RESEARCH INTERESTS

            My lab is interested in understanding the molecular mechanisms underlying the regulation of early development. We use the fruitfly, Drosophila melanogaster , as a model system because of its well defined genetics and short life cycle. Embryonic development in Drosophila is under complex genetic control requiring both maternal and zygotic transcriptional activity.  Maternal gene products are essential during oogenesis and early embryogenesis while zygotic gene activity becomes important at approximately the blastoderm stage. We have identified an essential, maternally-acting gene called lark , which encodes an RNA-binding protein. Embryos lacking maternally-expressed lark lay very few eggs which arrest in development prior to cellular blastoderm. We have spent the last few years characterizing the role of this gene during oogenesis and early embryogenesis.

                The lark gene was originally identified in a genetic screen for factors affecting the circadian clock-mediated regulation of adult eclosion (Newby and Jackson, 1993).  Whereas lark mutations have a dominant effect on the daily timing of eclosion, they are also associated with a recessive embryonic lethality; lark 1 homozygotes arrest in development at about embryonic stage 12, demonstrating that lark zygotic expression is essential at this stage. We have also shown that lark is maternally inherited, suggesting the possibility that it plays an additional role during early embryogenesis or oogenesis (McNeil et al., 1999). lark mRNA can be detected in 0-2 hour embryos, prior to the initiation of zygotic transcription, and is evenly distributed throughout the embryo. Lark protein is expressed in both developing egg chambers during oogenesis and in early embryos. Egg chambers show strong nuclear expression in both the germline nurse cells and the surrounding somatic follicle cells. Some cytoplasmic expression has been observed in nurse cells, follicle cells, and the developing oocyte.

            Since the lark 1 allele is a recessive lethal, we generated female germ-line chimeras (GLC), using the FLP-DFS method (Chou and Perrimon, 1996), to determine if lark maternal expression is important during oogenesis and/or embryogenesis.  Female GLCs lacking lark maternal expression did lay some eggs but they never hatched (McNeil et al., 1999). Most of the eggs deposited showed no signs of development. However, a small percentage did show some development and appear to arrest around cellular blastoderm. Several observations lead us to hypothesize that lark maternal expression was important during oogenesis. First, although some eggs were laid, the number was significantly fewer than controls (G.P. McNeil, unpublished results). This suggested an egg-laying defect. Second, although the eggs looked morphologically normal, they were shorter than normal and were very fragile. A similar phenotype has been observed in spaghetti squash GLCs where it has been shown to be due to a requirement during oogenesis (Wheatley et al., 1995). These results suggest that maternal lark expression is important, at least in part, during oogenesis. It is also possible that since some embryos attempted cellularization that lark is important for some aspect of cellularization as well.

            We have recently performed a detailed morphological analysis of ovaries of lark 1 GLCs (G.P. McNeil, manuscript in preparation). Although egg chambers look normal up to about stage 10, two defects are apparent later in oogenesis.  First, there is a ‘dumpless' phenotype. Rapid transport of nurse cell cytoplasm during stage 11 is defective in a large percentage of egg chambers. This correlates well with the observation that laid eggs are smaller than normal. In other ‘dumpless' mutants, laid eggs are usually smaller due to defects in nurse cell transport late in oogenesis (Spradling, 1993). Second, there is an egg laying defect. Normally, each ovariole contains no more than one mature, stage 14 egg. In contrast, lark 1 GLCs often contain as many as 6 late stage egg chambers. We have also shown that the number of eggs laid by these GLCs is far below that observed in controls. These data clearly suggest an egg laying defect.

            Molecular characterization of the lark gene indicates that it encodes a novel member of the RNA Recognition Motif (RRM) class of RNA-binding proteins (Newby and Jackson, 1996). It contains two consensus RRM domains and a C 2 HC motif known as a retroviral zinc-finger motif (RTZF). RRM domains have been shown to bind RNA in a number of different proteins (Burd and Dreyfuss, 1994). The RTZF, while not well characterized in eukaryotes, has been studied extensively in the retroviral nucleocapsid protein where it binds to and mediates the packaging of the RNA genome of the virus (Copeland et al., 1983; Summers, 1991). A similar but not identical motif is located with the Nanos protein where it is critical for translational repression of bicoid and hunchback mRNAs (Arrizabalaga and Lehmann, 1999). We completed an in vivo structure/function analysis of Lark and found that mutations in either of two RNA-binding domains (RRM 2 and/or RTZF) results in female sterility (McNeil et al., 2001). These results strengthen the hypothesis that Lark is required during oogenesis and indicate it functions as an RNA-binding protein. We are currently examining the ovaries of these mutants and preliminary results show similar defects to those observed in lark 1 GLCs. In addition, defects earlier in oogenesis have been observed. This maybe due to the fact that the mutant version of Lark is expressed in the somatic follicle cells as well. Interestingly, human Lark has been identified and recent evidence suggests it functions in the nucleus to regulate alternative splicing (Jackson et al., 1997; Lai et al., 2003). Drosophila Lark may also function in regulating the splicing of RNAs critical for cytoplasmic transport and egg laying behavior.

            We are currently trying to identify RNA targets of LARK during oogenesis in collaboration with Rob Jackson at Tufts University School of Medicine in Boston .  We have developed an immunoprecipitation protocol using anti-Lark antisera to purify in vivo RNA targets from a variety of tissues including ovaries. This approach has been successful using early embryos and is currently underway using late pupae and ovaries. Precipitated RNA will then be used to screen Drosophila microarrays to identify the targets and the associated gene sequence. The Department of Neuroscience at Tufts (where Rob Jackson is located and where I did my postdoctoral training) has the microarray facilities for these analyses.

REFERENCES

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Chou, T.B., and N. Perrimon (1996) The autosomal FLP-DFS technique for generating germ-line mosaic

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Jackson , F.R., Banfi, S., Guffanti, A., and E. Rossi (1997) A novel zinc finger-containing RNA-binding protein

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McNeil, G.P., Zhang, X., Roberts, M., and F.R. Jackson (1999) Maternal function of a retroviral-type

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McNeil, G.P., Schroeder, A.J., Roberts, M.A., and F.R. Jackson (2001) Genetic analysis of functional

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McNeil, G.P., Smith, F., and R. Galioto. Elimination of lark maternal expression results in defects in cytoplasmic

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