of polarity markers and spindle orientation over multiple divisions within intact brains, followed by molecular marker analysis of cell fate. We find that spindle alignment orthogonal to apical/basal polarity always segregates apical determinants into both siblings, which invariably assume a neuroblast identity. Basal determinants can all be localized into one sibling without inducing neuronal differentiation, but overexpression of the basal determinant Prospero can deplete neuroblasts. We conclude that the ratio of apical/basal determinants specifies neuroblast/GMC identity, and that apical/basal spindle orientation is required for neuroblast homeostasis and neuronal differentiation.

Engrailed controls the organization of the ventral nerve cord through frazzled regulation

In Drosophila, the ventral nerve cord (VNC) architecture is built from neuroblasts that are specified during embryonic development, mainly by transcription factors. Here we show that Engrailed, a homeodomain transcription factor known to be involved in the establishment of neuroblast identity, is also directly implicated in the regulation of axonal guidance cues. Posterior commissures (PC) are missing in engrailed mutant embryos, and axonal pathfinding defects are observed when Engrailed is ectopically expressed at early stages, prior to neuronal specification. We also show that frazzled, enabled, and trio, all of which are potential direct targets of Engrailed and are involved in axonal navigation, interact genetically with engrailed to form posterior commissures in the developing VNC. The regulation of frazzled expression in engrailed-expressing neuroblasts contributes significantly to the formation of the posterior commissures by acting on axon growth. Finally, we identified a small genomic fragment within intron 1 of frazzled that can mediate activation by Engrailed in vivo when fused to a GFP reporter. These results indicate that Engrailed's function during the segregation of the neuroblasts is crucial for regulating different actors that are later involved in axon guidance.

The Nk-2 box of the Drosophila homeodomain protein, Vnd, contributes to its repression activity in a Groucho-dependent manner

The transcription factor, Vnd, is a dual regulator that specifies ventral neuroblast identity in Drosophila by both repressing and activating target genes. Vnd and its homologues have a conserved amino acid sequence, the Nk-2 box or Nk specific domain, as well a conserved DNA-binding homeodomain and an EhI-type Groucho interaction domain. However, the function of the conserved Nk-2 box has not been fully defined. To explore its function, we deleted the Nk-2 box and compared the regulatory activity of mutant Vnd in transgenic over-expression assays to that of the wild-type protein. We were unable to assign regulatory activity to the Nk-2 box using an over-expression assay, because the mutant protein activated expression of endogenous Vnd, masking a requirement for the Nk-2 box. However, in transgenic rescue assays, Vnd lacking the Nk-2 box repressed ind expression at 30% lower levels than the wild-type protein. Moreover, in transient transfection assays using Gal4 DNA-binding domain-Vnd chimeras, the repression activity of Vnd lacking the Nk-2 box was compromised. Because Vnd represses target gene expression in conjunction with Groucho, we asked whether the Nk-2 box affects Vnd’s ability to interact with this co-repressor. Vnd lacking the Nk-2 box binds Groucho 30% less efficiently than wild-type Vnd in co-immunoprecipitations. These data suggest that the Nk-2 box contributes to the repression activity of Vnd by stabilizing its interaction with the co-repressor, Groucho.

Functions of the segment polarity genes midline and H15 in Drosophila melanogaster neurogenesis

The Drosophila melanogaster ventral nerve cord derives from neural progenitor cells called neuroblasts. Individual neuroblasts have unique gene expression profiles and give rise to distinct clones of neurons and glia. The specification of neuroblast identity provides a cell intrinsic mechanism which ultimately results in the generation of progeny which are different from each other. Segment polarity genes have a dual function in early neurogenesis: within distinct regions of the neuroectoderm, they are required both for neuroblast formation and for the specification of neuroblast identity. Previous studies of segment polarity gene function largely focused on neuroblasts that arise within the posterior part of the segment. Here we show that the segment polarity gene midline is required for neuroblast formation in the anterior-most part of the segment. Moreover, midline contributes to the specification of anterior neuroblast identity by negatively regulating the expression of Wingless and positively regulating the expression of Mirror. In the posterior-most part of the segment, midline and its paralog, H15, have partially redundant functions in the regulation of the NB marker Eagle. Hence, the segment polarity genes midline and H15 play an important role in the development of the ventral nerve cord in the anterior- and posterior-most part of the segment.

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.

The Drosophila EGF receptor controls the formation and specification of neuroblasts along the dorsal-ventral axis of the Drosophila embryo.

The segmented portion of the Drosophila embryonic central nervous system develops from a bilaterally symmetrical, segmentally reiterated array of 30 unique neural stem cells, called neuroblasts. The first 15 neuroblasts form about 30-60 minutes after gastrulation in two sequential waves of neuroblast segregation and are arranged in three dorsoventral columns and four anteroposterior rows per hemisegment. Each neuroblast acquires a unique identity, based on gene expression and the unique and nearly invariant cell lineage it produces. Recent experiments indicate that the segmentation genes specify neuroblast identity along the AP axis. However, little is known as to the control of neuroblast identity along the DV axis. Here, I show that the Drosophila EGF receptor (encoded by the DER gene) promotes the formation, patterning and individual fate specification of early forming neuroblasts along the DV axis. Specifically, I use molecular markers that identify particular neuroectodermal domains, all neuroblasts or individual neuroblasts, to show that in DER mutant embryos (1) intermediate column neuroblasts do not form, (2) medial column neuroblasts often acquire identities inappropriate for their position, while (3) lateral neuroblasts develop normally. Furthermore, I show that active DER signaling occurs in the regions from which the medial and intermediate neuroblasts will later delaminate. In addition, I demonstrate that the concomitant loss of rhomboid and vein yield CNS phenotypes indistinguishable from DER mutant embryos, even though loss of either gene alone yields minor CNS phenotypes. These results demonstrate that DER plays a critical role during neuroblast formation, patterning and specification along the DV axis within the developing Drosophila embryonic CNS.

Cell-cell signaling during neurogenesis: some answers and many questions.

Development of a multicellular organism requires that cells communicate with each other in order to regulate their growth, organize into tissues and coordinate their function. This cell-cell communication is mediated by signals cells receive (or send) between each other and from the environment. The signaling can be a short range remote signaling (through secreted signaling molecules), contact signaling (via plasma membrane bound molecules, gap junctions) or a long range signaling (through hormones). In this article, I have reviewed the recent advances on the role of cell-cell signaling in the development of the embryonic nervous system of the fruitfly Drosophila melanogaster and discussed some of the open questions raised by these studies. It discusses the contributions of the neurogenic genes Notch and Delta and the signaling pathways controlled by wingless, patched and hedgehog in neuroblast formation, neuroblast identity specification and neuroblast lineage elaboration.

Specification of neuroblast identity in the Drosophila embryonic central nervous system by gooseberry-distal.

The Drosophila central nervous system develops from a segmentally reiterated array of 30 unique neural precursors, called neuroblasts. Each neuroblast goes through a stereotyped cell lineage to produce an invariant clone of neural progeny. It is critical to identify the genes that specify neuroblast identity as these genes control the time of formation, gene expression profile, and cell lineage characteristics of each neuroblast. Here we show that the Pax-type gooseberry-distal gene specifies row 5 neuroblast identity. Initially, four rows of neuroblasts form per segment (1, 3, 5, 7) and gooseberry-distal is expressed in row 5 neuroblasts. By using 10 molecular markers, and by following the number and orientation of neuroblast divisions, we show that lack of gooseberry-distal transforms row 5 neuroblasts into row 3 neuroblasts, whereas ubiquitous gooseberry-distal generates the reciprocal transformation. Thus, gooseberry-distal is necessary and sufficient to specify row 5 neuroblast identity autonomously. The 10 genes coordinately regulated by gooseberry-distal are prime candidates for controlling specific aspects of neuroblast identity.

Heart Development in Drosophila Requires the Segment Polarity Gene wingless

Mesoderm induction has been studied in many systems and some of the factors involved have been identified. Although the heart is mesodermal in origin, the molecular basis of heart development is essentially unknown. The Drosophila heart is a simple tubular structure similar to the early heart tube in vertebrates. The homeobox gene, tinman, has been shown to be crucial for heart formation in Drosophila. Several genes with considerable sequence similarities to tinman are expressed in cardiac primordial tissue of vertebrates and are likely to be required for heart development of higher organisms as well. In addition to transcriptional control factors, heart development might also depend on inductive signals. Here, we demonstrate that the gene wingless ( wg), which is known to specify segmental polarity and neuroblast identity in Drosophila, has a novel role in mesoderm development: wg function is specifically required for heart development. A temperature-sensitive mutation of wg was used to inactivate wg function during precise developmental time periods. Elimination of wg function for a short time period after gastrulation results in the selective loss of heart precursors, without significantly affecting the formation of the body wall or visceral muscles, although some pattern defects are observed. This developmental requirement of wg for cardiac organogenesis is distinct from its function in segmentation and neurogenesis. We conclude that wg signaling is a crucial component of heart formation.