Early events in insect neurogenesis. II. The role of cell interactions and cell lineage in the determination of neuronal precursor cells

The insect central nervous system (CNS) is composed of a brain and a chain of segmental ganglia; each hemiganglion contains about 1000 individually identifiable neurons. How is the enormous neuronal diversity and specificity generated? Neurons of a hemiganglion largely arise during embryogenesis from a stereotyped pattern of individually identified neuronal precursor cells, called neuroblasts (NBs). The transition from ectoderm to individual neurons thus involves two major steps: first, an undifferentiated ectodermal cell sheet produces the stereotyped pattern of 30 NBs per hemisegment; second, each of these NBs contributes a specific family of neuronal progeny to the developing CNS. We have used a laser microbeam to ablate individual cells in the grasshopper embryo in order to study the initial events of neuronal determination. In particular, how does a layer of apparently equivalent ectodermal cells produce a highly stereotyped pattern of unique NBs? Our results suggest the following mechanism for NB determination. (1) Cell interactions between the approximately 150 equivalent ectodermal cells of a hemisegment allow 30 cells to enlarge into NBs. (2) As these young NBs enlarge they inhibit adjacent ectodermal cells from becoming NBs; the adjacent cells then either differentiate into nonneuronal support cells or die. (3) Each NB is assigned a unique identity due to its position of enlargement within the neuroepithelium. (4) The NB then generates its characteristic family of neurons by an invariant cell lineage. Development of the insect CNS depends on cell interactions and positional cues to create a pattern of NBs, and then on cell lineage to restrict the fate of the NB progeny.     

Neuronal determination during embryonic development of the grasshopper nervous system

We have examined the roles of cell lineage and interactions in the determination of individual identified neurons in the grasshopper embryo by selective ablations of individual cells and/or their neighbors at successive stages following their birth. The neurons in the grasshopper central nervous system (CNS) are produced by two types of identifiable neuronal precursor cells: neuroblasts (NBs), which generate most of the neurons, and midline precursors (MPs), which generate only a few. NBs divide asymmetrically in a stem cell fashion to generate a chain of ganglion mother cells (GMCs) which then divide once more symmetrically to produce pairs of sibling neurons: MPs cleave once to generate a single pair of sibling neurons. We analyzed the determination of (1) the pair of sibling progeny produced by midline precursor 3 (MP3) and the determination of (2) the pair of sibling progeny produced by the first GMC from neuroblast 1-1 (NB 1-1); in each case the siblings normally differentiate into morphologically distinct neurons. Our results indicate that both pairs of neuronal progeny (1) are born equivalent, (2) become determined by cell interactions early in their development before axonogenesis, and (3) demonstrate a hierarchy of fates with one fate dominant over the other. These results suggest a common pattern of neuronal determination in the grasshopper and possibly all insect embryos.     

Serial specification of diverse neuroblast identities from a neurogenic placode by Notch and Egfr signaling

We used the brain insulin-producing cell (IPC) lineage and its identified neuroblast (IPC NB) as a model to understand a novel example of serial specification of NB identities in the Drosophila dorsomedial protocerebral neuroectoderm. The IPC NB was specified from a small, molecularly identified group of cells comprising an invaginated epithelial placode. By progressive delamination of cells, the placode generated a series of NB identities, including the single IPC NB, a number of other canonical Type I NBs, and a single Type II NB that generates large lineages by transient amplification of neural progenitor cells. Loss of Notch function caused all cells of the placode to form as supernumerary IPC NBs, indicating that the placode is initially a fate equivalence group for the IPC NB fate. Loss of Egfr function caused all placodal cells to apoptose, except for the IPC NB, indicating a requirement of Egfr signaling for specification of alternative NB identities. Indeed, both derepressed Egfr activity in yan mutants and ectopic EGF activity produced supernumerary Type II NBs from the placode. Loss of both Notch and Egfr function caused all placode cells to become IPC NBs and survive, indicating that commitment to NB fate nullified the requirement of Egfr activity for placode cell survival. We discuss the surprising parallels between the serial specification of neural fates from this neurogenic placode and the fly retina.     

Cell lineage and cell fate specification in the embryonic CNS of Drosophila

The Drosophila CNS derives from a population of neural stem cells, called neuroblasts (NBs), which delaminate individually from the neurogenic region of the ectoderm. In the embryonic ventral nerve cord each NB can be uniquely identified and gives rise to a specific lineage consisting of neurons and/or glial cells. This 'NB identity' is dependent on the position of the progenitor cells in the neuroectoderm before delamination. The positional information is provided by the products of segment polarity and dorsoventral (D/V) patterning genes. Subsequently, 'cell fate genes' like huckebein (hkb) and eagle (eg) contribute to the generation of specific NB lineages. These genes act downstream of segment polarity and D/V patterning genes and regulate different processes such as the generation of glial cells and the determination of serotonergic neurons.     

On the roles of Notch, Delta, kuzbanian, and inscuteable during the development of Drosophila embryonic neuroblast lineages

The generation of cellular diversity in the nervous system involves the mechanism of asymmetric cell division. Besides an array of molecules, including the Par protein cassette, a heterotrimeric G protein signalling complex, Inscuteable plays a major role in controlling asymmetric cell division, which ultimately leads to differential activation of the Notch signalling pathway and correct specification of the two daughter cells. In this context, Notch is required to be active in one sibling and inactive in the other. Here, we investigated the requirement of genes previously known to play key roles in sibling cell fate specification such as members of the Notch signalling pathway, e.g., Notch (N), Delta (Dl), and kuzbanian (kuz) and a crucial regulator of asymmetric cell division, inscuteable (insc) throughout lineage progression of 4 neuroblasts (NB1-1, MP2, NB4-2, and NB7-1). Notch-mediated cell fate specification defects were cell-autonomous and were observed in all neuroblast lineages even in cells born from late ganglion mother cells (GMC) within the lineages. We also show that Dl functions non-autonomously during NB lineage progression and clonal cells do not require Dl from within the clone. This suggests that within a NB lineage Dl is dispensable for sibling cell fate specification. Furthermore, we provide evidence that kuz is involved in sibling cell fate specification in the central nervous system. It is cell-autonomously required in the same postmitotic cells which also depend on Notch function. This indicates that KUZ is required to facilitate a functional Notch signal in the Notch-dependent cell for correct cell fate specification. Finally, we show that three neuroblast lineages (NB1-1, NB4-2, and NB7-1) require insc function for sibling cell fate specification in cells born from early GMCs whereas insc is not required in cells born from later GMCs of the same lineages. Thus, there is differential requirement for insc for cell fate specification depending on the stage of lineage progression of NBs.     

The ladybird homeobox genes are essential for the specification of a subpopulation of neural cells

In Drosophila, neurons and glial cells are produced by neural precursor cells called neuroblasts (NBs), which can be individually identified. Each NB generates a characteristic cell lineage specified by a precise spatiotemporal control of gene expression within the NB and its progeny. Here we show that the homeobox genes ladybird early and ladybird late are expressed in subsets of cells deriving from neuroblasts NB 5-3 and NB 5-6 and are essential for their correct development. Our analysis revealed that ladybird in Drosophila, like their vertebrate orthologous Lbx1 genes, play an important role in cell fate specification processes. Among those cells that express ladybird are NB 5-6-derived glial cells. In ladybird loss-of-function mutants, the NB 5-6-derived exit glial cells are absent while overexpression of these genes leads to supernumerary glial cells of this type. Furthermore, aberrant glial cell positioning and aberrant spacing of axonal fascicles in the nerve roots observed in embryos with altered ladybird function suggest that the ladybird genes might also control directed cell movements and cell-cell interactions within the developing Drosophila ventral nerve cord.     

Mitochondrial fusion regulates proliferation and differentiation in the type II neuroblast lineage in Drosophila

Optimal mitochondrial function determined by mitochondrial dynamics, morphology and activity is coupled to stem cell differentiation and organism development. However, the mechanisms of interaction of signaling pathways with mitochondrial morphology and activity are not completely understood. We assessed the role of mitochondrial fusion and fission in the differentiation of neural stem cells called neuroblasts (NB) in the Drosophila brain. Depleting mitochondrial inner membrane fusion protein Opa1 and mitochondrial outer membrane fusion protein Marf in the Drosophila type II NB lineage led to mitochondrial fragmentation and loss of activity. Opa1 and Marf depletion did not affect the numbers of type II NBs but led to a decrease in differentiated progeny. Opa1 depletion decreased the mature intermediate precursor cells (INPs), ganglion mother cells (GMCs) and neurons by the decreased proliferation of the type II NBs and mature INPs. Marf depletion led to a decrease in neurons by a depletion of proliferation of GMCs. On the contrary, loss of mitochondrial fission protein Drp1 led to mitochondrial clustering but did not show defects in differentiation. Depletion of Drp1 along with Opa1 or Marf also led to mitochondrial clustering and suppressed the loss of mitochondrial activity and defects in proliferation and differentiation in the type II NB lineage. Opa1 depletion led to decreased Notch signaling in the type II NB lineage. Further, Notch signaling depletion via the canonical pathway showed mitochondrial fragmentation and loss of differentiation similar to Opa1 depletion. An increase in Notch signaling showed mitochondrial clustering similar to Drp1 mutants. Further, Drp1 mutant overexpression combined with Notch depletion showed mitochondrial fusion and drove differentiation in the lineage, suggesting that fused mitochondria can influence differentiation in the type II NB lineage. Our results implicate crosstalk between proliferation, Notch signaling, mitochondrial activity and fusion as an essential step in differentiation in the type II NB lineage.     

The CNS midline cells and spitz class genes are required for proper patterning of Drosophila ventral neuroectoderm.

The Drosophila embryonic central nervous system (CNS) develops from sets of neuroblasts (NBs) which segregate from the ventral neuroectoderm during early embryogenesis. It is not well established how each individual NB in the neuroectoderm acquires its characteristic identity along the dorsal-ventral axis. Since it is known that CNS midline cells and spitz class genes (pointed, rhomboid, single-minded, spitz and Star) are required for the proper patterning of ventral CNS and epidermis originated from the ventral neuroectoderm, this study was carried out to determine the functional roles of the CNS midline cells and spitz class genes in the fate determination of ventral NBs and formation of mature neurons and their axon pathways. Several molecular markers for the identified NBs, neurons, and axon pathways were employed to examine marker gene expression profile, cell lineage and axon pathway formation in the spitz class mutants. This analysis showed that the CNS midline cells specified by single-minded gene as well as spitz class genes are required for identity determination of a subset of ventral NBs and for formation of mature neurons and their axon pathways. This study suggests that the CNS midline cells and spitz class genes are necessary for proper patterning of the ventral neuroectoderm along the dorsal-ventral axis.     

The embryonic cell lineage of the nematode Rhabditophanes sp

One of the unique features of the model organism Caenorhabditis elegans is its invariant development, where a stereotyped cell lineage generates a fixed number of cells with a fixed cell type. It remains unclear how embryonic development evolved within the nematodes to give rise to the complex, invariant cell lineage of C. elegans. Therefore, we determined the embryonic cell lineage of the nematode, Rhabditophanes sp. (family Alloionematidae) and made detailed cell-by-cell comparison with the known cell lineages of C. elegans, Pellioditis marina and Halicephalobus gingivalis. This gave us a unique data set of four embryonic cell lineages, which allowed a detailed comparison between these cell lineages at the level of each individual cell. This lineage comparison revealed a similar complex polyclonal fate distribution in all four nematode species (85% of the cells have the same fate). It is striking that there is a conservation of a 'C. elegans' like polyclonal cell lineage with strong left-right asymmetry. We propose that an early symmetry-breaking event in nematodes of clade IV-V is a major developmental constraint which shapes their asymmetric cell lineage.     

Early events in insect neurogenesis. I. Development and segmental differences in the pattern of neuronal precursor cells

The earliest events in the development of the central nervous system in insect embryos involve the differentiation of a stereotyped pattern of individually identified neuronal precursor cells, called neuroblasts (NBs), each of which generates a stereotyped family of neuronal progeny. After gastrulation, the midventral region of the ectoderm becomes a neuroepithelium; it is from this sheet of seemingly uniform ectodermal cells that certain cells enlarge to become NBs while the rest of the cells either die or acquire other nonneuronal phenotypes. Here we focus on three aspects of neurogenesis. First, we examine the morphological changes associated with the differentiation of neuronal precursor cells and nonneuronal support cells. The differentiation of each cell type is reflected by its morphology; moreover, the identity of individual cells of certain cell types (e.g., a particular NB) is reflected by their position within the neuroepithelium. Second, we show that there is a characteristic temporal sequence of differentiation in the stereotyped pattern of NBs. Third, we show that this stereotyped pattern of NBs varies in a segment-specific way by the addition or deletion of particular neuronal precursor cells. These studies on the events of early neurogenesis set the stage for the experimental manipulations described in the following paper (C. Q. Doe and C.S. Goodman, 1985, Dev. Biol. 110, 206-219).     

Drosophila homologs of mammalian TNF/TNFR-related molecules regulate segregation of Miranda/Prospero in neuroblasts

During neuroblast (NB) divisions, cell fate determinants Prospero (Pros) and Numb, together with their adaptor proteins Miranda (Mira) and Partner of Numb, localize to the basal cell cortex at metaphase and segregate exclusively to the future ganglion mother cells (GMCs) at telophase. In inscuteable mutant NBs, these basal proteins are mislocalized during metaphase. However, during anaphase/telophase, these mutant NBs can partially correct these earlier localization defects and redistribute cell fate determinants as crescents to the region where the future GMC "buds" off. This compensatory mechanism has been referred to as "telophase rescue". We demonstrate that the Drosophila homolog of the mammalian tumor-necrosis factor (TNF) receptor-associated factor (DTRAF1) and Eiger (Egr), the homolog of the mammalian TNF, are required for telophase rescue of Mira/Pros. DTRAF1 localizes as an apical crescent in metaphase NBs and this apical localization requires Bazooka (Baz) and Egr. The Mira/Pros telophase rescue seen in inscuteable mutant NBs requires DTRAF1. Our data suggest that DTRAF1 binds to Baz and acts downstream of Egr in the Mira/Pros telophase rescue pathway.     

A critical role for cyclin E in cell fate determination in the central nervous system of Drosophila melanogaster

We have examined the process by which cell diversity is generated in neuroblast (NB) lineages in the central nervous system of Drosophila melanogaster. Thoracic NB6-4 (NB6-4t) generates both neurons and glial cells, whereas NB6-4a generates only glial cells in abdominal segments. This is attributed to an asymmetric first division of NB6-4t, localizing prospero (pros) and glial cell missing (gcm) only to the glial precursor cell, and a symmetric division of NB6-4a, where both daughter cells express pros and gcm. Here we show that the NB6-4t lineage represents the ground state, which does not require the input of any homeotic gene, whereas the NB6-4a lineage is specified by the homeotic genes abd-A and Abd-B. They specify the NB6-4a lineage by down-regulating levels of the G1 cyclin, DmCycE (CycE). CycE, which is asymmetrically expressed after the first division of NB6-4t, functions upstream of pros and gcm to specify the neuronal sublineage. Loss of CycE function causes homeotic transformation of NB6-4t to NB6-4a, whereas ectopic CycE induces reverse transformations. However, other components of the cell cycle seem to have a minor role in this process, suggesting a critical role for CycE in regulating cell fate in segment-specific neural lineages.     

bHLH-O proteins are crucial for Drosophila neuroblast self-renewal and mediate Notch-induced overproliferation

Drosophila larval neurogenesis is an excellent system for studying the balance between self-renewal and differentiation of a somatic stem cell (neuroblast). Neuroblasts (NBs) give rise to differentiated neurons and glia via intermediate precursors called GMCs or INPs. We show that E(spl)mγ, E(spl)mβ, E(spl)m8 and Deadpan (Dpn), members of the basic helix-loop-helix-Orange protein family, are expressed in NBs but not in differentiated cells. Double mutation for the E(spl) complex and dpn severely affects the ability of NBs to self-renew, causing premature termination of proliferation. Single mutations produce only minor defects, which points to functional redundancy between E(spl) proteins and Dpn. Expression of E(spl)mγ and m8, but not of dpn, depends on Notch signalling from the GMC/INP daughter to the NB. When Notch is abnormally activated in NB progeny cells, overproliferation defects are seen. We show that this depends on the abnormal induction of E(spl) genes. In fact E(spl) overexpression can partly mimic Notch-induced overproliferation. Therefore, E(spl) and Dpn act together to maintain the NB in a self-renewing state, a process in which they are assisted by Notch, which sustains expression of the E(spl) subset.     

Asymmetric cell division of thoracic neuroblast 6-4 to bifurcate glial and neuronal lineage in Drosophila

In the development of the Drosophila central nervous system, some of the neuroblasts designated as neuroglioblasts generate both glia and neurons. Little is known about how neuroglioblasts produce these different cell types. NB6-4 in the thoracic segment (NB6-4T) is a neuroglioblast, although the corresponding cell in the abdominal segment (NB6-4A) produces only glia. Here, we describe the cell divisions in the NB6-4T lineage, following changes in cell number and cell arrangement. We also examined successive changes in the expression of glial cells missing (gcm) mRNA and protein, activity of which is known to direct glial fate from the neuronal default state. The first cell division of NB6-4T occurred in the medial-lateral orientation, and was found to bifurcate the glial and neuronal lineage. After division, the medial daughter cell expressed GCM protein to produce three glial cells, while the lateral daughter cell with no GCM expression produced ganglion mother cells, secondary precursors of neurons. Although gcm mRNA was present evenly in the cytoplasm of NB6-4T before the first cell division, it became detected asymmetrically in the cell during mitosis and eventually only in the medial daughter cell. In contrast, NB6-4A showed a symmetrical distribution of gcm mRNA and GCM protein through division. Our observations suggest that mechanisms regulating gcm mRNA expression and its translation play an important role in glial and neuronal lineage bifurcation that results from asymmetric cell division.     

Upregulation of Mitimere and Nubbin acts through cyclin E to confer self-renewing asymmetric division potential to neural precursor cells

In the Drosophila CNS, neuroblasts undergo self-renewing asymmetric divisions, whereas their progeny, ganglion mother cells (GMCs), divide asymmetrically to generate terminal postmitotic neurons. It is not known whether GMCs have the potential to undergo self-renewing asymmetric divisions. It is also not known how precursor cells undergo self-renewing asymmetric divisions. Here, we report that maintaining high levels of Mitimere or Nubbin, two POU proteins, in a GMC causes it to undergo self-renewing asymmetric divisions. These asymmetric divisions are due to upregulation of Cyclin E in late GMC and its unequal distribution between two daughter cells. GMCs in an embryo overexpressing Cyclin E, or in an embryo mutant for archipelago, also undergo self-renewing asymmetric divisions. Although the GMC self-renewal is independent of inscuteable and numb, the fate of the differentiating daughter is inscuteable and numb-dependent. Our results reveal that regulation of Cyclin E levels, and asymmetric distribution of Cyclin E and other determinants, confer self-renewing asymmetric division potential to precursor cells, and thus define a pathway that regulates such divisions. These results add to our understanding of maintenance and loss of pluripotential stem cell identity.     

Ectopic Repo suppresses expression of castor gene in Drosophila central nervous system

The ventral nerve cord (VNC) of the Drosophila embryo is derived from neuroblasts (NBs). NBs divide in a stem cell lineage to generate a series of ganglion mother cells (GMCs), each of which divides once to produce a pair of neurons or glial cells. One of the NB genes, castor (cas), is expressed in a subset of NBs and has never been identified in neurons and the peripheral nervous system; cas plays a role in axonogenesis. But its limited expression along the dorsal-ventral axis within the central nervous system has not been investigated yet. In the present study, we examined the expression patterns of both genes using confocal microscopy to determine the effects of repo mutation on cas expression. Cas was mainly expressed in layers different from repo-expressed layers during early embryogenesis: repo was expressed mostly from deep to mid layers, while cas, from mid to superficial layers. Loss-of-function of repo did not result in an ectopic expression of cas, but rather, a scattering of cas-expressing cells. However, repo gain-of-function mutation caused repression of cas. In addition, repo-expressing cells seemed to block the migration of cas-expressing cells.