Magnus Baumgardt

Segment-specific neuronal subtype specification by the integration of anteroposterior and temporal cues.

To address the question of how neuronal diversity is achieved throughout the CNS, this study provides evidence of modulation of neural progenitor cell “output” along the body axis by integration of local anteroposterior and temporal cues.

Global programmed switch in neural daughter cell proliferation mode triggered by a temporal gene cascade.

During central nervous system (CNS) development, progenitors typically divide asymmetrically, renewing themselves while budding off daughter cells with more limited proliferative potential. Variation in daughter cell proliferation has a profound impact on CNS development and evolution, but the underlying mechanisms remain poorly understood. We find that Drosophila embryonic neural progenitors (neuroblasts) undergo a programmed daughter proliferation mode switch, from generating daughters that divide once (type I) to generating neurons directly (type 0). This typeI>0 switch is triggered by activation of Dacapo (mammalian p21(CIP1)/p27(KIP1)/p57(Kip2)) expression in neuroblasts. In the thoracic region, Dacapo expression is activated by the temporal cascade (castor) and the Hox gene Antennapedia. In addition, castor, Antennapedia, and the late temporal gene grainyhead act combinatorially to control the precise timing of neuroblast cell-cycle exit by repressing Cyclin E and E2f. This reveals a logical principle underlying progenitor and daughter cell proliferation control in the Drosophila CNS.

Seven up acts as a temporal factor during two different stages of neuroblast 5-6 development

Drosophila embryonic neuroblasts generate different cell types at different time points. This is controlled by a temporal cascade of Hb→Kr→Pdm→Cas→Grh, which acts to dictate distinct competence windows sequentially. In addition, Seven up (Svp), a member of the nuclear hormone receptor family, acts early in the temporal cascade, to ensure the transition from Hb to Kr, and has been referred to as a ‘switching factor’. However, Svp is also expressed in a second wave within the developing CNS, but here, the possible role of Svp has not been previously addressed. In a genetic screen for mutants affecting the last-born cell in the embryonic NB5-6T lineage, the Ap4/FMRFamide neuron, we have isolated a novel allele of svp. Expression analysis shows that Svp is expressed in two distinct pulses in NB5-6T, and mutant analysis reveals that svp plays two distinct roles. In the first pulse, svp acts to ensure proper downregulation of Hb. In the second pulse, which occurs in a Cas/Grh double-positive window, svp acts to ensure proper sub-division of this window. These studies show that a temporal factor may play dual roles, acting at two different stages during the development of one neural lineage.

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.

A genetic cascade involving klumpfuss, nab and castor specifies the abdominal leucokinergic neurons in the Drosophila CNS.

Identification of the genetic mechanisms underlying the specification of large numbers of different neuronal cell fates from limited numbers of progenitor cells is at the forefront of developmental neurobiology. In Drosophila, the identities of the different neuronal progenitor cells, the neuroblasts, are specified by a combination of spatial cues. These cues are integrated with temporal competence transitions within each neuroblast to give rise to a specific repertoire of cell types within each lineage. However, the nature of this integration is poorly understood. To begin addressing this issue, we analyze the specification of a small set of peptidergic cells: the abdominal leucokinergic neurons. We identify the progenitors of these neurons, the temporal window in which they are specified and the influence of the Notch signaling pathway on their specification. We also show that the products of the genes klumpfuss, nab and castor play important roles in their specification via a genetic cascade.

Lineage-unrelated neurons generated in different temporal windows and expressing different combinatorial codes can converge in the activation of the same terminal differentiation gene

It is becoming increasingly clear that the activation of specific terminal differentiation genes during neural development is critically dependent upon the establishment of unique combinatorial transcription factor codes within distinct neural cell subtypes. However, it is still unclear to which extent these codes are shared by lineage-unrelated neurons expressing the same terminal differentiation genes. Additionally, it is not known if the activation of a specific terminal differentiation gene is restricted to cells born at a particular developmental time point. Here, we utilize the terminal differentiation gene FMRFa which is expressed by the Ap4 and SE2 neurons in the Drosophila ventral nerve cord, to explore these issues in depth. We find that the Ap4 and SE2 neurons are generated by different neural progenitors and use different combinatorial codes to activate FMRFa expression. Additionally, we find that the Ap4 and SE2 neurons are generated in different temporal gene expression windows. Extending the investigation to include a second Drosophila terminal differentiation gene, Leucokinin, we find similar results, suggesting that neurons generated by different progenitors might commonly use different transcription factor codes to activate the same terminal differentiation gene. Furthermore, these results imply that the activation of a particular terminal differentiation gene in temporally unrestricted.

Development of the Drosophila Embryonic Ventral Nerve Cord: From Neuroectoderm to Unique Neurons and Glia

The Drosophila embryonic ventral nerve cord (VNC) is a powerful model system for addressing the basic mechanisms of nervous system development. Studies conducted in the last three decades have resulted in a detailed description of the basic anatomical features of the VNC, as well as an in-depth understanding of the molecular genetic mechanisms acting to control its development. This has revealed a complex multistep process, from early patterning events and the selection of neural progenitor cells (neuroblasts) with unique identities to lineage progression and the establishment of unique neuronal and glial identities. This chapter reviews the current understanding of these processes and highlights a number of outstanding issues.

From progenitor to unique neuron: Neuronal sub-type specification by the integration of positional and temporal cues

Recent evidence suggests that cell cycle proteins may have novel functions beyond the control of cell division. We have investigated the role of Rb/E2F pathway in the regulation of neuronal differentiation and migration during late embryonic development. We show that loss of Rb leads to terminal differentiation and radial migration defects as well as loss of specific interneuron subtypes in the developing forebrain and olfactory bulb. This phenotype is linked to a dramatic reduction in the levels of Dlx homeodomain genes that regulate ventral telencephalic development, most significantly Dlx2. To ask if Rb plays a direct role in controlling the induction of Dlx2, we examined the regulatory regions of the Dlx1/Dlx2 locus. Using chromatin immunoprecipitation experiments, we show that Rb modulates Dlx gene expression through interaction with the Dlx forebrain-specific enhancer, I12b, the Dlx2 proximal promoter and 3’UTR region in vivo. This interaction is mediated by E2F functional sites located in I12b that act as repressor sites. Deletion of E2F consensus sites on the I12bDlx1/Dlx2 enhancer results in increased reporter activity in the subventricular zone of the developing brain. We demonstrate that in the absence of Rb, E2F7, an Rb-independent repressor, is upregulated in the brain and could ectopically repress the I12b activity and Dlx2 transcription. In conclusion, our data provides the first evidence that cell cycle proteins such as Rb play an essential role to coordinate the transition from proliferation to differentiation and maintain terminal differentiation by regulating the levels of key transcription factors such as Dlx2 during neurogenesis. This work was supported by a grant from CIHR.