Ohad Shaham

Pax6: A multi-level regulator of ocular development ☆

Eye development has been a paradigm for the study of organogenesis, from the demonstration of lens induction through epithelial tissue morphogenesis, to neuronal specification and differentiation. The transcription factor Pax6 has been shown to play a key role in each of these processes. Pax6 is required for initiation of developmental pathways, patterning of epithelial tissues, activation of tissue-specific genes and interaction with other regulatory pathways. Herein we examine the data accumulated over the last few decades from extensive analyses of biochemical modules and genetic manipulation of the Pax6 gene. Specifically, we describe the regulation of Pax6s expression pattern, the proteins DNA-binding properties, and its specific roles and mechanisms of action at all stages of lens and retinal development. Pax6 functions at multiple levels to integrate extracellular information and execute cell-intrinsic differentiation programs that culminate in the specification and differentiation of a distinct ocular lineage.

Pax6 is essential for lens fiber cell differentiation

The developing ocular lens provides an excellent model system with which to study the intrinsic and extrinsic cues governing cell differentiation. Although the transcription factors Pax6 and Sox2 have been shown to be essential for lens induction, their later roles during lens fiber differentiation remain largely unknown. Using Cre/loxP mutagenesis, we somatically inactivated Pax6 and Sox2 in the developing mouse lens during differentiation of the secondary lens fibers and explored the regulatory interactions of these two intrinsic factors with the canonical Wnt pathway. Analysis of the Pax6-deficient lenses revealed a requirement for Pax6 in cell cycle exit and differentiation into lens fiber cells. In addition, Pax6 disruption led to apoptosis of lens epithelial cells. We show that Pax6 regulates the Wnt antagonist Sfrp2 in the lens, and that Sox2 expression is upregulated in the Pax6-deficient lenses. However, our study demonstrates that the failure of differentiation following loss of Pax6 is independent ofβ -catenin signaling or Sox2 activity. This study reveals that Pax6 is pivotal for initiation of the lens fiber differentiation program in the mammalian eye.

Pax6 Regulates Gene Expression in the Vertebrate Lens through miR-204

During development, tissue-specific transcription factors regulate both protein-coding and non-coding genes to control differentiation. Recent studies have established a dual role for the transcription factor Pax6 as both an activator and repressor of gene expression in the eye, central nervous system, and pancreas. However, the molecular mechanism underlying the inhibitory activity of Pax6 is not fully understood. Here, we reveal that Trpm3 and the intronic microRNA gene miR-204 are co-regulated by Pax6 during eye development. miR-204 is probably the best known microRNA to function as a negative modulator of gene expression during eye development in vertebrates. Analysis of genes altered in mouse Pax6 mutants during lens development revealed significant over-representation of miR-204 targets among the genes up-regulated in the Pax6 mutant lens. A number of new targets of miR-204 were revealed, among them Sox11, a member of the SoxC family of pro-neuronal transcription factors, and an important regulator of eye development. Expression of Trpm/miR-204 and a few of its targets are also Pax6-dependent in medaka fish eyes. Collectively, this study identifies a novel evolutionarily conserved mechanism by which Pax6 controls the down-regulation of multiple genes through direct up-regulation of miR-204.

Fibroblast Growth Factor 2 in the Dorsomedial Striatum Is a Novel Positive Regulator of Alcohol Consumption

Repeated alcohol intake leads to mesostriatal neuroadaptations, resulting in drinking escalation and addiction phenotypes. Fibroblast growth factor 2 (FGF2) has been shown to interact with the mesostriatal dopaminergic system, and has been implicated in the actions of psychostimulants in the brain, and in several psychiatric disorders. Here, we report on a positive regulatory feedback loop of alcohol and FGF2 in rodent models. Specifically, we found that acute alcohol exposure (2.5 g/kg, i.p.) increased the mRNA expression of Fgf2 in the dorsal hippocampus, nucleus accumbens, and dorsal striatum. Longer alcohol exposure (7 d × 2.5 g/kg, i.p.) restricted these increases to the dorsal striatum, and the latter effect was blocked by the dopamine D2-like receptor antagonist haloperidol. Voluntary prolonged and excessive alcohol consumption in a 2-bottle choice procedure increased Fgf2 expression selectively in dorsomedial striatum (DMS) of both mice and rats. Importantly, we found that systemic administration of recombinant FGF2 (rFGF2) in mice, or rFGF2 infusion into the dorsal striatum or DMS of rats, increased alcohol consumption and preference, with no similar effects on saccharin or sucrose consumption. Finally, we found that inhibition of the endogenous FGF2 function in the DMS, by an anti-FGF2 neutralizing antibody, suppressed alcohol consumption and preference. Together, our results suggest that alcohol consumption increases the expression of Fgf2 in the DMS, and that striatal FGF2 promotes alcohol consumption, suggesting that FGF2 in the DMS is a positive regulator of alcohol drinking. SIGNIFICANCE STATEMENT Long-term alcohol intake may lead to neuroadaptations in the mesostriatal reward system, resulting in addiction phenotypes. Fibroblast growth factor 2 (FGF2) is crucial for the development and maintenance of the mesostriatal dopaminergic system. Here, we provide evidence for the involvement of FGF2 in alcohol-drinking behaviors. We show that alcohol increases Fgf2 expression in the dorsal striatum, an effect mediated via dopamine D2-like receptors. Importantly, we show that infusion of recombinant FGF2 into the dorsomedial striatum increases alcohol consumption, whereas inhibiting the endogenous FGF2 function suppresses consumption. Thus, FGF2 is an alcohol-responsive gene constituting a positive regulatory feedback loop with alcohol. This loop leads to facilitation of alcohol consumption, marking FGF2 as a potential new therapeutic target for alcohol addiction.

09-P038 Pax6 roles in lens development

right ventricle and the outflow tract of the heart. Here we have investigated the role of tailup (tup), the Drosophila homologue of vertebrate islet-1 in the development of the dorsal vessel. In Drosophila, cardiac progenitor cells are specified bilaterally in the dorsal mesoderm, which requires complex interactions between the transcription factors Tinman/Nkx2.5, Pannier/GATA factor, and Dorsocross/Tbx factor. Additionally, the growth factors Wingless and Decapentaplegic provide crucial signals for the induction and maintenance of the cardiac regulatory network. Tup mutants are characterized by reduced expression of the essential transcription factors Tin, Pnr and Doc in early cardiogenesis. Conversely, Tup expression depends on each of these cardiac factors, as well as on the early inductive signals Dpp and Wg. Genetic interactions show that tup cooperates with tin, pnr and Doc in cardiogenesis. Lossand gain-of-function analyses for Tup and Pnr suggest that a proper balance of these factors is essential for cardiac specification. Inhibition of Tup in differentiated cardiac cells shows, that later in development, Tup is required to maintain cardiac identities. In summary, our data add tup as a critical factor to the early cardiac regulatory network that determines a cardiac fate.