The conserved barH-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by Sonic Hedgehog.

In this study, we investigated the gene regulatory network that governs formation of the Zona limitans intrathalamica (ZLI), a signaling center that secretes Sonic Hedgehog (Shh) to control the growth and regionalization of the caudal forebrain. Using loss- and gain-of-function, explants and grafting experiments in amphibians, we demonstrate that barhl2 acts downstream of otx2 and together with the iroquois (irx)-3 gene in establishment of the ZLI compartment initiated by Shh influence. We find that the presumptive (pre)-ZLI domain expresses barhl2, otx2 and irx3, whereas the thalamus territory caudally bordering the pre-ZLI expresses barhl2, otx2 and irx1/2 and early on irx3. We demonstrate that Barhl2 activity is required for determination of the ZLI and thalamus fates and that within the p2 alar plate the ratio of Irx3 to Irx1/2 contributes to ZLI specification and size determination. We show that when continuously exposed to Shh, neuroepithelial cells coexpressing barhl2, otx2 and irx3 acquire two characteristics of the ZLI compartment-the competence to express shh and the ability to segregate from anterior neural plate cells. In contrast, neuroepithelial cells expressing barhl2, otx2 and irx1/2, are not competent to express shh. Noteworthy in explants, under Shh influence, ZLI-like cells segregate from thalamic-like cells. Our study establishes that Barhl2 activity plays a key role in p2 alar plate patterning, specifically ZLI formation, and provides new insights on establishment of the signaling center of the caudal forebrain.

Obesity-associated variants within FTO form long-range functional connections with IRX3.

Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D). Although the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes. However, no direct connection between the obesity-associated variants and FTO expression or function has been made. Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition.

Coenzyme Q10 and alpha-tocopherol protect against amitriptyline toxicity.

Since amitriptyline is a very frequently prescribed antidepressant drug, it is not surprising that amitriptyline toxicity is relatively common. Amitriptyline toxic systemic effects include cardiovascular, autonomous nervous, and central nervous systems. To understand the mechanisms of amitriptyline toxicity we studied the cytotoxic effects of amitriptyline treatment on cultured primary human fibroblasts and zebrafish embryos, and the protective role of coenzyme Q(10) and alpha-tocopherol, two membrane antioxidants. We found that amitriptyline treatment induced oxidative stress and mitochondrial dysfunction in primary human fibroblasts. Mitochondrial dysfunction in amitriptyline treatment was characterized by reduced expression levels of mitochondrial proteins and coenzyme Q(10), decreased NADH:cytochrome c reductase activity, and a drop in mitochondrial membrane potential. Moreover, and as a consequence of these toxic effects, amitriptyline treatment induced a significant increase in apoptotic cell death activating mitochondrial permeability transition. Coenzyme Q(10) and alpha-tocopherol supplementation attenuated ROS production, lipid peroxidation, mitochondrial dysfunction, and cell death, suggesting that oxidative stress affecting cell membrane components is involved in amitriptyline cytotoxicity. Furthermore, amitriptyline-dependent toxicity and antioxidant protection were also evaluated in zebrafish embryos, a well established vertebrate model to study developmental toxicity. Amitriptyline significantly increased embryonic cell death and apoptosis rate, and both antioxidants provided a significant protection against amitriptyline embryotoxicity.

The Xenopus Irx genes are essential for neural patterning and define the border between prethalamus and thalamus through mutual antagonism with the anterior repressors Fezf and Arx.

The Iroquois (Irx) genes encode homeoproteins conserved during evolution. Vertebrate genomes contain six Irx genes organized in two clusters, IrxA (which harbors Irx1, Irx2 and Irx4) and IrxB (which harbors Irx3, Irx5 and Irx6). To determine the precise role of these genes during development and their putative redundancies, we conducted a comparative expression analysis and a comprehensive loss-of-function study of all the early expressed Irx genes (Irx1-5) using specific morpholinos in Xenopus. We found that the five Irx genes display largely overlapping expression patterns and contribute to neural patterning. All Irx genes are required for proper formation of posterior forebrain, midbrain, hindbrain and, to a lesser an extent, spinal cord. Nevertheless, Irx1 and Irx3 seem to have a predominant role during regionalization of the neural plate. In addition, we find that the common anterior limit of Irx gene expression, which will correspond to the future border between the prethalamus and thalamus, is defined by mutual repression between Fezf and Irx proteins. This mutual repression is likely direct. Finally, we show that Arx, another anteriorly expressed repressor, also contribute to delineate the anterior border of Irx expression.

Xiro homeoproteins coordinate cell cycle exit and primary neuron formation by upregulating neuronal-fate repressors and downregulating the cell-cycle inhibitor XGadd45-gamma.

The iroquois (iro) homeobox genes participate in many developmental processes both in vertebrates and invertebrates, among them are neural plate formation and neural patterning. In this work, we study in detail Xenopus Iro (Xiro) function in primary neurogenesis. We show that misexpression of Xiro genes promotes the activation of the proneural gene Xngnr1 but suppresses neuronal differentiation. This is probably due to upregulation of at least two neuronal-fate repressors: XHairy2A and XZic2. Accordingly, primary neurons arise at the border of the Xiro expression domains. In addition, we identify XGadd45-gamma as a new gene repressed by Xiro. XGadd45-gamma encodes a cell-cycle inhibitor and is expressed in territories where cells will exit mitosis, such as those where primary neurons arise. Indeed, XGadd45-gamma misexpression causes cell cycle arrest. We conclude that, during Xenopus primary neuron formation, in Xiro expressing territories neuronal differentiation is impaired, while in adjacent cells, XGadd45-gamma may help cells stop dividing and differentiate as neurons.