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.     

From epithelium to neuroblasts to neurons: the role of cell interactions and cell lineage during insect neurogenesis

The grasshopper central nervous system is composed of a brain and a chain of segmental ganglia. Each hemiganglion contains about 1000 neurons, most of which can be individually identified by their unique morphology and synaptic connectivity. Shortly after gastrulation the ventral ectoderm becomes a neurogenic region. In each hemisegment, ca. 150 neurogenic ectodermal cells (nECs) give rise to a stereotyped pattern of 30 identified neuroblasts (NBs, neuronal stem cells); the remaining nECs become various non-neuronal cells or die. The 30 NBs then give rise to about 1000 neurons as each NB initiates an invariant lineage, generating a stereotyped chain of ganglion mother cells (GMCs), each of which in turn divides once to generate two identified neurons. We have used a laser microbeam or microelectrode to ablate individual cells in ovo and in vitro at various stages of embryogenesis to study how neuronal diversity and specificity are generated during development. Our results suggest that cell interactions between ca. 150 equivalent nECs allow 30 cells to enlarge into NBs, the dominant fate in a hierarchy; the NBs inhibit adjacent nECs and thus cause them to differentiate into various non-neuronal cells; each NB is assigned its unique identity according to its position of enlargement within the neurogenic epithelium; each NB then generates its characteristic chain of GMCs by an invariant cell lineage; and each GMC generates a pair of equivalent progeny, the fate of each individual neuron being determined by both its GMC of origin and interactions with its sibling.     

A targeted genetic screen identifies crucial players in the specification of the Drosophila abdominal Capaergic neurons

The central nervous system contains a wide variety of neuronal subclasses generated by neural progenitors. The achievement of a unique neural fate is the consequence of a sequence of early and increasingly restricted regulatory events, which culminates in the expression of a specific genetic combinatorial code that confers individual characteristics to the differentiated cell. How the earlier regulatory events influence post-mitotic cell fate decisions is beginning to be understood in the Drosophila NB 5-6 lineage. However, it remains unknown to what extent these events operate in other lineages. To better understand this issue, we have used a very highly specific marker that identifies a small subset of abdominal cells expressing the Drosophila neuropeptide Capa: the ABCA neurons. Our data support the birth of the ABCA neurons from NB 5-3 in a cas temporal window in the abdominal segments A2-A4. Moreover, we show that the ABCA neuron has an ABCA-sibling cell which dies by apoptosis. Surprisingly, both cells are also generated in the abdominal segments A5-A7, although they undergo apoptosis before expressing Capa. In addition, we have performed a targeted genetic screen to identify players involved in ABCA specification. We have found that the ABCA fate requires zfh2, grain, Grunge and hedgehog genes. Finally, we show that the NB 5-3 generates other subtype of Capa-expressing cells (SECAs) in the third suboesophageal segment, which are born during a pdm/cas temporal window, and have different genetic requirements for their specification.     

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.     

Integrating bits and pieces: synapse structure and formation in Drosophila embryos

During the development of the nervous system, numerous neurons connect to form complex networks. In order to build a functional network each neuron has to establish contacts with appropriate target cells, and at these contacts synapses of the right quality and strength have to be formed. Gaining insight into the mechanisms underlying this complex development is an important step towards a better understanding of how the nervous system is formed and behaviour generated. One model system in which synapse formation can be studied at the morphological, physiological and molecular level is that of the fruitfly Drosophila, and insights gained from Drosophila embryos are reviewed here. The first part of this review deals with the neuromuscular junction as the best-known synaptic contact in Drosophila. It describes: (1) its structure, (2) mechanisms underlying the formation of the neuromuscular cell junction and the arborisation of the presynaptic terminal, and (3) our present understanding of signal-dependent and -independent processes during synapse formation at the neuromuscular junction. The last part of this review deals with the question of how particular neurons can adopt specific synaptic properties, stating as an example the development of the neural lineage of NB7-3, which gives rise to two serotonergic neurons.     

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.     

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.     

Successive specification of Drosophila neuroblasts NB 6-4 and NB 7-3 depends on interaction of the segment polarity genes wingless, gooseberry and naked cuticle

The Drosophila central nervous system derives from neural precursor cells, the neuroblasts (NBs), which are born from the neuroectoderm by the process of delamination. Each NB has a unique identity, which is revealed by the production of a characteristic cell lineage and a specific set of molecular markers it expresses. These NBs delaminate at different but reproducible time points during neurogenesis (S1-S5) and it has been shown for early delaminating NBs (S1/S2) that their identities depend on positional information conferred by segment polarity genes and dorsoventral patterning genes. We have studied mechanisms leading to the fate specification of a set of late delaminating neuroblasts, NB 6-4 and NB 7-3, both of which arise from the engrailed (en) expression domain, with NB 6-4 delaminating first. In contrast to former reports, we did not find any evidence for a direct role of hedgehog in the process of NB 7-3 specification. Instead, we present evidence to show that the interplay of the segmentation genes naked cuticle (nkd) and gooseberry (gsb), both of which are targets of wingless (wg) activity, leads to differential commitment to NB 6-4 and NB 7-3 cell fate. In the absence of either nkd or gsb, one NB fate is replaced by the other. However, the temporal sequence of delamination is maintained, suggesting that formation and specification of these two NBs are under independent control.     

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.     

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.     

Control of neuronal cell fate and number by integration of distinct daughter cell proliferation modes with temporal progression

During neural lineage progression, differences in daughter cell proliferation can generate different lineage topologies. This is apparent in the Drosophila neuroblast 5-6 lineage (NB5-6T), which undergoes a daughter cell proliferation switch from generating daughter cells that divide once to generating neurons directly. Simultaneously, neural lineages, e.g. NB5-6T, undergo temporal changes in competence, as evidenced by the generation of different neural subtypes at distinct time points. When daughter proliferation is altered against a backdrop of temporal competence changes, it may create an integrative mechanism for simultaneously controlling cell fate and number. Here, we identify two independent pathways, Prospero and Notch, which act in concert to control the different daughter cell proliferation modes in NB5-6T. Altering daughter cell proliferation and temporal progression, individually and simultaneously, results in predictable changes in cell fate and number. This demonstrates that different daughter cell proliferation modes can be integrated with temporal competence changes, and suggests a novel mechanism for coordinately controlling neuronal subtype numbers.     

Mechanism of glia-neuron cell-fate switch in the Drosophila thoracic neuroblast 6-4 lineage

During development of the Drosophila central nervous system, neuroblast 6-4 in the thoracic segment (NB6-4T) divides asymmetrically into a medially located glial precursor cell and a laterally located neuronal precursor cell. In this study, to understand the molecular basis for this glia-neuron cell-fate decision, we examined the effects of some known mutations on the NB6-4T lineage. First, we found that prospero (pros) mutations led to a loss of expression of Glial cells missing, which is essential to trigger glial differentiation, in the NB6-4T lineage. In wild-type embryos, Pros protein was localized at the medial cell cortex of dividing NB6-4T and segregated to the nucleus of the glial precursor cell. miranda and inscuteable mutations altered the behavior of Pros, resulting in failure to correctly switch the glial and neuronal fates. Our results suggested that NB6-4T used the same molecular machinery in the asymmetric cell division as other neuroblasts in cell divisions producing ganglion mother cells. Furthermore, we showed that outside the NB6-4T lineage most glial cells appeared independently of Pros.     

Drosophila Polycomb complexes restrict neuroblast competence to generate motoneurons

Similar to mammalian neural progenitors, Drosophila neuroblasts progressively lose competence to make early-born neurons. In neuroblast 7-1 (NB7-1), Kruppel (Kr) specifies the third-born U3 motoneuron and Kr misexpression induces ectopic U3 cells. However, competence to generate U3 cells is limited to early divisions, when the Eve(+) U motoneurons are produced, and competence is lost when NB7-1 transitions to making interneurons. We have found that Polycomb repressor complexes (PRCs) are necessary and sufficient to restrict competence in NB7-1. PRC loss of function extends the ability of Kr to induce U3 fates and PRC gain of function causes precocious loss of competence to make motoneurons. PRCs also restrict competence to make HB9(+) Islet(+) motoneurons in another neuroblast that undergoes a motoneuron-to-interneuron transition, NB3-1. In contrast to the regulation of motoneuron competence, PRC activity does not affect the production of Eve(+) interneurons by NB3-3, HB9(+) Islet(+) interneurons by NB7-3, or Dbx(+) interneurons by multiple neuroblasts. These findings support a model in which PRCs establish motoneuron-specific competence windows in neuroblasts that transition from motoneuron to interneuron production.     

Sloppy paired acts as the downstream target of wingless in the Drosophila CNS and interaction between sloppy paired and gooseberry inhibits sloppy paired during neurogenesis

Wingless (Wg) and other Wnt proteins play a crucial role in a number of developmental decisions in a variety of organisms. In the ventral nerve cord of the Drosophila embryo, Wg is non-autonomously required for the formation and specification of a neuronal precursor cell, NB4-2. NB4-2 gives rise to a well-studied neuronal lineage, the RP2/sib lineage. While the various components of the Wg-signaling pathway are also required for generating NB4-2, the target gene(s) of this pathway in the signal-receiving cell is not known. In this paper, we show that sloppy paired 1 and sloppy paired 2 function as the downstream targets of the Wg signaling to generate the NB4-2 cell. Thus, while the loss-of-function mutations in wg and slp have the same NB4-2 formation and specification defects, these defects in wg mutants can be rescued by expressing slp genes from a heterologous promoter. That slp genes function downstream of the Wg signaling is also indicated by the result that expression of slp genes is lost from the neuroectoderm in wg mutants and that ectopic expression of wg induces ectopic expression of slp. Finally, previous results show that Gooseberry (Gsb) prevents Wg from specifying NB4-2 identity to the wg-expressing NB5-3. In this paper, we also show that gsb interacts with slp and prevents Slp from specifying NB4-2 identity. Overexpression of slp overcomes this antagonistic interaction and respecifies NB5-3 as NB4-2. This respecification, however, can be suppressed by a simultaneous overexpression of gsb at high levels. This mechanism appears to be responsible for specifying NB5-3 identity to a row 5 neuroblast and preventing Wg from specifying NB4-2 identity to that cell.