Developmental programs that coordinate neuronal precursor proliferation and cell cycle exit are poorly understood. However they are key links between proper differentiation and disregulation of cell proliferation leading to cancer. Secreted signaling proteins such as Sonic hedgehog (Shh) are key players in patterning various tissues during animal development. Disregulation of this pathway is implicated in congenital defects, cancers and other pathologic states in humans. In fact in normal development and in adult tissues that constantly regenerate, a delicate balance is established between cell death and cell division. Several types of cancers have been linked to the disruption the SHH signaling pathway which is crucial to the normal development of many tissues. Cancers of the digestive systems can be caused by excessive production of SHH by the tumors themselves. Taken together these data suggest that SHH can trigger the activation of certain genes involved in growth. Shh signaling promotes proliferation of cerebellar granule neurons by controlling transcription of Cyclin D1 and Nmyc1 and disregulation of this function may be cause of medulloblastoma. A link between these functions in the Shh signaling pathway and stem cell regulation in the adult was recently suggested in reports showing that Shh is required in postnatal and adult neurogenesis. In fact, in synergism with EGF, Shh regulates neocortical precursors as demonstrated also in Shh mutant mice that are deficient in neural stem cells (1). Shh also regulates retinal progenitors (2). Furthermore, its activity in modulating proliferation of progenitor cells was also demonstrated in regeneration of the chick retina from the ciliary marginal zone (3). In the retina Shh is expressed in ganglion and amacrine cells (4) regulating ganglion cell production and outgrowth (5, 6) and retinal cell differentiation and laminar organization (7, 8). Therefore the retina is a very good model for the study of Shh pathway affecting neuronal proliferation and differentiation.The intricate Shh signaling pathway has been characterized by component 2 and 3 who collaborated in the identification of the receptor Ptch (9, 10). Interestingly, the receptor does not directly transduce the signal inside the cell but this function is mediated by another protein, called Smo, which is expressed on the surface of the target cells. The signal is transduced inside the cell by a complex of proteins bound to microtubules controlling the function of the Gli family of transcription factors. Component 2 (Marigo) and 3 (Tabin) showed that Shh positively controls expression of Ptch and Gli1 themselves and can either positively or negatively control expression of Gli3 (11-13). Gli3 itself can act as activator or repressor in the developing nervous system. There are three Gli proteins that participate in the mediation and interpretation of the response to the Shh signal. Identification of the intracellular events upstream and downstream to the Shh signaling pathway is crucial for a comprehensive understanding of growth control in both normal and neoplastic neurons. The regulation of Shh transcription is still not well characterized. Moreover, transcription factors directly controlling spatial and tissue specific expression of Shh have not been well characterized yet. Transcription factors belonging to the Hand (15) or Hox (16) gene families, for example, have been implicated in the regulation of Shh expression, but their direct involvement in Shh expression has never been addressed experimentally. The genetic pathways lying downstream of Shh signaling are similarly poorly characterized. Gli genes are key factors downstream to the Shh signal however their function is as yet not fully understood. In fact, genetic analyses have shown that Gli1 function is redundant (17), whereas Gli2 and Gli3 show specific defects in mouse mutants but also overlapping functions (18). Thus, the response of distinct cells in their activation of Gli genes either as transcriptional activators or as transcriptional repressors can vary according to the environmental context or to cell-intrinsic genetic cues. Given the strong impact of Shh expression and activation of its signaling pathway in neuronal proliferation, differentiation and neoplastic transition we propose to study how Shh expression is regulated and how Shh mediates these different biological events. Although much is known about the genetic interactions of the genes belonging to this network, only a few regulatory interactions have been analyzed to the level of DNA-protein interactions.Our main objectives to address these key questions are:1) To identify and characterize within the Shh promoter relevant regulatory elements that are necessary for the correct spatio-temporal and tissue-specific expression of Shh in neural retinal cells. We intend to apply bioinformatic approaches to study the Shh genomic region in the human and murine genome. The analysis will cover non-genic sequences of the Shh locus. The collaboration and laboratory exchanges of Component 1 (Zappavigna) with the laboratory of Component 3 Tabin will be essential to accomplish this objective. In fact, he, together with Dr. Marigo (Component 2), studied the human SHH gene and characterized SHH locus in the human genome (19).2) To identify transcription factors that directly control activation of Shh in the neuronal tissue. We will study the direct involvement of candidate transcriptional regulators in the function of these cis-regulatory elements in controlling the expression of Shh in different neuronal cell types and in particular in retinal progenitors and neurons. We will focus on homeodomain and bHLH transcription factors. In fact, in the retina bHLH repressors suppress bHLH activators and promote maintenance of progenitors and generation of glial cells. Conversely, bHLH activators override activities of bHLH repressors and promote neuronal differentiation. bHLH activators, however, are not sufficient since homeodomain factors are additionally required for neuronal subtype specification. Homeodomain factors likely regulate the layer specificity but not the neuronal fate, while bHLH activators determine the neuronal fate within the homedomain factor-specified layers. Thus, combinations of proper bHLH and homeodomain factors are required for neuronal subtype specification (20). Both Prof. Tabin and Prof. Zappavigna are experts in transcription regulated by homeodomain genes. The collaboration with Prof. Tabin will be beneficial in sharing technical expertise and reagents for these studies.3) To dissect the signaling pathway in adult retinal stem cells exposed to Shh. Retinal stem cells derived from the adult eye show phenotypic and molecular characteristics of retinal progenitors and therefore are an excellent tool for in vitro studies of retinal neuronal proliferation and differentiation. We propose the use of this cell type to study the response to Shh signal of retinal neurons before and after differentiation. We will evaluate which Gli protein is activated by Shh in the retinal stem cell system and whether retinal stem cells respond to Shh upregulating Nmyc and CyclinD1as shown in other neuronal cell populations. Prof. Tabin and Dr. Marigo have a long term collaboration in the study of the Shh signaling pathway. The animal models recently generated by Prof. Tabin (21) will allow an in vivo study of this objective.Building on our long-standing collaboration with Prof. Tabin's group at the Harvard Medical School, should the present project be funded, we are going to expand our mutual scientific exchanges by scheduling a series of meetings and progress reports on a regular basis to reinforce the sharing of information and reagents among our groups.
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|Titolo:||Studio delle vie di regolazione a monte e a valle del segnale di Sonic hedgehog nel tessuto neurale della retina|
|Autori:||V. Zappavigna; V. Marigo|
|Data di pubblicazione:||2004|
|Appare nelle tipologie:||Partecipazione a progetti di ricerca|
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