We investigate Drosophila CNS development. Current interests are (1) how stem cell-like neural precursors (neuroblasts) establish cell polarity and divide asymmetrically; (2) how neuroblasts maintain stem cell-like features as they divide to produce differentiating progeny; (3) how transcription factors regulate temporal identity within neuroblast lineages; and (4) the genetic program governing the production of motoneurons, serotonergic interneurons, or glia.
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 shown 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 , as well as the precise birth-order of all progeny for selected neuroblasts. (http://www.neuro.uoregon.edu/doelab/lineages/)
We recently showed that nearly all of the 30 different Drosophila neuroblasts in each segment sequentially express the transcription factors Hunchback -> Krüppel -> Pdm -> 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 -> Krüppel -> Pdm -> 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 -> Krüppel -> Pdm -> Castor? And, do hunchback and Krüppel orthologs have similar functions during vertebrate neurogenesis or hematopoiesis?
Generation of motoneuron, interneuron, and glial cell fates
A long-term interest of the lab has been to understand how neural diversity is generated. A graduate student in the lab, Joanne Odden, is investigating how specific types of motoneurons are produced. Her initial work has been on the Drosophila homologue of homeodomain transcription factor HB9/MNR2. Drosophila HB9 is expressed in a subset of motoneurons that project to the lateral body wall muscles; these are distinct from the pool of Eve+ motoneurons that project to dorsal body wall muscles and from a small pool of motoneurons that project to the ventral-most muscles. RNAi and misexpression experiments are consistent with a model that HB9 is necessary and sufficient for motoneuron targeting to lateral muscles. Additional studies on other transcription factors expressed in some or all motoneurons are ongoing.
A postdoctoral fellow in the lab, Marc Freeman, has begun a comprehensive analysis of glial development. Marc is using a novel computational method, microarray technology, and saturation mutagenesis to identify new genes involved in glial development. The computational method identifies putative target genes for the glial cells missing transcription factor, a "master regulator" of glial development. The microarray method looks for genes upregulated following Gcm overexpression in the CNS. These two approaches have already given us over 40 new genes that are involved in glial specification, migration, differentiation, or function. Most of these genes have murine or human orthologs, so it will be interesting to see if they play similar roles in Drosophila and vertebrate gliogenesis.
(pulled from pubmed)
(pulled from pubmed)