The pattern of proliferation of the neuroblasts in the wild-type embryo of Drosophila melanogaster

Summary The pattern of neuroblast divisions was studied in thoracic and abdominal neuromeres of wild-type Drosophila melanogaster embryos stained with a monoclonal antibody directed against a chromatin-associated antigen. Since fixed material was used, our conclusions are based upon the statistical evaluation of a large number of accurately staged embryos, covering the stages between the formation of the cephalic furrow up to shortened germ band. Our observations point to a rather stereotypic pattern of proliferation, consisting of several parasynchronous cycles of division. The data suggest that all SI neuroblasts divide at least eight times, all SII neuroblasts six or seven times and all SIII neuroblasts at least five times. This conclusion is based on the mapping of mitotic neuroblasts and is supported by the progressive reduction of the neuroblast volume and by the results of cell countings performed on embryos of increasing age. No conclusive evidence was obtained concerning the fate of the neuroblasts after their last mitosis, i.e. it cannot be decided whether the neuroblasts degenerate or become incorporated as inconspicuous cells in the larval ventral cord. The duration of the cycles of division of the neuroblasts was found to be 40–50 min each, while in the case of ganglion mother cells about 100 min are required to complete one cell cycle.     

Caenorhabditis elegans anillin (ani-1) regulates neuroblast cytokinesis and epidermal morphogenesis during embryonic development

The formation of tissues is essential for metazoan development. During Caenorhabditis elegans embryogenesis, ventral epidermal cells migrate to encase the ventral surface of the embryo in a layer of epidermis by a process known as ventral enclosure. This process is regulated by guidance cues secreted by the underlying neuroblasts. However, since the cues and their receptors are differentially expressed in multiple cell types, the role of the neuroblasts in ventral enclosure is not fully understood. Furthermore, although F-actin is required for epidermal cell migration, it is not known if nonmuscle myosin is also required. Anillin (ANI-1) is an actin and myosin-binding protein that coordinates actin–myosin contractility in the early embryo. Here, we show that ANI-1 localizes to the cleavage furrows of dividing neuroblasts during mid-embryogenesis and is required for their division. Embryos depleted of ani-1 display a range of ventral enclosure phenotypes, where ventral epidermal cells migrate with similar speeds to control embryos, but contralateral neighbors often fail to meet and are misaligned. The ventral enclosure phenotypes in ani-1 RNAi embryos suggest that the position or shape of neuroblasts is important for directing ventral epidermal cell migration, although does not rule out an autonomous requirement for ani-1 in the epidermal cells. Furthermore, we show that rho-1 and other regulators of nonmuscle myosin activity are required for ventral epidermal cell migration. Interestingly, altering nonmuscle myosin contractility alleviates or strengthens ani-1's ventral enclosure phenotypes. Our findings suggest that ventral enclosure is a complex process that likely relies on inputs from multiple tissues.     

Cell cycle independent role of Cyclin E during neural cell fate specification in Drosophila is mediated by its regulation of Prospero function

During development, neural progenitor cells or neuroblasts generate a great intra- and inter-segmental diversity of neuronal and glial cell types in the nervous system. In thoracic segments of the embryonic central nervous system of Drosophila, the neuroblast NB6-4t undergoes an asymmetric first division to generate a neuronal and a glial sublineage, while abdominal NB6-4a divides once symmetrically to generate only 2 glial cells. We had earlier reported a critical function for the G1 cyclin, CyclinE (CycE) in regulating asymmetric cell division in NB6-4t. Here we show that (i) this function of CycE is independent of its role in cell cycle regulation and (ii) the two functions are mediated by distinct domains at the protein level. Results presented here also suggest that CycE inhibits the function of Prospero and facilitates its cortical localization, which is critical for inducing stem cell behaviour, i.e. asymmetric cell division of NB6-4t. Furthermore our data imply that CycE is required for the maintenance of stem cell identity of most other neuroblasts.     

Embryonic development of the pars intercerebralis/central complex of the grasshopper

 We have studied the embryonic development of the pars intercerebralis/central complex in the brain of the grasshopper using immunocytochemical and histochemical techniques. Expression of the cell-surface antigen lachesin reveals that the neuroblasts of the pars intercerebralis first differentiate from the neuroectoderm at around 26% of embryogenesis. Differentiation of medial and lateral neuroblasts occurs first. By the 28% stage a more or less uniform sheet of 20 neuroblasts has formed. As a result of both cell proliferation and cell translocation, the pars intercerebralis proliferative cluster in each hemisphere expands so that at 30% the most medial neuroblasts lie apposed at the midline. We followed the further development of the pars intercerebralis of each brain hemisphere using bromo-deoxy-uridine incorporation and osmium-ethyl-gallate staining. Within the pars intercerebralis itself, the neuroblasts redistribute into discrete subsets. The neuroblasts of each subset generate clusters of progeny which extend in a stereotypic, subset-specific direction in the brain. We have used this feature to identify one subset of four neuroblasts as being the likely progenitor cells for four clusters of embryonic neurons (W, X, Y, Z) which develop at around 55% of embryogenesis. We show that these progeny project axons via four discrete fascicles (w, x, y, z) into the embryonic central complex. At the single cell level, Golgi impregnation reveals that the axons from these neighbouring cell clusters remain discrete, and those from the same cluster tightly fasciculated, as they project into the central complex, consistent with a modular organization for this brain region. Received: 16 June 1997 / Accepted: 25 June 1997     

Development of neural lineages derived from the sine oculis positive eye field of Drosophila

The Anlage of the Drosophila visual system, called eye field, comprises a domain in the dorso-medial neurectoderm of the embryonic head and is defined by the expression of the early eye gene sine oculis (so). Beside the eye and optic lobe, the eye field gives rise to several neuroblasts that contribute their lineages to the central brain. Since so expression is only very short lived, the later development of these neuroblasts has so far been elusive. Using the P-element replacement technique [Genetics, 151 (1999) 1093] we generated a so-Gal4 line driving the reporter gene LacZ that perdures in the eye field derived cells throughout embryogenesis and into the larval period. This allowed us to reconstruct the morphogenetic movements of the eye field derived lineages, as well as the projection pattern of their neurons. The eye field produces a dorsal (Pc1/2) and a ventral (Pp3) group of three to four neuroblasts each. In addition, the target neurons of the larval eye, the optic lobe pioneers (OLPs) are derived from the eye field. The embryonically born (primary) neurons of the Pp3 lineages spread out at the inner surface of the optic lobe. Together with the OLPs, their axons project to the dorsal neuropile of the protocerebrum. Pp3 neuroblasts reassume expression of so-Gal4 in the larval period and produce secondary neurons whose axonal projection coincides with the pattern formed by the primary Pp3 neurons. Several other small clusters of neurons that originate from outside the eye field, but have axonal connections to the dorsal protocerebrum, also express so and are labeled by so-Gal4 driven LacZ. We discuss the dynamic pattern of the so-positive lineages as a tool to reconstruct the morphogenesis of the larval brain.     

Actin modulation of a MARCKS phosphorylation site located outside the effector domain

MARCKS (Myristoylated alanine-rich C kinase substrate) is a ubiquitous actin regulating protein, especially abundant in the nervous system. This protein may be phosphorylated by other enzymes, particularly by proline-directed kinases, at serine and threonine residues located at different sites along its chain. We demonstrate here that the phosphorylation of chick MARCKS at serine 25, which only takes place in the nervous tissue, does not impair its association with particular plasma membrane regions such as the “detergent resistant microdomains” that also contain actin. This phosphorylated form of MARCKS is able to bind actin, and the integrity of actin filaments in cells (retina neuroblasts) is a necessary condition to sustain this phosphorylation. Taken together, these results indicate the existence of a functional interaction between actin filaments and MARCKS in cells, and particularly of an action in maintaining a phosphorylation in a region of the N-terminal moiety of MARCKS.     

Where does asymmetry come from? Illustrating principles of polarity and asymmetry establishment in Drosophila neuroblasts

Asymmetric cell division (ACD) is the fundamental process through which one cell divides into two cells with different fates. In animals, it is crucial for the generation of cell-type diversity and for stem cells, which use ACD both to self-renew and produce one differentiating daughter cell. One of the most prominent model systems of ACD, Drosophila neuroblasts, relies on the PAR complex, a conserved set of proteins governing cell polarity in animals. Here, we focus on recent advances in our understanding of the mechanisms that control the orientation of the neuroblast polarity axis, how the PAR complex is positioned, and how its activity may regulate division orientation and cell fate determinant localization and discuss how important findings about the composition polarity complexes in other models may apply to neuroblasts.     

Proliferative activity in the cerebellar external granular layer evaluated by bromodeoxyuridine labeling

We have optimised an indirect immunoperoxidase technique demonstrating bromodeoxyuridine (BrdU) incorporation into dividing cells for cerebellar tissue sections of four-day-old rats injected with this marker. This permits confident identification of granule-cell precursors engaged in DNA synthesis in the external granular layer of the developing cerebellum. Preservation of BrdU immunoreactivity is attained using methanol/acetic acid fixation and different pretreatments before immunostaining, while unlabeled nuclei can be recognized clearly after Feulgen or hematoxylin counterstaining. We established conditions to ensure satisfactory BrdU uptake without affecting cell-cycle progression during the postlabeling time period. The dose of BrdU employed provides saturation S-phase labeling from at least 1 h after BrdU delivery. Various kinetic parameters and phase durations have been determined in experiments involving a single injection or cumulative labeling sequences, and the cycle time was calculated based on two models of generative behavior: steady-state and exponential growth. The working hypothesis of steadystate kinetics can be adopted successfully if the existence of neuroblasts with different proliferation rates is taken into account.     

Mutually exclusive expression of sex-specific and non-sex-specific fruitless gene products in the Drosophila central nervous system

The fruitless gene of Drosophila produces multiple protein isoforms, which are classified into two major classes, sex-specific Fru proteins (FruM) and non-sex specific proteins (FruCOM). Whereas FruM proteins are expressed in ∼2000 neurons to masculinize their structure and function, little is known about FruCOM's roles. As an attempt to obtain clues to the roles of FruCOM, we compared expression patterns of FruCOM and FruM in the central nervous system at the late larval stage. We found that nearly all neuroblasts express FruCOM but not FruM, whereas a subset of ganglion mother cells and differentiated neurons express FruM but not FruCOM. It is inferred that FruCOM proteins support fundamental stem cell functions, contrasting to FruM proteins, which play major roles in sex-specific differentiation of neurons.     

Expression, regulation and function of the homeobox gene empty spiracles in brain and ventral nerve cord development of Drosophila

We analyse the role of the empty spiracles (ems) gene in embryonic brain and ventral nerve cord development. ems is differentially expressed in the neurectoderm of the anterior head versus the trunk region of early embryos. A distal enhancer region drives expression in the deutocerebral brain anlage and a proximal enhancer region drives expression in the VNC and tritocerebral brain anlage. Mutant analysis indicates that in the anterior brain ems is necessary for regionalized neurogenesis in the deutocerebral and tritocerebral anlagen. In the posterior brain and VNC ems is necessary for correct axonal pathfinding of specific interneurons. Rescue experiments indicate that the murine Emx2 gene can partially replace the fly ems gene in CNS development.