Page 356 - 53rd Annual Drosophila Research Conference

Poster Full Abstracts - Stem Cells
Poster board number is above title. The first author is the presenter
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Institute, University of Michigan Medical School, Ann Arbor, MI 48109.
Stem cells in numerous tissues give rise to transit-amplifying cells that divide a limited number of times to produce post-mitotic progeny. Homeostasis of
these tissues requires that following stem cell division a fraction of the daughter cells are specified as transit-amplifying cells that go on to maintain their
restricted identity. Failure of this process results in unconstrained expansion of the stem cell pool at the expense of differentiated cells. Neural stem cell
lineages (Type II Neuroblasts) that produce transit-amplifying cells (intermediate neural progenitors (INPs)) in the Drosophila larval brain provide an
extremely robust biological system for genetic analysis of this process. Previous studies have identified two classes of genes that are required to functionally
distinguish INPs from there parental neuroblasts. In brat or numb mutants newly born INPs rapidly reacquire a neuroblast identity, thus establishment of an
INP fate is dependent on these genes. In erm mutants INPs are properly established, but fail to maintain their restricted developmental potential and de-
differentiate into neuroblasts. We find that heterozygous mutation of the transcription factor pntP1 dominantly enhances both erm and brat sensitized mutant
backgrounds, indicating pntP1 is involved in both the establishment and maintenance of INP identity. PntP1 protein is enriched specifically within newly
born INPs just prior to activation of erm expression. In addition, epistatic analysis reveals that pntP1 functions downstream of brat and likely functions
upstream or in parallel to erm. This demonstrates that a signaling network, initiated in the neuroblast, persists within INPs to intrinsically establish and
maintain their fate.
833B
The Landscape of Drosophila cis-Regulation as Revealed by EvoPrinter Analysis. Mukta R. Kundu1, Alexander Kuzin1, Tzu-Yang Lin2, Chi-Hon Lee2 ,
Thomas Brody1, Ward F. Odenwald1 . 1) Neural Cell Fate Determinants, NINDS, Bethesda, MD; 2) Section on Neuronal Connectivity, NICHD, NIH,
Bethesda, MD.
To gain insights into the diversity and structural complexity of cis-regulatory DNA within intergenic regions that are not nearby flanking genes, we have
tested the cis-regulatory activities of 20 consecutive conserved sequence clusters (CSCs), identified by EvoPrint analysis (Yavatkar et al., 2008, BMC
Genomics 9: 106), within a 28kb genomic region beginning 23kb downstream of ventral veins lacking/drifter gene and separated from the flanking gene by
67kb. Enhancer/reporter analysis reveals a diversity of cis-regulatory functions associated with these CSCs, including CNS, PNS, ventral midline, epidermal
and trachea enhancers that are active in the embryo, larva and/or adult. Two of the neural enhancers share conserved POU-homeodomain binding sites, a
common signature of late temporal neuroblast network enhancers such as those regulating castor and grainyhead, and each drives expression in distinct sets
of bilaterally symmetrical larval CNS neurons. Another identified enhancer drives reporter expression in the embryonic and larval ventral cord CNS midline
glia. Analysis of this CSC revealed the presence of four conserved binding sites for the midline determinant Single-minded. Both tracheal and midline
enhancers appear to consist of two sub-modules, revealed by the clustered distribution of repeat sequences within these enhancers and the observation that
these modules exhibit DNaseI accessible embryonic genomic DNA (Thomas et al., 2011, Genome Biol. 12: R43) within a restricted region of their CSCs.
Based on these findings, we conclude that most of the ~100,000 CSCs within the Drosophila genome that flank transcribed DNA represent cis-regulatory
modules, and that phylogenetic footprinting via EvoPrinter is an invaluable tool for the discovery and further analysis of these sequences. We are currently
using these observations to test the relationship of DNaseI hypersensitivity to CSC enhancer function.
834C
The Drosophila gene clueless is required for mitochondrial function and dynamics in larval neuroblasts. Aditya Sen, Vanessa Damm, Rachel Cox.
Biochemisty and Mol. Biology, Uniformed Services Univ., Bethesda, MD.
Mitochondria are double membranous organelles responsible for making ATP. They are highly dynamic, changing location, shape and numbers,
characteristics that are important to maintain the structure and functional integrity of the organelles, as well as maintain cellular health. There appears to be a
link between mitochondrial dysfunction and neurodegenerative diseases, such as Alzheimer’s diseases (AD) and Parkinson disease (PD). To study genes
important for mitochondrial function and dynamics, we have taken a candidate gene approach and identified and characterized the Drosophila gene clueless
(clu). Drosophila Clueless protein shares 53% overall identity to its uncharacterized human homolog (KIAA0664), and shows even greater (85%) identity in
one of its hypothetical domains, called the ‘Clu’ domain. In female germ cells, clu mutants have mislocalized mitochondria, and are sterile. Because clu
mutants are uncoordinated and clu genetically interacts with parkin, we are examining mitochondrial dynamics during neurogenesis in wildtype and clu
mutant brains. We have found that NBs contain high levels of Clu and large numbers of small, spherical mitochondria. The normal wildtype distribution
pattern of mitochondria during the cell cycle is disturbed in clu mutant NBs. Mitochondria tend to clump, a phenotype also seen in female germ cells. We are
currently creating maternal/zygotic clu mutants to determine if they will experience NB loss. To identify Clu’s molecular mechanism, we are testing Clu
function in S2R+ insect cells and have found that mitochondrial distribution is affected in clu knockdown cells, similar to NBs and germ cells. We are
carrying out a structure function study using both transgenic flies and knockdown S2R+ cells and have found that the ‘Clu’ and the‘TPR’ (tetratricopeptide
repeat) domains, are important for Clu function. Due to Clu’s high sequence identity among species, we believe that our findings about Clu function and
mitochondrial dynamics in neuroblasts could be of general importance for stem cells.
835A
Klumpfuss (klu) encodes a novel regulator of neuroblast identity during larval brain neurogenesis. Qi Xiao1,3, Cheng-Yu Lee 1,2,3. 1) Department of Cell
and Developmental Biology; 2) Division of Molecular Medicine and Genetics, Department of Internal Medicine; 3) Center for Stem Cell Biology, Life
Sciences Institute, University of Michigan Medical School, Ann Arbor, MI 48109.
Precise distinction of the daughter cell potential following asymmetric stem cell divisions is essential for preserving the stem cell pool and generating
sufficient post-mitotic progeny, but the mechanisms are unclear. We study how the daughter neuroblast and progenitor cell are functionally distinguished
during asymmetric divisions of type II neuroblasts in which the basal protein Brain tumor (Brat) plays a central role. We identified klu as a dominant
suppressor of the ectopic type II neuroblast phenotype in a sensitized brat mutant genetic background. klu encodes a C2H2 zinc-finger transcription factor,
and the Klu protein is detectable in all brain neuroblasts. Neuroblasts in klu mutant brains undergo premature differentiation, indicating that klu is necessary
for maintenance of neuroblast identity. Immature intermediate neural progenitor cells over-expressing Klu fail to acquire restricted potential and assume the
progenitor cell fate, and instead, rapidly reacquire the type II neuroblast identity, strongly suggesting that Klu is sufficient to promote the neuroblast fate.
Domain analyses reveal that both a novel central domain and the zinc fingers are necessary for Klu to promote the neuroblast fate. This result suggests that