Alvaro Glavic

BAX Inhibitor-1 Is a Negative Regulator of the ER Stress Sensor IRE1α

Adaptation to endoplasmic reticulum (ER) stress depends on the activation of an integrated signal transduction pathway known as the unfolded protein response (UPR). Bax inhibitor-1 (BI-1) is an evolutionarily conserved ER-resident protein that suppresses cell death. Here we have investigated the role of BI-1 in the UPR. BI-1 expression suppressed IRE1alpha activity in fly and mouse models of ER stress. BI-1-deficient cells displayed hyperactivation of the ER stress sensor IRE1alpha, leading to increased levels of its downstream target X-box-binding protein-1 (XBP-1) and upregulation of UPR target genes. This phenotype was associated with the formation of a stable protein complex between BI-1 and IRE1alpha, decreasing its ribonuclease activity. Finally, BI-1 deficiency increased the secretory activity of primary B cells, a phenomenon regulated by XBP-1. Our results suggest a role for BI-1 in early adaptive responses against ER stress that contrasts with its known downstream function in apoptosis.

Xiro, a Xenopus homolog of the Drosophila Iroquois complex genes, controls development at the neural plate

The Drosophila homeoproteins Ara and Caup are members of a combination of factors (prepattern) that control the highly localized expression of the proneural genes achaete and scute. We have identified two Xenopus homologs of ara and caup, Xiro1 and Xiro2. Similarly to their Drosophila counterparts, they control the expression of proneural genes and, probably as a consequence, the size of the neural plate. Moreover, Xiro1 and Xiro2 are themselves controlled by noggin and retinoic acid and, similarly to ara and caup, they are overexpressed by expression in Xenopus embryos of the Drosophila cubitus interruptus gene. These and other findings suggest the conservation of at least part of the genetic cascade that regulates proneural genes, and the existence in vertebrates of a prepattern of factors important to control the differentiation of the neural plate.

Interplay between Notch signaling and the homeoprotein Xiro1 is required for neural crest induction in Xenopus embryos

The neural crest is a population of cells that originates at the interface between the neural plate and non-neural ectoderm. Here, we have analyzed the role that Notch and the homeoprotein Xiro1 play in the specification of the neural crest. We show that Xiro1, Notch and the Notch target gene Hairy2A are all expressed in the neural crest territory, whereas the Notch ligands Delta1 and Serrate are expressed in the cells that surround the prospective crest cells. We have used inducible dominant-negative and activator constructs of both Notch signaling components and Xiro1 to analyze the role of these factors in neural crest specification without interfering with mesodermal or neural plate development. Activation of Xiro1 or Notch signaling led to an enlargement of the neural crest territory, whereas blocking their activity inhibited the expression of neural crest markers. It is known that BMPs are involved in the induction of the neural crest and, thus, we assessed whether these two elements might influence the expression of Bmp4. Activation of Xiro1 and of Notch signaling upregulated Hairy2A and inhibited Bmp4 transcription during neural crest specification. These results, in conjunction with data from rescue experiments, allow us to propose a model wherein Xiro1 lies upstream of the cascade regulating Delta1 transcription. At the early gastrula stage, the coordinated action of Xiro1, as a positive regulator, and Snail, as a repressor, restricts the expression of Delta1 at the border of the neural crest territory. At the late gastrula stage, Delta1 interacts with Notch to activate Hairy2A in the region of the neural fold. Subsequently, Hairy2A acts as a repressor of Bmp4 transcription, ensuring that levels of Bmp4 optimal for the specification of the neural plate border are attained in this region. Finally, the activity of additional signals (WNTs, FGF and retinoic acid) in this newly defined domain induces the production of neural crest cells. These data also highlight the different roles played by BMP in neural crest specification in chick and Xenopus or zebrafish embryos.

BAX inhibitor‐1 regulates autophagy by controlling the IRE1α branch of the unfolded protein response

Both autophagy and apoptosis are tightly regulated processes playing a central role in tissue homeostasis. Bax inhibitor 1 (BI‐1) is a highly conserved protein with a dual role in apoptosis and endoplasmic reticulum (ER) stress signalling through the regulation of the ER stress sensor inositol requiring kinase 1 α (IRE1α). Here, we describe a novel function of BI‐1 in the modulation of autophagy. BI‐1‐deficient cells presented a faster and stronger induction of autophagy, increasing LC3 flux and autophagosome formation. These effects were associated with enhanced cell survival under nutrient deprivation. Repression of autophagy by BI‐1 was dependent on cJun‐N terminal kinase (JNK) and IRE1α expression, possibly due to a displacement of TNF‐receptor associated factor‐2 (TRAF2) from IRE1α. Targeting BI‐1 expression in flies altered autophagy fluxes and salivary gland degradation. BI‐1 deficiency increased flies survival under fasting conditions. Increased expression of autophagy indicators was observed in the liver and kidney of bi‐1‐deficient mice. In summary, we identify a novel function of BI‐1 in multicellular organisms, and suggest a critical role of BI‐1 as a stress integrator that modulates autophagy levels and other interconnected homeostatic processes.

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

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.

A Gain-of-Function Screen Identifying Genes Required for Growth and Pattern Formation of the Drosophila melanogaster Wing

The Drosophila melanogaster wing is a model system for analyzing the genetic control of organ size, shape, and pattern formation. The formation of the wing involves a variety of processes, such as cell growth, proliferation, pattern formation, and differentiation. These developmental processes are under genetic control, and many genes participating in specific aspects of wing development have already being characterized. In this work, we aim to identify novel genes regulating wing growth and patterning. To this end, we have carried out a gain-of-function screen generating novel P-UAS (upstream activating sequences) insertions allowing forced gene expression. We produced 3340 novel P-UAS insertions and isolated 300 that cause a variety of wing phenotypes in combination with a Gal4 driver expressed exclusively in the central domain of the presumptive wing blade. The mapping of these P-UAS insertion sites allowed us to identify the gene that causes the gain-of-function phenotypes. We show that a fraction of these phenotypes are related to the induction of cell death in the domain of ectopic gene expression. Finally, we present a preliminary characterization of a gene identified in the screen, the function of which is required for the development of the L5 longitudinal vein.

Xiro-1 controls mesoderm patterning by repressing bmp-4 expression in the Spemann organizer.

The Iroquois genes code for homeodomain proteins that have been implicated in the neural development of Drosophila and vertebrates. We show here for the first time that Xiro‐1, one of the Xenopus Iroquois genes, is expressed in the Spemann organizer from the start of gastrulation and that its overexpression induces a secondary axis as well as the ectopic expression of several organizer genes, such as chordin, goosecoid, and Xlim‐1. Our results also indicate that Xiro‐1 normally functions as a transcriptional repressor in the mesoderm. Overexpression of Xiro‐1 or a chimeric form fused to the repressor domain of Engrailed cause similar phenotypes while overexpression of functional derivatives of Xiro‐1 fused with transactivation domains (VP16 or E1A) produce the opposite effects. Finally, we show that Xiro‐1 works as a repressor of bmp‐4 transcription and that its effect on organizer development is dependent on BMP‐4 activity. We propose that the previously observed down regulation of bmp‐4 in the dorsal mesoderm during gastrulation can be explained by the repressor activity of Xiro‐1 described here. Thus, Xiro‐1 seems to have at least two different functions: control of neural plate and organizer development, both of which could be mediated by repression of bmp‐4 transcription.

Global translational impacts of the loss of the tRNA modification t6A in yeast

The universal tRNA modification t6A is found at position 37 of nearly all tRNAs decoding ANN codons. The absence of t6A37 leads to severe growth defects in baker’s yeast, phenotypes similar to those caused by defects in mcm5s2U34 synthesis. Mutants in mcm5s2U34 can be suppressed by overexpression of tRNALysUUU, but we show t6A phenotypes could not be suppressed by expressing any individual ANN decoding tRNA, and t6A and mcm5s2U are not determinants for each other’s formation. Our results suggest that t6A deficiency, like mcm5s2U deficiency, leads to protein folding defects, and show that the absence of t6A led to stress sensitivities (heat, ethanol, salt) and sensitivity to TOR pathway inhibitors. Additionally, L-homoserine suppressed the slow growth phenotype seen in t6A-deficient strains, and proteins aggregates and Advanced Glycation End-products (AGEs) were increased in the mutants. The global consequences on translation caused by t6A absence were examined by ribosome profiling. Interestingly, the absence of t6A did not lead to global translation defects, but did increase translation initiation at upstream non-AUG codons and increased frame-shifting in specific genes. Analysis of codon occupancy rates suggests that one of the major roles of t6A is to homogenize the process of elongation by slowing the elongation rate at codons decoded by high abundance tRNAs and I34:C3 pairs while increasing the elongation rate of rare tRNAs and G34:U3 pairs. This work reveals that the consequences of t6A absence are complex and multilayered and has set the stage to elucidate the molecular basis of the observed phenotypes.

Extracellular signals, cell interactions and transcription factors involved in the induction of the neural crest cells

The neural crest is induced at the border between the neural plate and the epidermis. A complex set of signals is required for the specification of the crest cells between the epidermis and the neural plate. Here we discuss evidence supporting a model for neural crest induction in which different signals contribute in a sequential order. First, a gradient of bone morphogenic proteins (BMPs) is established in the ectoderm that results in segregation into neural plate, neural folds and epidermis at increasing levels of BMP activity. Thus, the neural folds are induced at a precise threshold concentration of BMP, but this neural fold has an anterior character. In a second step, these anterior neural folds are transformed into prospective neural crest by posteriorizing signals due to fibroblast growth factor, Wnts and retinoic acid. Finally, the induced cells interact to complete neural crest induction by a process that requires Notch/Delta signaling. Once neural crest formation has been induced by this combination of extracellular and intracellular signals, a cascade of transcription factors is activated in these cells that culminates in the ultimate steps of neural crest differentiation.

Cystein-serine-rich nuclear protein 1, Axud1/Csrnp1, is essential for cephalic neural progenitor proliferation and survival in zebrafish

The CSRNP (cystein‐serine‐rich nuclear protein) family has been conserved from Drosophila to human. Although knockout mice for each of the mammalian proteins have been generated, their function during vertebrate development has remained elusive. As an alternative to obtain insights on CSRNPs role in development, we have analysed the expression pattern and function of one member of this family, axud1, during zebrafish development. Our expression analysis indicates that axud1 is expressed from cleavage to larval stages in a dynamic pattern, becoming restricted after gastrulation to anterior regions of the developing neuraxis and later on concentrated predominantly in proliferating domains of the brain. Knockdown analysis using antisense morpholinos shows that reducing Axud1 levels impairs neural progenitor cell proliferation and survival, revealing an essential function of this gene for the growth of cephalic derivatives. The brain growth phenotypes elicited by decreasing Axud1 expression are specific and independent of anterior‐posterior patterning events, initial establishment of neural progenitors, or neural differentiation occurring in this tissue. However, Axud1 is necessary for six3.1 expression and is positively regulated by sonic hedgehog. Phylogenetic examination shows that axud1 is likely to be the ortholog of the only member of this family present in Drosophila, as well as to the previously described mouse CSRNP1 and to human AXUD1 (Axin upregulated‐1). Thus, we provide evidence as to the role of axud1 in brain growth in vertebrates. Developmental Dynamics, 2009.