Karen A. Schachter

Mutations in CDON, Encoding a Hedgehog Receptor, Result in Holoprosencephaly and Defective Interactions with Other Hedgehog Receptors

Holoprosencephaly (HPE), a common human congenital anomaly defined by a failure to delineate the midline of the forebrain and/or midface, is associated with diminished Sonic hedgehog (SHH)-pathway activity in development of these structures. SHH signaling is regulated by a network of ligand-binding factors, including the primary receptor PTCH1 and the putative coreceptors, CDON (also called CDO), BOC, and GAS1. Although binding of SHH to these receptors promotes pathway activity, it is not known whether interactions between these receptors are important. We report here identification of missense CDON mutations in human HPE. These mutations diminish CDONs ability to support SHH-dependent gene expression in cell-based signaling assays. The mutations occur outside the SHH-binding domain of CDON, and the encoded variant CDON proteins do not display defects in binding to SHH. In contrast, wild-type CDON associates with PTCH1 and GAS1, but the variants do so inefficiently, in a manner that parallels their activity in cell-based assays. Our findings argue that CDON must associate with both ligand and other hedgehog-receptor components, particularly PTCH1, for signaling to occur and that disruption of the latter interactions is a mechanism of HPE.

Cdc42 Induces Activation Loop Phosphorylation and Membrane Targeting of Mixed Lineage Kinase 3

Mixed lineage kinase 3 (MLK3) functions as a mitogen-activated protein kinase kinase kinase to activate multiple mitogen-activated protein kinase pathways. Our current studies demonstrate that lack of MLK3 blocks signaling of activated Cdc42 to c-Jun N-terminal kinase, giving strong support for the idea that Cdc42 is a physiological activator of MLK3. We show herein that Cdc42, in a prenylation-dependent manner, targets MLK3 from a perinuclear region to membranes, including the plasma membrane. Cdc42-induced membrane targeting of MLK3 is independent of MLK3 catalytic activity but depends upon an intact Cdc42/Rac-interactive binding motif, consistent with MLK3 membrane translocation being mediated through direct binding of Cdc42. Phosphorylation of the activation loop of MLK3 requires MLK3 catalytic activity and is induced by Cdc42 in a prenylation-independent manner, arguing that Cdc42 binding is sufficient for activation loop autophosphorylation of MLK3. However, membrane targeting is necessary for full activation of MLK3 and maximal signaling to JNK. We previously reported that MLK3 is autoinhibited through an interaction between its N-terminal SH3 domain and a proline-containing sequence found between the leucine zipper and the CRIB motif of MLK3. Thus we propose a model in which GTP-bound Cdc42/Rac binds MLK3 and disrupts SH3-mediated autoinhibition leading to dimerization and activation loop autophosphorylation. Targeting of this partially active MLK3 to membranes likely results in additional phosphorylation events that fully activate MLK3 and its ability to maximally signal through the JNK pathway.

β-Catenin Regulates Primitive Streak Induction through Collaborative Interactions with SMAD2/SMAD3 and OCT4

Canonical Wnt and Nodal signaling are both required for induction of the primitive streak (PS), which guides organization of the early embryo. The Wnt effector β-catenin is thought to function in these early lineage specification decisions via transcriptional activation of Nodal signaling. Here, we demonstrate a broader role for β-catenin in PS formation by analyzing its genome-wide binding in a human embryonic stem cell model of PS induction. β-catenin occupies regulatory regions in numerous PS and neural crest genes, and direct interactions between β-catenin and the Nodal effectors SMAD2/SMAD3 are required at these regions for PS gene activation. Furthermore, OCT4 binding in proximity to these sites is likewise required for PS induction, suggesting a collaborative interaction between β-catenin and OCT4. Induction of neural crest genes by β-catenin is repressed by SMAD2/SMAD3, ensuring proper lineage specification. This study provides mechanistic insight into how Wnt signaling controls early cell lineage decisions.

Differences in expression of two endoxylanase genes (xynA and xynB) from Penicillium purpurogenum.

A number of xylanolytic microorganisms secrete to the medium several molecular forms of endoxylanases. The physiological function of these isoforms is not clear; one possibility is that they are produced under different growth conditions. To study this problem, we have used two endoxylanases (XynA and XynB) produced by the fungus Penicillium purpurogenum. These enzymes have been previously purified and characterized; they belong to family 10 and 11 of the glycosyl hydrolases, respectively. The promoters of the xynA and xynB genes have been sequenced; both present consensus sequences for the binding of the carbon catabolite repressor CreA, but otherwise show substantial differences. The xynB promoter has eight boxes in tandem for the binding of the XlnR activator and lacks the consensus sequence for the PacC pH regulator. On the other hand, the xynA promoter contains one XlnR box and three PacC consensus sequences. To investigate if these differences are reflected in gene expression, Northern blot assays were carried out. The xynA gene is transiently expressed when oat spelt xylan is used as carbon source, but negligible expression was observed with birchwood xylan, xylose or xylitol. In contrast, xynB is broadly induced by all these carbon sources; this may be related to the presence of several XlnR boxes. Similar results were obtained by zymogram analysis of the expressed proteins. The different induction capabilities of birchwood and oat spelt xylan may be due to differences in their composition and structure. Expression assays carried out at different pH reflects that, despite the lack of PacC binding sites in the xynB promoter, this gene is tightly regulated by pH. The findings described here illustrate new and important differences between endoxylanases from families 10 and 11 in P. purpurogenum. They may help explain the production of multiple endoxylanase forms by this organism.

Chapter 3 Murine Models of Holoprosencephaly

Holoprosencephaly (HPE), the most common developmental defect of the forebrain and midface, is caused by a failure to delineate the midline in these structures. Both genetic and environmental etiologies exist for HPE, and clinical presentation is highly variable. HPE occurs in sporadic and inherited forms, and even HPE in pedigrees is characterized by incomplete penetrance and variable expressivity. Heterozygous mutations in eight different genes have been identified in human HPE, and disruption of Sonic hedgehog expression and/or signaling in the rostroventral region of the embryo is a major common effect of these mutations. An understanding of the mechanisms whereby genetic defects and teratogenic exposures become manifest as developmental anomalies of varying severity requires experimental models that accurately reproduce the spectrum of defects seen in human HPE. The mouse has emerged as such a model, because of its ease of genetic manipulation and similarity to humans in development of the forebrain and face. HPE is generally observed in mice homozygous for mutations in orthologs of human HPE genes though, unlike humans, rarely in mice with heterozygous mutations. Moreover, reverse genetics in the mouse has provided a wealth of new candidate human HPE genes. Construction of hypomorphic alleles, interbreeding to produce double mutants, and analysis of these mutations on different genetic backgrounds has generated multiple models of HPE and begun to provide insight into the conundrum of the HPE spectrum. Here, we review forebrain development with an emphasis on the pathways known to be defective in HPE and describe the strengths and weaknesses of various murine models of HPE.

Dynamic Positive Feedback Phosphorylation of Mixed Lineage Kinase 3 by JNK Reversibly Regulates Its Distribution to Triton-soluble Domains

MLK3 (mixed lineage kinase 3) is a widely expressed, mammalian serine/threonine protein kinase that activates multiple MAPK pathways. Previously our laboratory used in vivo labeling/mass spectrometry to identify phosphorylation sites of activated MLK3. Seven of 11 identified sites correspond to the consensus motif for phosphorylation by proline-directed kinases. Based on these results, we hypothesized that JNK, or another proline-directed kinase, phosphorylates MLK3 as part of a feedback loop. Herein we provide evidence that MLK3 can be phosphorylated by JNK in vitro and in vivo. Blockade of JNK results in dephosphorylation of MLK3. The hypophosphorylated form of MLK3 is inactive and redistributes to a Triton-insoluble fraction. Recovery from JNK inhibition restores MLK3 solubility and activity, indicating that the redistribution process is reversible. This work describes a novel mode of regulation of MLK3, by which JNK-mediated feedback phosphorylation of MLK3 regulates its activation and deactivation states by cycling between Triton-soluble and Triton-insoluble forms.

Structure analysis of the endoxylanase. A gene from penicillium purpurogenum

Penicillium purpurogenum produces several endoxylanases, two of which (XynA and XynB) have been purified and characterized. XynB has been sequenced, and it belongs to glycosyl hydrolase family 11. In this publication we report the structure of the xynA gene. The amino terminal sequence of the protein was determined and this allowed the design of oligonucleotides for use in polymerase chain reactions. Different polymerase chain reaction strategies were used to amplify and sequence the entire cDNA and the gene. The gene has an open reading frame of 1450 base pairs, including 8 introns with an average length of 56 base pairs each. Only one copy of this gene is present in the P. purpurogenum genome as shown by Southern blot. The gene encodes a protein of 329 residues (including the signal peptide), and the calculated molecular mass of the mature protein is 31,668 Da. Immunodetection assays of the expressed gene positively identified it as xynA, and sequence alignments indicate a high degree of similarity with family 10 endoxylanases. It is concluded that P. purpurogenum produces endoxylanases of family 10 and 11. The complementary action of endoxylanases of both families may be important for an efficient degradation of xylan by the fungus.

The acetyl xylan esterase II gene from Penicillium purpurogenum is differentially expressed in several carbon sources, and tightly regulated by pH

The expression of the acetyl xylan esterase II (axeII) gene from Penicillium purpurogenum is repressed by glucose and induced by xylan, as well as to a small degree by xylose and xylitol. This gene is expressed at neutral pH, but not under alkaline or acidic conditions, in agreement with previous findings for other xylanolytic genes of this organism. This is the first report showing pH regulation of an axe gene.

Neogenin regulates sonic hedgehog pathway activity during digit patterning

Background: Digit patterning integrates signaling by the Sonic Hedgehog (SHH), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) pathways. GLI3, a component of the SHH pathway, is a major regulator of digit number and identity. Neogenin (encoded by Neo1) is a cell surface protein that serves to transduce signals from several ligands, including BMPs, in various developmental contexts. Although neogenin is implicated in BMP signaling, it has not been linked to SHH signaling and its role in digit patterning is unknown. Results: We report that Neo1 mutant mice have preaxial polydactyly with low penetrance. Expression of SHH target genes, but not BMP target genes, is altered in Neo1 mutant limb buds. Analysis of mice carrying mutations in both Neo1 and Gli3 reveals that, although neogenin plays a role in constraint of digit numbers, suppressing polydactyly, it is also required for the severe polydactyly caused by loss of GLI3. Furthermore, embryo fibroblasts from Neo1 mutant mice are sensitized to SHH pathway activation in vitro. Conclusions: Our findings indicate that neogenin regulates SHH signaling in the limb bud to achieve proper digit patterning. Developmental Dynamics 241:627–637, 2012.