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Gene regulation and morphogenesis
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Specific Projects
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The Casares lab Projects1- Control of the progenitor to precursor transition during eye development
I. Transcriptional control of the mitotic trigger cdc25/string (stg). When progenitors are recruited as precursors they undergo a rapid mitotic cycle, the first mitotic wave (FMW). This FMW is driven by the localized transcription of the universal mitotic trigger cdc25/stg right at the progenitor-precursor transition. Thereby, stg transcription at the FMW represents a proxy for the whole progenitor-to-precursor transition. To study it at a molecular level, we are currently characterizing the stg FMW enhancer (Carla S. Lopes).
II. Determination of the GRN and its dynamics. After obtaining the transcriptional profile of eye progenitors and precursors using microarrays, we are now trying to knit these genes (nodes) into a GRN. This requires determining the collection of transcription factors (TFs) expressed in both cell states plus the cis-regulatory elements (CREs) they regulate. Currently, we are using chromatin immunoprecipitation (ChIP), bioinformatics and transgenesis to find some of the key CREs. The TF homothorax (Hth), a Meis family TALE-homeodomain class gene, plays a pivotal role in the transition from progenitors into precursors. Therefore, we are placing many of our efforts in solving Hth’s role at a molecular, genome-wide level in collaboration with R. Mann (Columbia U, NY). Another aspect we will include in our analysis is how the progenitor-to-precursor transition is made irreversible through chromatin modifications. If the key transitions from progenitor to precursors were known, in principle it should be possible to revert it (Carla S. Lopes, Asun Lago, Carlos M. Luque, Marta Neto).
2- Quantitative analysis of the eye GRN. The Drosophila eye GRN is a paradigm of organ specific GRN. Most of its nodes are evolutionarily conserved transcription factors or nuclear proteins whose loss leads to reduced or absent eyes, and which, if expressed ectopically, induce ectopic eye tissue. These genes are collectively called Retinal Determination (RD) genes and include members of the Pax6, Eya, Six and Dach gene families. In addition, this GRN, or variations of it, seem to be at work in several other organs, both in insects and in vertebrates. However, a large fraction of the experiments used to build the fly eye network were ectopic, gain-of-function experiments. We are currently reassessing the regulatory relationships between RD genes in the context of normal eye development. In addition, we are probing the network quantitatively by perturbing it with means of gene-specific RNAis and analyzing the effects on other GRN components (Max Sanchez).
3- Modeling Drosophila eye growth and size. Technically, Drosophila offers the possibility of obtaining quantitative data on aspects such as proliferation rates and probability distributions, pace of differentiation or shapes of tissue growth. Using this information, together with the current knowledge of the major signaling pathways and their impact, we have initiated the building of a phenomenological model, in collaborations with colleagues at Universidad de Sevilla. This model will serve us to detect flaws in our description, and to predict missing phenomena. In addition, the size and shape of eyes vary greatly within insects and even within dipterans (flies and mosquitoes). Likely, at least within the flies, molecular and celluar mechanisms are likely to be extremely similar. Playing with the model should give us hints on how different forms and sizes arise during evolution (Carla S. Lopes and Daniel Aguilar with Antonio Cordoba (US)).
4- An intraspecific comparative approach to identify the “minimal” visual GRN. A common and useful way of determining essential components of a given general process is to search for those that are evolutionarily conserved. This strategy has been applied to the eye GRN using as points of comparison eyes belonging to different groups, sometimes really far away phylogenetically. However, Drosophila adults possess two sets of eyes: the compound eyes and the simpler ocelli. Therefore, we can study two different eye types within a single species. We are now exploring the GRN governing ocellar development with similar approaches to those we apply to the compound eye. In addition, we have recently become interested in the lamina. The lamina is the first optic neuropil in the fly’s brain, and is in charge of processing visual motion information. Lamina neurons develop from a proliferating neuroepithelium that divides symmetrically and are innervated by the compound eye outer photoreceptors. Recent work in the lab highlights similarities between the lamina and the other visual system components (compound eyes and ocelli) that we are exploiting to understand globally the visual GRN and the coordination of specification with patterned cell proliferation. Comparisons between eye, ocelli and lamina GRNs should lead to that “minimal” visual GRN, but also to pinpoint eye-type specific differences (M. Angeles Dominguez, Cris Pineiro, D. Aguilar and G. Amore, (SZN, Naples)).