Zn-Finger Transcription Factor Genes as Regulatory Targets of Drosophila Prospero and Cyclin E During Neurogenesis
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Date
2010-06
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The Ohio State University
Abstract
Neurogenesis within Drosophila begins with the separation of neural progenitor cells (neuroblasts) form the ventral neuroectoderm and the procephalic neuroectoderm. To do this the neuroblasts must move inward to the interior of the ectoderm so that they may build up the primordium of the central nervous system (Campos-Ortega, 1993). The other cells of the neuroectoderm take on a different developmental state as they develop as epidermoblasts. The determination of whether cells follow the neural pathway to form the central nervous system or the peripheral nervous system (PNS) requires cell to cell communication (Hartenstein, 1984). This communication depends on the function neurogenic genes and proneural genes. This model for cell determination by cell to cell communication is substantiated by the function of the AS-C genes in neural determination and the effects of E(spl)-C in epidermogenesis. These and other neurogenic and proneural genes are regulated by certain transcription factor genes.
The development of the Drosophila nervous system requires the regulatory functions of numerous transcriptional regulators. Drosophila Prospero (Pros) is a well studied transcription factor regulator whose function is crucial for normal nervous system development in Drosophila (Vaessin et al., 1991). Pros was found in the Vaessin laboratory to coordinate the developmental decision between cell cycle and neuronal differentiation (Li and Vaessin, 2000). Specifically, it was shown to regulate the mitotic activity and transcriptional expression of a range of target genes. More recently, the Vaessin laboratory showed that Pros cooperatively interacts with Cyclin E (CycE) in the regulation of various target genes, such as nerfin-1, during Drosophila neurogenesis (Vaessin, unpublished observation). While the dynamics of Pros dependent nerfin-1 (a zinc nervous finger transcription factor encoding gene) expression have been researched and determined by in situ hybridization of Drosophila embryonic tissues, other zinc-finger transcription factors who may represent Pros regulatory targets, have yet to be well studied. nerfin-1 regulates the development of Drosophila central nervous system at several levels, including cell fate and early axon guidance decisions. (Kuzin et al., 2005). Several additional Zn-finger transcription factor genes have been identified in the Vaessin laboratory by microarray analysis as potential regulatory targets of Pros and CycE. Zinc finger transcription factor genes are genes that code for proteins which bind to a specific segment of DNA and control the transcription of that segment of DNA into mRNA (Ashraf, 2004). They gain the zinc finger part of their name because they coordinate one or more zinc ions to help the protein fold in a certain way.
Since these are transcription factors and affect transcription of DNA, it becomes easy to see that a biological system might target these genes to control neurogenesis. My goal is to determine whether three of these genes (snail, worniu, and cubitus interruptus, Cai et al., 2001, Culi et al., 2006) are indeed regulatory targets of Pros and CycE and to further characterize the regulatory and functional interactions, and/or phenotypic consequences of these interactions, between these genes and Pros/CycE during Drosophila neurogenesis. By learning more about the function of these genes during the development of the central nervous system [CNS] and peripheral nervous system [PNS] of the model system Drosophila, we can gain insight about how these genes function in humans and the possible regulatory effect of human Prospero (Prox1) and CycE in the regulation of cell differentiation and organogenesis.
The main question at the basis of my research addresses on how cell differentiation and proliferation is regulated during neurogenesis of Drosophila. While this question is very broad, my immediate research in the context of my honors thesis focused on whether the three Zn-finger transcription factor genes mentioned above are indeed regulatory targets of Pros and CycE.
Zn-finger transcription factor genes were determined as probable regulatory targets of Drosophila Pros and CycE, I performed a series of in situ hybridization experiments. This allowed me to localize and detect the specific transgenic mRNA sequences in the embryonic tissue of Drosophila embryos at different stages of their development. As the name suggests, this is done by hybridizing labeled complementary RNA to the sequence of interest. In our case, in situ hybridization reactions were performed over a three day period. A probe was generated for in situ hybridization, the DNA of the target gene will be amplified by a polymerase chain reaction (see figure 2) so that enough of the target DNA (modified to contain a T7 RNA polymerase recognition site) is acquired to make an antisense RNA probe. In making the probe, I used Digoxigenin labeled UTP which will allow me to tag and trace that specific RNA probe within the embryo and visually see it once the color reaction of the in situ hybridization reaction was performed.
Figure 3:
Figure 2. Diagram revealing how PCR can amplify a target gene exponentially. Cycling DNA in certain temperatures within an environment that promotes DNA replication (DNA Primers, dNTP mix, amplifications buffer, and genomic DNA). See Materials and Methods Section for a further description of PCR. ( Andy Vierstraete, 1999)
Figure 4:
(modified from Rudolf Amann & Bernhard M. Fuchs, 2008)
However, just doing in-situ hybridizations of wild type Drosophila simply showed me where these genes end up being expressed normally and will not enable me to see how transcription factor genes affect neurogenesis and their potential as regulatory targets of Pros and CycE. To see if these genes do possess these qualities, I performed a series of tests. First, I compared the in situ hybridizations done with wild type Drosophila embryos with those of mutant Drosophila embryos where either Pros or CycE alone, or both together have been up regulated by ectopic expression to see how this affects the expression of these Zn-finger candidate genes. These experiments allowed me to determine whether these three candidate genes are indeed target genes of Pros and the coordinating function of Pros and CycE. To determine the biological relevance of the observed change in Zn-finger transcription factor gene expression, I set up crosses of Drosophila with mutant loss of function alleles of the Zn-finger transcription factors with Drosophila that are either wild type, or carry mutant alleles of Pros, CycE, or both Pros and CycE. Immunohistochemistry with various antibodies that allow the visualization of CNS/PNS, mitotic activity, and neuronal differentiation steps, was used to analyze the embryos recovered from these crosses. Microscopy was used to image the antibody labeled embryos.
In summary, I attempted in the following to show the expression of a set of three candidate Zn-finger transcription factors in various genetic backgrounds to determine whether they are regulating targets of Pros and CycE. While, due to technical problems and time limitations, this could not be accomplished for all three genes, I will show a full set of data for one gene (worniu) that indicate that it may indeed be a regulatory target of Prospero and Cyclin E. In addition, I will present additional, incomplete data sets for the other two genes (cubitus interruptus and snail).