Ben-Zion Shilo

Insulin induces transcription of target genes through the hypoxia-inducible factor HIF-1alpha/ARNT.

Hypoxic stress induces the expression of genes associated with increased energy flux, including the glucose transporters Glut1 and Glut3, several glycolytic enzymes, nitric oxide synthase, tyrosine hydroxylase, erythropoietin and vascular endothelial growth factor (VEGF). Induction of these genes is mediated by a common basic helix–loop–helix‐PAS transcription complex, the hypoxia‐inducible factor‐1α (HIF‐1α)/aryl hydrocarbon nuclear translocator (ARNT). Insulin also induces some of these genes; however, the underlying mechanism is unestablished. We report here that insulin shares with hypoxia the ability to induce the HIF‐1α/ARNT transcription complex in various cell types. This induction was demonstrated by electrophoretic mobility shift of the hypoxia response element (HRE), and abolished by specific antisera to HIF‐1α and ARNT, and by transcription activation of HRE reporter vectors. Furthermore, basal and insulin‐induced expression of Glut1, Glut3, aldolase A, phosphoglycerate kinase and VEGF was reduced in cells having a defective ARNT. Similarly, the insulin‐induced activation of HRE reporter vectors and VEGF was impaired in these cells and was rescued by re‐introduction of ARNT. Finally, insulin‐like growth factor‐I (IGF‐I) also induced the HIF‐1α/ARNT transcription complex. These observations establish a novel signal transduction pathway of insulin and IGF‐I and broaden considerably the scope of activity of HIF‐1α/ARNT.

Robustness of the BMP morphogen gradient in Drosophila embryonic patterning.

Developmental patterning relies on morphogen gradients, which generally involve feedback loops to buffer against perturbations caused by fluctuations in gene dosage and expression. Although many gene components involved in such feedback loops have been identified, how they work together to generate a robust pattern remains unclear. Here we study the network of extracellular proteins that patterns the dorsal region of the Drosophila embryo by establishing a graded activation of the bone morphogenic protein (BMP) pathway. We find that the BMP activation gradient itself is robust to changes in gene dosage. Computational search for networks that support robustness shows that transport of the BMP class ligands (Scw and Dpp) into the dorsal midline by the BMP inhibitor Sog is the key event in this patterning process. The mechanism underlying robustness relies on the ability to store an excess of signalling molecules in a restricted spatial domain where Sog is largely absent. It requires extensive diffusion of the BMP–Sog complexes, coupled with restricted diffusion of the free ligands. We show experimentally that Dpp is widely diffusible in the presence of Sog but tightly localized in its absence, thus validating a central prediction of our theoretical study.

A thousand and one roles for the Drosophila EGF receptor

In the Drosophila genome there is a single member of the EGF receptor tyrosine kinase family. This receptor fulfills multiple roles during development, as reflected by the many designations given to mutant alleles in the locus (Egfr, DER, faint little ball, torpedo and Ellipse). The full scope of EGFR functions became apparent only in recent years: receptor activation was shown to have an instructive role in successive cell fate determination events during oogenesis, embryogenesis, and the proliferation and differentiation of imaginal discs. To ensure the fidelity of these processes, the precise place and time of receptor activation are tightly regulated by the localized presentation of activating ligands, in conjunction with a negative-feedback loop generated by an inhibitory secreted factor. The cellular mechanisms that translate EGFR activation to discrete cell fates are now the focus of intense studies.

Self-Enhanced Ligand Degradation Underlies Robustness of Morphogen Gradients

Morphogen gradients provide long-range positional information by extending across a developing field. To ensure reproducible patterning, their profile is invariable despite genetic or environmental fluctuations. Common models assume a morphogen profile that decays exponentially. Here, we show that exponential profiles cannot, at the same time, buffer fluctuations in morphogen production rate and define long-range gradients. To comply with both requirements, morphogens should decay rapidly close to their source but at a significantly slower rate over most of the field. Numerical search revealed two network designs that support robustness to fluctuations in morphogen production rate. In both cases, morphogens enhance their own degradation, leading to a higher degradation rate close to their source. This is achieved through reciprocal interactions between the morphogen and its receptor. The two robust networks are consistent with properties of the Wg and Hh morphogens in the Drosophila wing disc and provide novel insights into their function.

The Drosophila EGF receptor homolog (DER) gene is allelic to faint little ball, a locus essential for embryonic development

Recessive lethal mutations in the genetic locus of the Drosophila EGF receptor homolog (DER) were isolated. Identification of mutations in the gene is based on assays of DER protein autophosphorylation activity. Most DER alleles show little or no in vivo autophosphorylation. The ability to monitor these activities in vivo and in vitro offers a preliminary insight into the functional defects in the different mutant proteins. The identification of the DER locus was also confirmed by partial rescue of the mutant phenotype with a DER P-element construct. Homozygous DER mutants display a complex embryonic phenotype. Most notably, the anterior structures deteriorate, ventral denticle bands are missing, the germ band does not retract, and the central nervous system shows a collapse of commissure and midline pattern. Mutations in DER were shown to be allelic to the previously described locus faint little ball.

Scaling of the BMP activation gradient in Xenopus embryos

In groundbreaking experiments, Hans Spemann demonstrated that the dorsal part of the amphibian embryo can generate a well-proportioned tadpole, and that a small group of dorsal cells, the ‘organizer’, can induce a complete and well-proportioned twinned axis when transplanted into a host embryo. Key to organizer function is the localized secretion of inhibitors of bone morphogenetic protein (BMP), which defines a graded BMP activation profile. Although the central proteins involved in shaping this gradient are well characterized, their integrated function, and in particular how pattern scales with size, is not understood. Here we present evidence that in Xenopus, the BMP activity gradient is defined by a ‘shuttling-based’ mechanism, whereby the BMP ligands are translocated ventrally through their association with the BMP inhibitor Chordin. This shuttling, with feedback repression of the BMP ligand Admp, offers a quantitative explanation to Spemann’s observations, and accounts naturally for the scaling of embryo pattern with its size.

Regulation of Cell Number by MAPK-Dependent Control of Apoptosis: A Mechanism for Trophic Survival Signaling

Trophic mechanisms in which neighboring cells mutually control their survival by secreting extracellular factors play an important role in determining cell number. However, how trophic signaling suppresses cell death is still poorly understood. We now show that the survival of a subset of midline glia cells in Drosophila depends upon direct suppression of the proapoptotic protein HID via the EGF receptor/RAS/MAPK pathway. The TGFalpha-like ligand SPITZ is activated in the neurons, and glial cells compete for limited amounts of secreted SPITZ to survive. In midline glia that fail to activate the EGFR pathway, HID induces apoptosis by blocking a caspase inhibitor, Diap1. Therefore, a direct pathway linking a specific extracellular survival factor with a caspase-based death program has been established.

The Drosophila EGF receptor gene homolog: Conservation of both hormone binding and kinase domains

Chicken v-erB probe was used to isolate a unique clone of Drosophila melanogaster DNA. It maps by in situ hybridization to position 57F on chromosome 2. A complete nucleotide sequence of the coding region has been obtained. The putative Drosophila EGF receptor protein is similar in overall organization to the human homolog. It shows three distinct domains: an extracellular putative EGF binding domain, a hydrophobic transmembrane region, and a cytoplasmic kinase domain. The overall amino acid homology is 41% in the extracellular domain and 55% in the kinase domain. Two cysteine-rich regions, a hallmark of the human ligand-binding domain, have also been conserved. Fusion of the coding sequences of the kinase and extracellular domains generating the receptor gene must have occurred over 800 million years ago.

Regulating the dynamics of EGF receptor signaling in space and time

The epidermal growth factor receptor (EGFR) signaling cascade represents one of the cardinal pathways that transmits information between cells during development in a broad range of multicellular organisms. Most of the elements that constitute the core EGFR signaling module, as well as a variety of negative and positive modulators, have been identified. Although this molecular pathway is utilized multiple times during development, the spatial and temporal features of its signaling can be modified to fit a particular developmental setting. Recent work has unraveled the various mechanisms by which the EGFR pathway can be modulated.

Pre-Steady-State Decoding of the Bicoid Morphogen Gradient

Morphogen gradients are established by the localized production and subsequent diffusion of signaling molecules. It is generally assumed that cell fates are induced only after morphogen profiles have reached their steady state. Yet, patterning processes during early development occur rapidly, and tissue patterning may precede the convergence of the gradient to its steady state. Here we consider the implications of pre-steady-state decoding of the Bicoid morphogen gradient for patterning of the anterior–posterior axis of the Drosophila embryo. Quantitative analysis of the shift in the expression domains of several Bicoid targets (gap genes) upon alteration of bcd dosage, as well as a temporal analysis of a reporter for Bicoid activity, suggest that a transient decoding mechanism is employed in this setting. We show that decoding the pre-steady-state morphogen profile can reduce patterning errors caused by fluctuations in the rate of morphogen production. This can explain the surprisingly small shifts in gap and pair-rule gene expression domains observed in response to alterations in bcd dosage.