Cell Polarity and Asymmetric Cell Division
Drosophila neural precursors (called neuroblasts) repeatedly divide along their apical/basal axis to regenerate an apical neuroblast and bud off a smaller basal daughter cell (called a ganglion mother cell, or GMC) that differentiates into two neurons or glia. Work from our lab and others has identified apically localized proteins (e.g., Par complex, Inscuteable, Pins, Gai) and basally localized proteins (e.g., Miranda, Prospero, and Numb).
Establishing cortical polarity is a key step in regulating asymmetric cell division, cell migration, and differentiated cell function—and neuroblasts are a good model system for studying cell polarity. Graduate student Sarah Siegrist has identified two signaling pathways that induce apical cortical polarity. The first is an extrinsic cue that localizes Par-complex polarity proteins at the site of contact between neuroectoderm and neuroblast. Without this signal, neuroblasts form Par-complex protein crescents at random positions and bud off GMCs at incorrect sites, leading to a disruption in central nervous system tissue polarity. The second is an intrinsic cue from the astral microtubules of the mitotic spindle acting at metaphase. Siegrist's model is that microtubules are required to deliver the microtubule motor protein kinesin Khc-73 to the cortex, where it binds and "opens" the tumor-suppressor protein Discs large (Dlg), which induces Dlg oligomerization and recruitment of the polarity proteins Pins and Gai. Microtubule-induced cortical polarity is functional for generating spindle asymmetry, daughter cell size asymmetry, and distinct sibling fates.
Former postdoctoral fellow Melissa Rolls (now at Pennsylvania State University) has been investigating neuronal polarity. She is performing gene-trap and MARCM (mosaic analysis with a repressible cell marker) mutant screens for genes involved in axon/dendrite specification.
Stem Cell Self-Renewal
Larval neuroblasts divide asymmetrically to generate hundreds of neurons while maintaining their proliferative, undifferentiated state. Thus, larval neuroblasts—like germline stem cells—can be used as a model system to study stem cell self-renewal versus differentiation.
Former postdoctoral fellow Cheng-Yu Lee (now at the University of Michigan) initiated a genetic screen to identify mutants that affect self-renewal of larval central brain neuroblasts. Lee identified mutants that have ectopic self-renewal at the expense of differentiation, resulting in a "brain full of neuroblasts" phenotype. These mutants encode proteins that are normally partitioned into the differentiating GMC (the transcription factor Prospero, the translational repressor Brat, the Notch inhibitor Numb) or proteins required for proper localization of those factors (the Prospero/Brat scaffolding protein Miranda, and the tumor-suppressor proteins Lgl, Dlg, Scrib, and the kinase Aurora-A). Our work on Brat was done in collaboration with Robin Wharton (Duke University Medical Center).
Lee has also identified mutants that fail to self-renew neuroblasts, resulting in a "small brain" phenotype. The most important of these is atypical protein kinase C (aPKC), an evolutionarily conserved polarity protein that is partitioned into the neuroblast at every asymmetric cell division. Loss of aPKC depletes neuroblasts, and misexpression of aPKC in GMCs leads to a severe "brain full of neuroblasts" phenotype. Graduate student Chiswili Chabu has identified aPKC-interacting proteins required for promoting neuroblast self-renewal by immunoprecipitation–mass spectrometry (IP-MS) analysis. Chabu and graduate student Ryan Andersen are also screening for enhancers and suppressors of the aPKC misexpression phenotype to identify additional genes in the aPKC self-renewal pathway.
Graduate student Karsten Siller and postdoctoral fellow Clemens Cabernard have been studying how the mitotic spindle is aligned with neuroblast cortical polarity to ensure asymmetric cell division. They find that reductions in Lis1, dynactin, Mud (fly NuMA), and Aurora-A proteins lead to spindle orientation defects and symmetric cell division, resulting in an expansion of the neuroblast population. They conclude that regulation of spindle orientation is a mechanism for changing progenitor pool size and brain morphology.
Temporal Regulation of Cell Fate Within Neuroblast Cell Lineages
Producing the right cells at the right time is essential for normal mammalian brain development, and for the development of many other embryonic tissues, yet it is not well understood how progenitors or stem cells reproducibly generate a characteristic sequence of different cell types. We previously showed that nearly all of the 30 different Drosophila neuroblasts in each segment sequentially express the transcription factors Hunchback (Ikaros class) —> Kruppel (zinc finger class) —> Pdm (Pou/homeodomain class) —> Castor (zinc finger class), suggesting the possibility of a molecular "clock" that distinguishes GMC identity based on their birth order. Mutant and misexpression studies show that Hunchback is necessary and sufficient for first-born cell fates, whereas Kruppel is necessary and sufficient for second-born cell fates. We postulate that hunchback-Kruppel-pdm-castor are "temporal identity" genes that act together with neuroblast "spatial identity" genes to specify the unique fate of every neuron in the CNS.
Hunchback also regulates neuroblast competence. Extending Hunchback expression in a neuroblast lineage not only leads to extra first-born neurons, but the neuroblast resumes its normal lineage after Hunchback is gone. Thus, Hunchback can maintain neuroblasts in a fully multipotent state. We are identifying Hunchback target genes to explore both functions and searching for temporal identity genes acting in postembryonic neuroblast lineages. We are also investigating the function of Pdm and Castor in specifying later-born neuronal identity.
Ruth Grosskortenhaus (a postdoctoral fellow) is investigating the "clock" that times the sequential Hunchback/Kruppel/Pdm/Castor expression. She has shown that this sequence of gene expression occurs normally in single neuroblasts cultured in vitro and that these transitions in gene expression are regulated at the transcriptional level. The Hunchback-Kruppel transition requires neuroblast cytokinesis to advance (suggesting a feedback signal from the GMC), whereas the later Kruppel-Pdm-Castor transitions can occur normally in G2-arrested single neuroblasts (suggesting a cell-intrinsic timing mechanism).
Generation of Neural Cell Diversity
A long-term interest of our lab has been to understand neuron and glial subtype specification. A former postdoctoral fellow, Marc Freeman (now at the University of Massachusetts Medical School), used genomic, genetic, and bioinformatic methods to identify more than 80 glial expressed genes, many of them identifying glial subtypes. Graduate student Michael Layden has characterized two motor neuron genes, Hb9 and zfh1, that regulate motor neuron–subtype specification and axon exit from the CNS, respectively. Layden is currently mapping dozens of transcription factor profiles to identified neurons as part of a larger project to create a Web-based, searchable database (NeuroAtlas) to link cell lineage, gene expression, axon projections, and neural circuit information. This work is in collaboration with Julie Simpson (HHMI, Janelia Farm Research Campus) and Eugene Meyers (also HHMI, JFRC).