Wnt9a deficiency discloses a repressive role of Tcf7l2 on endocrine differentiation in the embryonic pancreas.

Transcriptional and signaling networks establish complex cross-regulatory interactions that drive cellular differentiation during development. Using microarrays we identified the gene encoding the ligand Wnt9a as a candidate target of Neurogenin3, a basic helix-loop-helix transcription factor that functions as a master regulator of pancreatic endocrine differentiation. Here we show that Wnt9a is expressed in the embryonic pancreas and that its deficiency enhances activation of the endocrine transcriptional program and increases the number of endocrine cells at birth. We identify the gene encoding the endocrine transcription factor Nkx2-2 as one of the most upregulated genes in Wnt9a-ablated pancreases and associate its activation to reduced expression of the Wnt effector Tcf7l2. Accordingly, in vitro studies confirm that Tcf7l2 represses activation of Nkx2-2 by Neurogenin3 and inhibits Nkx2-2 expression in differentiated ß-cells. Further, we report that Tcf7l2 protein levels decline upon initiation of endocrine differentiation in vivo, disclosing the downregulation of this factor in the developing endocrine compartment. These findings highlight the notion that modulation of signalling cues by lineage-promoting factors is pivotal for controlling differentiation programs.

Independent regulation of Sox3 and Lmx1b by FGF and BMP signaling influences the neurogenic and non-neurogenic domains in the chick otic placode.

The development of neural tissue starts with the activation of early neural genes such as the SoxB1 transcription factors, which are expressed in response to signaling molecules. Neural progenitors in the inner ear are only generated in the anterior placodal domain, but the mechanisms that determine when and how otic neural fate is acquired are still unknown. Here, we show that Sox3 expression becomes restricted to the anterior territory of the chick otic field and that misexpression of Sox3 induces Sox2 and Delta1 in the non-neurogenic otic territory. This suggests that Sox3 plays a central role in the establishment of an otic neural fate. Furthermore, Sox3 down-regulates the expression of Lmx1b, a marker of the posterior non-neurogenic otic epithelium. The expression of Sox3 is maintained by the positive action of FGF8 derived from the otic ectoderm. On the contrary, BMP signaling does not have a major influence on neural commitment but instead regulates Lmx1b expression in the otic placode. Together, the data support the notion that Sox3 is critical for the development of the neural elements of the inner ear, and they highlight the importance of localized signaling from the ectoderm in establishing the neurogenic vs. non-neurogenic anteroposterior asymmetry that characterizes the early otic placode.

Insulin-like growth factor 1 is required for survival of transit-amplifying neuroblasts and differentiation of otic neurons.

Neurons that connect mechanosensory hair cell receptors to the central nervous system derive from the otic vesicle from where otic neuroblasts delaminate and form the cochleovestibular ganglion (CVG). Local signals interact to promote this process, which is autonomous and intrinsic to the otic vesicle. We have studied the expression and activity of insulin-like growth factor-1 (IGF-1) during the formation of the chick CVG, focusing attention on its role in neurogenesis. IGF-1 and its receptor (IGFR) were detected at the mRNA and protein levels in the otic epithelium and the CVG. The function of IGF-1 was explored in explants of otic vesicle by assessing the formation of the CVG in the presence of anti-IGF-1 antibodies or the receptor competitive antagonist JB1. Interference with IGF-1 activity inhibited CVG formation in growth factor-free media, revealing that endogenous IGF-1 activity is essential for ganglion generation. Analysis of cell proliferation cell death, and expression of the early neuronal antigens Tuj-1, Islet-1/2, and G4 indicated that IGF-1 was required for survival, proliferation, and differentiation of an actively expanding population of otic neuroblasts. IGF-1 blockade, however, did not affect NeuroD within the otic epithelium. Experiments carried out on isolated CVG showed that exogenous IGF-1 induced cell proliferation, neurite outgrowth, and G4 expression. These effects of IGF-1 were blocked by JB1. These findings suggest that IGF-1 is essential for neurogenesis by allowing the expansion of a transit-amplifying neuroblast population and its differentiation into postmitotic neurons. IGF-1 is one of the signals underlying autonomous development of the otic vesicle.

Visualizing synapse formation in arborizing optic axons in vivo: dynamics and modulation by BDNF.

Dynamic developmental changes in axon arbor morphology may directly reflect the formation, stabilization and elimination of synapses. We used dual-color imaging to study, in the live, developing animal, the relationship between axon arborization and synapse formation at the single cell level, and to examine the participation of brain-derived neurotrophic factor (BDNF) in synaptogenesis. Green fluorescent protein (GFP)-tagged synaptobrevin II served as a marker to visualize synaptic sites in individual fluorescently labeled Xenopus optic axons. Time-lapse confocal microscopy revealed that although most synapses remain stable, synapses are also formed and eliminated as axons branch and increase their complexity. Most new branches originated at GFP-labeled synaptic sites. Increasing BDNF levels significantly increased both axon arborization and synapse number, with BDNF increasing synapse number per axon terminal. The ability to visualize central synapses in real time provides insights about the dynamic mechanisms underlying synaptogenesis, and reveals BDNF as a modulator of synaptogenesis in vivo.

The Drosophila selenophosphate synthetase (selD) gene is required for development and cell proliferation.

To study the function of selenoproteins in development and growth we have used a lethal mutation (selD(ptuf)) of the Drosophila homologous selenophosphate synthetase (selD) gene. This enzyme is involved in the selenoprotein biosynthesis. The selD(ptuf) loss-of-function mutation causes aberrant cell proliferation and differentiation patterns in the brain and imaginal discs, as deduced from genetic mosaics, patterns of gene expression and analysis of cell cycle markers. In addition to that, selenium metabolism is also necessary for the ras/MAPKinase signal tansduction pathway. Therefore, the use of Drosophila imaginal discs and brain and in particular the selD(ptuf) mutation, provide an excellent model to investigate the role of selenoproteins in the regulation of cell proliferation, growth and differentiation.

Disruption of selenoprotein biosynthesis affects cell proliferation in the imaginal discs and brain of Drosophila melanogaster.

The patufet gene encodes the Drosophila melanogaster homologue of selenophosphate synthetase, an enzyme required for selenoprotein synthesis, and appears to have a role in cell proliferation. In this paper we analyse the expression pattern of patufet during the development of imaginal discs and brain as well as the function of this gene in relation to cell proliferation. Wild-type organisms showed a highly dynamic pattern of ptuf mRNA expression during larval and pupal development. Co-localization analysis of ptuf mRNA expression and BrdU incorporation showed high levels of ptuf mRNA in dividing cells and low or undetectable levels in non-dividing cells. In addition, [(75)Se] incorporation revealed a major selenoprotein band of 42 kDa. Mutant organisms showed no selenoprotein synthesis, lower levels of cell proliferation, a higher proportion of cells arrested in G(2) as seen by cyclin B labeling and increased levels of reactive oxygen species (ROS). Because most selenoproteins identified so far are antioxidants, the role of ptuf in cell proliferation through the control of the cellular redox balance is discussed.

Screening of larval/pupal P-element induced lethals on the second chromosome in Drosophila melanogaster: clonal analysis and morphology of imaginal discs.

We have carried out screens for lethal mutations on the second chromosome of Drosophila melanogaster that are associated with abnormal imaginal disc morphologies, particularly in the wing disc. From a collection of 164 P element-induced mutations with a late larva/pupa lethal phase we have identified 56 new loci whose gene products are required for normal wing disc development and for normal morphology of other larval organs. Genetic mosaics of these 56 mutant lines show clonal mutant phenotypes for 23 cell-viable mutations. These phenotypes result from altered cell parameters. Causal relationships between disc and clonal phenotypes are discussed.

patufet, the gene encoding the Drosophila melanogaster homologue of selenophosphate synthetase, is involved in imaginal disc morphogenesis.

Proliferation in imaginal discs requires cell growth and is linked to patterning processes controlled by secreted cell-signalling molecules. To identify new genes involved in the control of cell proliferation we have screened a collection of P-lacW insertion mutants that result in lethality in the larval/pupal stages, and characterized a novel gene, patufet (ptuf). Inactivation of ptuf by a P element insertion in the 5' untranslated region leads to aberrant imaginal disc morphology characterized by a reduction in mass of discs and disorganization of disc cells where no folding or patterning can be detected. Moreover, apoptotic cells can be observed in these small and abnormal mutant discs. To examine the role of ptuf we have studied its clonal behaviour in genetic mosaics generated by mitotic recombination. The mutation causes reduced cell viability, smaller cell size and stops vein differentiation. Non-autonomous effects, such as abnormal differentiation of wild-type cells surrounding the clones, are also observed. We have cloned the ptuf gene of Drosophila melanogaster and found that it encodes a selenophosphate synthetase, which is the first identified in insects. Mutant flies transformed with the full-length cDNA show complete reversion of lethality and disc phenotype. Northern blot analysis and in situ hybridization indicate that the ptuf gene is expressed in imaginal discs as well as at different stages of development. The synthesis of selenoproteins by the selenophosphate synthetase, the role of selenoproteins in the maintenance of the oxidant/antioxidant balance of the cell and its possible implications in imaginal disc morphogenesis are discussed.