Chris Q. Doe Professor, Department of Biology B.S., New College Ph.D., Stanford University Research Interests Generation of neuron and glial diversity in the Drosophila CNS Asymmetric cell division in Drosophila Email IMB Web Page HHMI Web Page

Generation of Cell Polarity, Temporal Identity, and Neural Diversity in Drosophila

Asymmetric cell division of neural precursors. 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 GMC) that differentiates into a neurons or glia. Normal asymmetric division requires alignment of the mitotic spindle along the apical/basal axis as well as polarized localization of cell fate determinants to the apical or basal poles of the cell -- which allows two molecularly distinct daughter cells to be produced.

We are interested how neuroblasts establish cell polarity, and how cell polarity is used to generate two different cell types at each cell division. Work from our lab and others has identified basally-localized mRNA and proteins (e.g. prospero RNA and Miranda, Prospero, and Numb proteins) as well as apically-localized proteins (e.g. Baz, Par-6, and aPKC). We have done genetic screens to identify new genes involved in apical protein localization, spindle orientation, and basal protein localization, and have identified 12 loci that are required for one or more of these events. A graduate student in the lab, Sarah Siegrist, has developed methods for timelapse imaging of asymmetric neuroblast division both in vivo and in vitro, which is providing new insights into wild type and mutant cell division phenotypes.

Two former graduate students, Chian-Yu Peng and Roger Albertson, have characterized three basal localization mutants, the previously identified "tumor suppressor genes" lethal giant larvae ( lgl ), discs large ( dlg ), and scribble . All three mutants show normal apical protein localization and spindle orientation, but a loss of basal protein targeting. Interestingly, these phenotypes can be suppressed by reducing the level of non-muscle myosin II protein, and mimicked by a pan-myosin inhibitor, leading to a model in which both positive and negative myosins regulate basal transport of Miranda and Numb proteins. A third graduate student, Karsten Siller, is working on the role of the dynactin complex and Lis1 in regulating basal protein targeting and spindle orientation in neuroblasts. Karsten has show that Lis1 is essential for normal asymmetric division (both basal targeting and spindle orientation). His results are likely to aid in our understanding of the human Lissencephaly phenotype, which has yet to be characterized at the cellular level. Our work on cell polarity and asymmetric cell division has been supported by HHMI and the NIH.

Temporal regulation of cell fate within neuroblast cell lineages. Producing the right cells at the right time is essential for normal development, yet it is not well understood how an embryonic precursor cell or a stem cell reproducibly generates a characteristic sequence of different cell types. To begin to study this question, we have done comprehensive cell lineage studies to identify the clone of neurons and glia produced by all 30 different embryonic neuroblasts ( http://www.neuro.uoregon.edu/doelab/lineages/ ), as well as the precise birth-order of all progeny for selected neuroblasts.

We recently showed that nearly all of the 30 different Drosophila neuroblasts in each segment sequentially express the transcription factors Hunchback ö Krüppel ö Pdm ö Castor, raising the possibility of a molecular "clock" for distinguishing GMC birth-order (Isshiki et al., 2001, Cell 106:511). Interestingly, while neuroblast only transiently expressed each gene, the daughter GMCs born during each window of expression maintained expression of that gene as they differentiated. Thus, first-born GMCs maintain Hunchback as they differentiate, whereas second-born GMCs maintain Kruppel as they differentiate. Mutant and misexpression studies show that Hunchback is necessary and sufficient for first-born cell fates, whereas Krüppel is necessary and sufficient for second-born cell fates; we observe this in multiple neuroblast lineages and is independent of the cell type involved. We postulate that Hunchback ö Krüppel ö Pdm ö Castor are "temporal coordinate genes" that act together with "spatial coordinate genes" known to specify each neuroblast identity to uniquely specify the identity of each neuron or glia in the CNS.

More recently, Bret Pearson in the lab has shown that Hunchback has the potential to "restart" the lineage of older neuroblasts, revealing a surprising degree of plasticity in neuroblast developmental potential. Bret has also shown that transient expression of Hunchback can produce long-term heritable specification of first-born cell fate, suggesting that Hunchback-mediated chromatin remodeling may be involved in the specification of neuronal temporal identity, similar to the role of Hunchback in establishing heritable HOX gene expression.

Other questions that we are interested in are: Do Pdm and Castor have similar functions in specifying later-born fates? What regulates the timing of the gene expression "clock" that controls Hunchback ö Krüppel ö Pdm ö Castor? And, do hunchback and Krüppel orthologs have similar functions during vertebrate neurogenesis or hematopoiesis?

Generation of motor neuron identity. A long-term interest of the lab has been to understand how neural diversity is generated. Two graduate students in the lab, Joanne Odden and Mike Layden, are investigating how specific types of motor neurons are produced. Joanne has shown that the Drosophila HB9 homeodomain transcription factor is expressed in a subset of motor neurons that project to the lateral body wall muscles; these are distinct from the pool of Eve+ motor neurons that project to dorsal body wall muscles and from a small pool of motor neurons that project to the ventral-most muscles. RNAi and misexpression experiments show that HB9 is necessary and sufficient for motor neuron targeting to lateral muscles. Mike is working on a pan-neuronal transcription factor called Zfh1. This transcriptional repressor is also expressed in a subset of glia that are associated with motor nerve roots. Mike is currently doing mutant and misexpression analysis of Zfh1 to test its role in motor neuron specification. Additional studies on other transcription factors expressed in some or all motor neurons are ongoing.

Key words stem cell, neuroblast, asymmetric cell division, neural patterning, cell lineage, motor neuron, glia, neurogenesis, neuro, development

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