Neural stem cells are multipotent primary progenitors that proliferate and initiate lineages comprising the differentiated neuronal and glial cell types of the brain.  The differentiated neural cells are, however, not always generated directly by neural stem cells; they can also be produced by intermediate neural progenitors (INPs) that act as transit amplifying cells to generate the enormous number and diversity of neuronal cells required for the formation of complex brain circuitry.  Indeed, recent studies on mammalian cortical neurogenesis indicate that most neural cells are produced by INPs implying that the primary role of neural stem cells in cortical development is to generate extended lineages of INPs.  Analysis of neural stem cells and INPs as well as elucidation of the mechanisms by which they give rise to lineages of specific neural cells are, hence, essential for understanding the basis of neural diversity and circuit complexity in the brain.  Drosophila neural stem cells, called neuroblasts, are similar to vertebrate neural stem cells in many aspects of asymmetric cell division, self-renewal, multipotency and cell fate determination, and are currently one of the best understood models for neural stem cell biology.  Approximately 100 neuroblast pairs generate the central brain of Drosophila, and among these are 8 identified neuroblast pairs that amplify proliferation through INPs.  These so-called type II neuroblasts generate approximately 8000 neural cells of many different types; this corresponds to about one-fourth of the total number of neural cells in the central brain.  How this limited number of neuroblasts acting through transit amplifying INPs can generate this number and diversity of neural cell types in the developing brain is not known.

Here we propose to analyse the cellular and molecular mechanisms by which amplifying type II neuroblasts and their INPs generate the remarkably large and diverse lineages of neural cells in the brain.  Our analysis will be based on targeted genetic access to identified cell types and the powerful analysis methods offered by current Gal4/UAS technology including single-cell, twin-spot, and dual MARCM as well as UAS-RNAi knockdown.  This proposal consists of two highly interrelated research projects.  The first is a cellular and molecular analysis of the type II neuroblasts during embryonic and postembryonic brain development.  For this we will determine the specific combination of key developmental control genes that are expressed in each of the 8 type II neuroblasts and investigate the roles of these genes in the specification of neuroblast identity and of lineage-specific neural cell fate.  Moreover, we will determine if the mechanisms that underlie cell fate specification and those that control proliferation are coupled in the amplifying type II lineages.  We will also investigate if an experimental transformation of type II neuroblasts to type I neuroblasts results in alterations in the genetic mechanisms for proliferation and cell fate control.  The second project is a developmental genetic analysis of the number and diversity of adult-specific neural cell types generated by the amplifying type II neuroblasts.  For this we will characterize these different types of adult-specific neuronal cells neuroanatomically and relate each of these cell types to their lineage/sublineage of origin with clonal MARCM and lineage tracing methods.  Subsequently we will analyse the cellular and molecular mechanisms that underlie the lineage-specific diversity of these neural cell types.  We will investigate the role of birth order and temporal genes in neural cell type specification, characterize the role of programmed cell death in lineage-specific neural differentiation, and analyse the developmental mechanisms that give rise to novel type II-derived glial cells of the optic lobe.  Moreover, we will investigate the roles of so-called “embryonic patterning genes” and “proliferation control genes” in neural cell type specification in these type II lineages.

This work should contribute to our understanding of lineage-specific brain development in quantitative terms and also increase our mechanistic insight into the development of the many qualitatively different neuronal and glial cell types that these lineages comprise.  Moreover, our investigation should help establish the type II neuroblasts and their lineages in Drosophila as an excellent genetic model for understanding the mechanisms of INP-based amplification of proliferation in the mammalian brain.  Taken together, our studies should provide fundamental insight into general and conserved aspects of neural stem cell-based mechanisms that give rise to complex circuit assemblies in complex brains.