Multipotent neural stem cells generate glial cells of the central complex through transit amplifying intermediate progenitors in Drosophila brain development

The neural stem cells that give rise to the neural lineages of the brain can generate their progeny directly or through transit amplifying intermediate neural progenitor cells (INPs). The INP-producing neural stem cells in Drosophila are called type II neuroblasts, and their neural progeny innervate the central complex, a prominent integrative brain center. Here we use genetic lineage tracing and clonal analysis to show that the INPs of these type II neuroblast lineages give rise to glial cells as well as neurons during postembryonic brain development. Our data indicate that two main types of INP lineages are generated, namely mixed neuronal/glial lineages and neuronal lineages. Genetic loss-of-function and gain-of-function experiments show that the gcm gene is necessary and sufficient for gliogenesis in these lineages. The INP-derived glial cells, like the INP-derived neuronal cells, make major contributions to the central complex. In postembryonic development, these INP-derived glial cells surround the entire developing central complex neuropile, and once the major compartments of the central complex are formed, they also delimit each of these compartments. During this process, the number of these glial cells in the central complex is increased markedly through local proliferation based on glial cell mitosis. Taken together, these findings uncover a novel and complex form of neurogliogenesis in Drosophila involving transit amplifying intermediate progenitors. Moreover, they indicate that type II neuroblasts are remarkably multipotent neural stem cells that can generate both the neuronal and the glial progeny that make major contributions to one and the same complex brain structure.     

Brat is a Miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal

An important question in stem cell biology is how a cell decides to self-renew or differentiate. Drosophila neuroblasts divide asymmetrically to self-renew and generate differentiating progeny called GMCs. Here, we report that the Brain tumor (Brat) translation repressor is partitioned into GMCs via direct interaction with the Miranda scaffolding protein. In brat mutants, another Miranda cargo protein (Prospero) is not partitioned into GMCs, GMCs fail to downregulate neuroblast gene expression, and there is a massive increase in neuroblast numbers. Single neuroblast clones lacking Prospero have a similar phenotype. We conclude that Brat suppresses neuroblast stem cell self-renewal and promotes neuronal differentiation.     

Long-Term Live Cell Imaging and Automated 4D Analysis of Drosophila Neuroblast Lineages

The developing Drosophila brain is a well-studied model system for neurogenesis and stem cell biology. In the Drosophila central brain, around 200 neural stem cells called neuroblasts undergo repeated rounds of asymmetric cell division. These divisions typically generate a larger self-renewing neuroblast and a smaller ganglion mother cell that undergoes one terminal division to create two differentiating neurons. Although single mitotic divisions of neuroblasts can easily be imaged in real time, the lack of long term imaging procedures has limited the use of neuroblast live imaging for lineage analysis. Here we describe a method that allows live imaging of cultured Drosophila neuroblasts over multiple cell cycles for up to 24 hours. We describe a 4D image analysis protocol that can be used to extract cell cycle times and growth rates from the resulting movies in an automated manner. We use it to perform lineage analysis in type II neuroblasts where clonal analysis has indicated the presence of a transit-amplifying population that potentiates the number of neurons. Indeed, our experiments verify type II lineages and provide quantitative parameters for all cell types in those lineages. As defects in type II neuroblast lineages can result in brain tumor formation, our lineage analysis method will allow more detailed and quantitative analysis of tumorigenesis and asymmetric cell division in the Drosophila brain.     

Nuclear Prospero allows one-division potential to neural precursors and post-mitotic status to neurons via opposite regulation of Cyclin E

In Drosophila embryonic CNS, the multipotential stem cells called neuroblasts (NBs) divide by self-renewing asymmetric division and generate bipotential precursors called ganglion mother cells (GMCs). GMCs divide only once to generate two distinct post-mitotic neurons. The genes and the pathways that confer a single division potential to precursor cells or how neurons become post-mitotic are unknown. It has been suggested that the homeodomain protein Prospero (Pros) when localized to the nucleus, limits the stem-cell potential of precursors. Here we show that nuclear Prospero is phosphorylated, where it binds to chromatin. In NB lineages such as MP2, or GMC lineages such as GMC4-2a, Pros allows the one-division potential, as well as the post-mitotic status of progeny neurons. These events are mediated by augmenting the expression of Cyclin E in the precursor and repressing the expression in post-mitotic neurons. Thus, in the absence of Pros, Cyclin E is downregulated in the MP2 cell. Consequently, MP2 fails to divide, instead, it differentiates into one of the two progeny neurons. In progeny cells, Pros reverses its role and augments the downregulation of Cyclin E, allowing neurons to exit the cell cycle. Thus, in older pros mutant embryos Cyclin E is upregulated in progeny cells. These results elucidate a long-standing problem of division potential of precursors and post-mitotic status of progeny cells and how fine-tuning cyclin E expression in the opposite direction controls these fundamental cellular events. This work also sheds light on the post-translational modification of Pros that determines its cytoplasmic versus nuclear localization.     

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.     

Molecular markers for identified neuroblasts and ganglion mother cells in the Drosophila central nervous system.

The first step in generating cellular diversity in the Drosophila central nervous system is the formation of a segmentally reiterated array of neural precursor cells, called neuroblasts. Subsequently, each neuroblast goes through an invariant cell lineage to generate neurons and/or glia. Using molecular lineage markers, I show that (1) each neuroblast forms at a stereotyped time and position; (2) the neuroblast pattern is indistinguishable between thoracic and abdominal segments; (3) the development of individual neuroblasts can be followed throughout early neurogenesis; (4) gene expression in a neuroblast can be reproducibly modulated during its cell lineage; (5) identified ganglion mother cells form at stereotyped times and positions; and (6) the cell lineage of four well-characterized neurons can be traced back to two identified neuroblasts. These results set the stage for investigating neuroblast specification and the mechanisms controlling neuroblast cell lineages.     

The bHLH Repressor Deadpan Regulates the Self-renewal and Specification of Drosophila Larval Neural Stem Cells Independently of Notch

Neural stem cells (NSCs) are able to self-renew while giving rise to neurons and glia that comprise a functional nervous system. However, how NSC self-renewal is maintained is not well understood. Using the Drosophila larval NSCs called neuroblasts (NBs) as a model, we demonstrate that the Hairy and Enhancer-of-Split (Hes) family protein Deadpan (Dpn) plays important roles in NB self-renewal and specification. The loss of Dpn leads to the premature loss of NBs and truncated NB lineages, a process likely mediated by the homeobox protein Prospero (Pros). Conversely, ectopic/over-expression of Dpn promotes ectopic self-renewing divisions and maintains NB self-renewal into adulthood. In type II NBs, which generate transit amplifying intermediate neural progenitors (INPs) like mammalian NSCs, the loss of Dpn results in ectopic expression of type I NB markers Asense (Ase) and Pros before these type II NBs are lost at early larval stages. Our results also show that knockdown of Notch leads to ectopic Ase expression in type II NBs and the premature loss of type II NBs. Significantly, dpn expression is unchanged in these transformed NBs. Furthermore, the loss of Dpn does not inhibit the over-proliferation of type II NBs and immature INPs caused by over-expression of activated Notch. Our data suggest that Dpn plays important roles in maintaining NB self-renewal and specification of type II NBs in larval brains and that Dpn and Notch function independently in regulating type II NB proliferation and specification.     

The role of centrosomes and astral microtubules during asymmetric division of Drosophila neuroblasts

Drosophila neuroblasts are stem cells that divide asymmetrically to produce another large neuroblast and a smaller ganglion mother cell (GMC). During neuroblast division, several cell fate determinants, such as Miranda, Prospero and Numb, are preferentially segregated into the GMC, ensuring its correct developmental fate. The accurate segregation of these determinants relies on proper orientation of the mitotic spindle within the dividing neuroblast, and on the correct positioning of the cleavage plane. In this study we have analyzed the role of centrosomes and astral microtubules in neuroblast spindle orientation and cytokinesis. We examined neuroblast division in asterless (asl) mutants, which, although devoid of functional centrosomes and astral microtubules, form well-focused anastral spindles that undergo anaphase and telophase. We show that asl neuroblasts assemble a normal cytokinetic ring around the central spindle midzone and undergo unequal cytokinesis. Thus, astral microtubules are not required for either signaling or positioning cytokinesis in Drosophila neuroblasts. Our results indicate that the cleavage plane is dictated by the positioning of the central spindle midzone within the cell, and suggest a model on how the central spindle attains an asymmetric position during neuroblast mitosis. We have also analyzed the localization of Miranda during mitotic division of asl neuroblasts. This protein accumulates in morphologically regular cortical crescents but these crescents are mislocalized with respect to the spindle orientation. This suggests that astral microtubules mediate proper spindle rotation during neuroblast division.     

Combinatorial expression of Prospero,Seven-up,and Elav identifies progenitor cell types during sense-organ differentiation in the Drosophila antenna

The Drosophila antenna has a diversity of chemosensory organs within a single epidermal field. We have some idea from recent studies of how the three broad categories of sense-organs are specified at the level of progenitor choice. However, little is known about how cell fates within single sense-organs are specified. Selection of individual primary olfactory progenitors is followed by organization of groups of secondary progenitors, which divide in a specific order to form a differentiated sensillum. The combinatorial expression of Prospero Elav, and Seven-up allows us to distinguish three secondary progenitor fates. The lineages of these cells have been established by clonal analysis and marker distribution following mitosis. High Notch signaling and the exclusion of these markers identifies PIIa; this cell gives rise to the shaft and socket. The sheath/neuron lineage progenitor PIIb, expresses all three markers; upon division, Prospero asymmetrically segregates to the sheath cell. In the coeloconica, PIIb undergoes an additional division to produce glia. PIIc is present in multiinnervated sense-organs and divides to form neurons. An understanding of the lineage and development of olfactory sense-organs provides a handle for the analysis of how olfactory neurons acquire distinct terminal fates.     

Embryonic development of the insect central complex: insights from lineages in the grasshopper and Drosophila

The neurons of the insect brain derive from neuroblasts which delaminate from the neuroectoderm at stereotypic locations during early embryogenesis. In both grasshopper and Drosophila, each developing neuroblast acquires an intrinsic capacity for neuronal proliferation in a cell autonomous manner and generates a specific lineage of neural progeny which is nearly invariant and unique. Maps revealing numbers and distributions of brain neuroblasts now exist for various species, and in both grasshopper and Drosophila four putatively homologous neuroblasts have been identified whose progeny direct axons to the protocerebral bridge and then to the central body via an equivalent set of tracts. Lineage analysis in the grasshopper nervous system reveals that the progeny of a neuroblast maintain their topological position within the lineage throughout embryogenesis. We have taken advantage of this to study the pioneering of the so-called w, x, y, z tracts, to show how fascicle switching generates central body neuroarchitecture, and to evaluate the roles of so-called intermediate progenitors as well as programmed cell death in shaping lineage structure. The novel form of neurogenesis involving intermediate progenitors has been demonstrated in grasshopper, Drosophila and mammalian cortical development and may represent a general strategy for increasing brain size and complexity. An analysis of gap junctional communication involving serotonergic cells reveals an intrinsic cellular organization which may relate to the presence of such transient progenitors in central complex lineages.     

Asymmetric localisation of Miranda and its cargo proteins during neuroblast division requires the anaphase-promoting complex/cyclosome

Asymmetric cell divisions generate cell fate diversity during both invertebrate and vertebrate development. Drosophila neural progenitors or neuroblasts (NBs) each divide asymmetrically to produce a larger neuroblast and a smaller ganglion mother cell (GMC). The asymmetric localisation of neural cell fate determinants and their adapter proteins to the neuroblast cortex during mitosis facilitates their preferential segregation to the GMC upon cytokinesis. In this study we report a novel role for the anaphase-promoting complex/cyclosome (APC/C) during this process. Attenuation of APC/C activity disrupts the asymmetric localisation of the adapter protein Miranda and its associated cargo proteins Staufen, Prospero and Brat, but not other components of the asymmetric division machinery. We demonstrate that Miranda is ubiquitylated via its C-terminal domain; removal of this domain disrupts Miranda localisation and replacement of this domain with a ubiquitin moiety restores normal asymmetric Miranda localisation. Our results demonstrate that APC/C activity and ubiquitylation of Miranda are required for the asymmetric localisation of Miranda and its cargo proteins to the NB cortex.