Role of Dlx6 in regulation of an endothelin-1-dependent, dHAND branchial arch enhancer.

Neural crest cells play a key role in craniofacial development. The endothelin family of secreted polypeptides regulates development of several neural crest sublineages, including the branchial arch neural crest. The basic helix-loop-helix transcription factor dHAND is also required for craniofacial development, and in endothelin-1 (ET-1) mutant embryos, dHAND expression in the branchial arches is down-regulated, implicating it as a transcriptional effector of ET-1 action. To determine the mechanism that links ET-1 signaling to dHAND transcription, we analyzed the dHAND gene for cis-regulatory elements that control transcription in the branchial arches. We describe an evolutionarily conserved dHAND enhancer that requires ET-1 signaling for activity. This enhancer contains four homeodomain binding sites that are required for branchial arch expression. By comparing protein binding to these sites in branchial arch extracts from endothelin receptor A (EdnrA) mutant and wild-type mouse embryos, we identified Dlx6, a member of the Distal-less family of homeodomain proteins, as an ET-1-dependent binding factor. Consistent with this conclusion, Dlx6 was down-regulated in branchial arches from EdnrA mutant mice. These results suggest that Dlx6 acts as an intermediary between ET-1 signaling and dHAND transcription during craniofacial morphogenesis.

A GATA-dependent right ventricular enhancer controls dHAND transcription in the developing heart.

Heart formation in vertebrates is believed to occur in a segmental fashion, with discreet populations of cardiac progenitors giving rise to different chambers of the heart. However, the mechanisms involved in specification of different chamber lineages are unclear. The basic helix-loop-helix transcription factor dHAND is expressed in cardiac precursors throughout the cardiac crescent and the linear heart tube, before becoming restricted to the right ventricular chamber at the onset of looping morphogenesis. dHAND is also expressed in the branchial arch neural crest, which contributes to craniofacial structures and the aortic arch arteries. Using a series of dHAND-lacZ reporter genes in transgenic mice, we show that cardiac and neural crest expression of dHAND are controlled by separate upstream enhancers and we describe a composite cardiac-specific enhancer that directs lacZ expression in a pattern that mimics that of the endogenous dHAND gene throughout heart development. Deletion analysis reduced this enhancer to a 1.5 kb region and identified subregions responsible for expression in the right ventricle and cardiac outflow tract. Comparison of mouse regulatory elements required for right ventricular expression to the human dHAND upstream sequence revealed two conserved consensus sites for binding of GATA transcription factors. Mutation of these sites abolished transgene expression in the right ventricle, identifying dHAND as a direct transcriptional target of GATA factors during right ventricle development. Since GATA factors are not chamber-restricted, these findings suggest the existence of positive and/or negative coregulators that cooperate with GATA factors to control right ventricular-specific gene expression in the developing heart.

The bHLH transcription factor dHAND controls Sonic hedgehog expression and establishment of the zone of polarizing activity during limb development.

Limb outgrowth and patterning of skeletal elements are dependent on complex tissue interactions involving the zone of polarizing activity (ZPA) in the posterior region of the limb bud and the apical ectodermal ridge. The peptide morphogen Sonic hedgehog (SHH) is expressed specifically in the ZPA and, when expressed ectopically, is sufficient to mimic its functions, inducing tissue growth and formation of posterior skeletal elements. We show that the basic helix-loop-helix transcription factor dHAND is expressed posteriorly in the developing limb prior to Shh and subsequently occupies a broad domain that encompasses the Shh expression domain. In mouse embryos homozygous for a dHAND null allele, limb buds are severely underdeveloped and Shh is not expressed. Conversely, misexpression of dHAND in the anterior region of the limb bud of transgenic mice results in formation of an additional ZPA, revealed by ectopic expression of Shh and its target genes, and resulting limb abnormalities that include preaxial polydactyly with duplication of posterior skeletal elements. Analysis of mouse mutants in which Hedgehog expression is altered also revealed a feedback mechanism in which Hedgehog signaling is required to maintain the full dHAND expression domain in the developing limb. Together, these findings identify dHAND as an upstream activator of Shh expression and important transcriptional regulator of limb development.

Transducing positional information to the Hox genes: critical interaction of cdx gene products with position-sensitive regulatory elements.

Studies of pattern formation in the vertebrate central nervous system indicate that anteroposterior positional information is generated in the embryo by signalling gradients of an as yet unknown nature. We searched for transcription factors that transduce this information to the Hox genes. Based on the assumption that the activity levels of such factors might vary with position along the anteroposterior axis, we devised an in vivo assay to detect responsiveness of cis-acting sequences to such differentially active factors. We used this assay to analyze a Hoxb8 regulatory element, and detected the most pronounced response in a short stretch of DNA containing a cluster of potential CDX binding sites. We show that differentially expressed DNA binding proteins are present in gastrulating embryos that bind to these sites in vitro, that cdx gene products are among these, and that binding site mutations that abolish binding of these proteins completely destroy the ability of the regulatory element to drive regionally restricted expression in the embryo. Finally, we show that ectopic expression of cdx gene products anteriorizes expression of reporter transgenes driven by this regulatory element, as well as that of the endogenous Hoxb8 gene, in a manner that is consistent with them being essential transducers of positional information. These data suggest that, in contrast to Drosophila Caudal, vertebrate cdx gene products transduce positional information directly to the Hox genes, acting through CDX binding sites in their enhancers. This may represent the ancestral mode of action of caudal homologues, which are involved in anteroposterior patterning in organisms with widely divergent body plans and modes of development.

Hoxb-8 gain-of-function transgenic mice exhibit alterations in the peripheral nervous system.

To understand the developmental role of Hoxb-8, this relatively 5' Hoxb gene was ectopically expressed in embryonic regions where only more 3' Hox genes are normally expressed. Hoxb-8 coding sequences driven by a retinoic acid receptor beta2 promoter fragment were introduced in the mouse germ line by pronuclear injection. The promoter was chosen with the aim to extend rostrally the expression domain of the gene in neurectoderm and mesoderm at the time of development when Hox gene expression domains are being established. Embryos developing from DNA-injected zygotes, and from transgenic mouse lines were analyzed. Pattern alterations were observed in transgenic embryos, some of which involved the peripheral nervous system. Spinal ganglia in the mouse are first detectable around embryonic day 9.5. By day 11.5, the first of these ganglia (C1, Froriep's ganglion) has degenerated in the mouse and other amniotes. In contrast, this first ganglion did persist in the Hoxb-8 gain-of-function transgenic mice. We have started to take advantage of the phenotype of transgenic versus wild-type embryos to understand the mechanisms underlying the ontogeny and degeneration of Froriep's ganglion in wild-type mice, and the role of Hoxb-8 in C1 maintenance in transgenic embryos. The present work describes a morphological, histological and immunocytological analysis of both the degenerating and the permanent C1, and a preliminary characterization of the axonal extensions from the transgenic C1. We discuss the methodology of generating gain-of-function transgenic mice to study the genetics of pattern formation along the antero-posterior axis, and the usefulness of analyzing these particular Hoxb-8 transgenic embryos to understand some aspects of the ontogenesis and development of the upper cervical dorsal root ganglia.

Regulation of the Hoxb-8 gene: synergism between multimerized cis-acting elements increases responsiveness to positional information.

Hox genes play a key role in the specification of regional development in the vertebrate embryo. They are expressed in regionally restricted domains along the anterior-posterior axis, generally extending from a sharp rostral boundary toward the posterior end. We have studied the regulation of the murine Hoxb-8 gene in vivo using reporter constructs and found that 11 kb of genomic sequences upstream of Hoxb-8 confer a Hox-like pattern of expression on a lacZ reporter gene fused in-frame to the first exon of Hoxb-8. Reporter gene expression was detectable from early stages onwards, but reached rostral expression boundaries in mesoderm and neurectoderm that were more posterior than those of the endogenous gene. Within the upstream region, we have identified several cis-acting elements which are individually capable of driving regionally restricted expression in combination with the Hoxb-8 promoter, and we have investigated their relative contributions to the expression pattern. The results suggest that, in this experimental context, these upstream elements, as well as previously identified elements located within the Hoxb-8 gene, cooperate in setting the rostral expression boundaries. Furthermore, we show that multiple identical copies of cis-acting elements can cooperate, causing a pronounced anterior shift in the expression boundaries. This indicates that the rostral extent of expression is limited by the activity of a transcription factor binding to these elements and that this activity was, at some point in development, higher in precursors of more posterior structures than in precursors of anterior structures. This factor is thus very likely to be involved in the transduction of positional signals.

Specification of multiple vertebral identities by ectopically expressed Hoxb-8.

We have recently generated Hoxb-8 gain-of-function mutant embryos, using a Hoxb-8 transgene driven by a retinoic acid receptor beta 2 promoter to extend the expression domain to more anterior regions of the embryo (Charité et al. [1994] Cell 78:589-601). Here we describe the phenotype in the axial skeleton of transgenic embryos. The severity of the phenotype was variable, and cervical vertebrae and the base of the skull were affected in different ways. We observed fusion of the anterior arch of the atlas to the dens of the axis, partial splitting of the vertebral body and the neural arch of the axis, and abnormal morphology of the basioccipital and exoccipital bones. The basioccipital bone projected into the atlas, sometimes fusing to the dens of the axis; the exoccipitial bones appeared to be transformed towards neural arch-like structures. A novel pattern of posterior homeotic transformations was observed, involving cervical vertebrae C3 to C7: the ventral aspect of vertebrae C5 to C7 could acquire different morphologies characteristic of more posterior vertebrae: C5 could be transformed into C6, C7, or T1, C6 into C7 or T1, and C7 into T1. Phenotypes of different severity could be arranged into a phenotypic series, starting with the transformation of C7 to T1 and involving transformation of increasingly more anterior vertebrae into increasingly more posterior identities; no vertebra acquired a more posterior morphology than that of the vertebra immediately caudal to it. Ribs appeared to be formed relatively independently of rib heads; cervical ribs (but not rib heads) could be observed as anterior as C3. The results suggest that higher levels of ectopically expressed Hoxb-8 result in specification of more posterior vertebral identities.

Ectopic expression of Hoxb-8 causes duplication of the ZPA in the forelimb and homeotic transformation of axial structures.

Transgenic embryos were generated carrying a Hoxb-8 transgene under control of the mouse RAR beta 2 promoter, which extends the normal expression domain to more anterior regions of the embryo. These embryos showed mirror-image duplications in the forelimb, analogous to the duplications observed in chick in response to transplantation of a ZPA to the anterior margin of the limb bud. Examination of Sonic hedgehog, Fgf-4, and Hoxd-11 gene expression confirmed that a second ZPA had been generated at the anterior side of the limb bud. Besides other alterations, posterior homeotic transformations of axial structures were observed, involving the first spinal (Froriep's) ganglion and several cervical vertebrae.

Proximal cis-acting elements cooperate to set Hoxb-7 (Hox-2.3) expression boundaries in transgenic mice.

The Hox genes have been proved to be instrumental in establishing the positional identity of cells along the embryonic anteroposterior (A-P) axis. Studying the regulation of these genes is a first step toward elucidating the molecular basis of regionalization during embryogenesis. We report here on the identification of cis-acting elements controlling the expression of Hoxb-7 (Hox-2.3). We show that elements driving A-P restricted gene expression are located within the 3.5 kb proximal upstream sequences of the Hoxb-7 gene. A deletion analysis provides evidence for at least three cis-acting control elements upstream from Hoxb-7, and for cooperative interactions between some of these elements in generating the A-P restricted transgenic pattern. One element, conferring by itself Hox-like expression boundaries to the transgene, has been studied in more detail and found to act in an orientation-and promoter-dependent manner. Together the 3.5 kb sequences proximal to Hoxb-7 mediate A-P restricted Hoxb-7/lacZ gene expression in a domain showing rostral boundaries more posterior than those of Hoxb-7. The evolution throughout embryogenesis of the expression pattern of a transgene carrying these sequences has been analysed and shown to mimick that of the endogenous gene, except for a slight delay in the initial expression. We conclude that the transgenes that we tested, spanning a total of 27 kb genomic sequences, do not reproduce all the features of the Hoxb-7 expression pattern. The differences in expression between Hoxb-7 and the transgenes may reveal an aspect of the Hox regulation for which either remote cis-acting control elements and/or gene clustering is required. Additional features that may have favoured maintenance of clustered organisation during evolution are partial overlap of transcription units with the regulatory regions of the neighbouring genes, and cis-regulatory interactions between multiple Hox genes: not only do cis-acting control elements of the Hoxb-7 gene map in the 3' untranslated sequences of the Hoxb-8 (Hox-2.4) gene, but our experiments suggest that Hoxb-7 control sequences modulate expression of the Hoxb-8 gene as well.

Glu-69 of the D2 protein in photosystem II is a potential ligand to Mn involved in photosynthetic oxygen evolution.

To probe the involvement of amino acid residues of the D2 protein in the water-splitting process in photosystem II, site-directed mutagenesis was applied to identify D2 residues that might contribute to binding the Mn cluster involved in oxygen evolution. Mutation of Glu-69 to Gln or Val in D2 of the cyanobacterium Synechocystis sp. PCC 6803 was found to lead to a loss of photoautotrophic growth. However, in cells of the Gln mutant (E69Q) a significant Hill reaction rate could be observed upon the start of illumination, but the oxygen evolution rate declined with a half-time of approximately 1 min. Addition of 1 mM Mn2+ stabilized oxygen evolution in E69Q thylakoids. Other divalent cations were ineffective in specific stabilization. When the water-splitting system was bypassed, the rate of electron transport remained stable during illumination, indicating that the inactivation of oxygen evolution is localized in the water-splitting complex. We interpret these observations to indicate that Glu-69 is a Mn ligand and that the loss of oxygen evolution in the E69Q mutant upon turnover of PS II is initiated by changes in the Mn cluster, possibly leading to Mn release from the water-splitting complex. The addition of exogenous Mn to E69Q thylakoids may help to keep the Mn cluster active for a longer time, perhaps by providing Mn to rebind in the cluster after release of one Mn and before the Mn cluster had disintegrated.(ABSTRACT TRUNCATED AT 250 WORDS)

The primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus MHV-A59; a highly conserved polymerase is expressed by an efficient ribosomal frameshifting mechanism.

Sequence analysis of a substantial part of the polymerase gene of the murine coronavirus MHV-A59 revealed the 3' end of an open reading frame (ORF1a) overlapping with a large ORF (ORF1b; 2733 amino acids) which covers the 3' half of the polymerase gene. The expression of ORF1b occurs by a ribosomal frameshifting mechanism since the ORF1a/ORF1b overlapping nucleotide sequence is capable of inducing ribosomal frameshifting in vitro as well as in vivo. A stem-loop structure and a pseudoknot are predicted in the nucleotide sequence involved in ribosomal frameshifting. Comparison of the predicted amino acid sequence of MHV ORF1b with the amino acid sequence deduced from the corresponding gene of the avian coronavirus IBV demonstrated that in contrast to the other viral genes this ORF is extremely conserved. Detailed analysis of the predicted amino acid sequence revealed sequence elements which are conserved in many DNA and RNA polymerases.

The psbC start codon in Synechocystis sp. PCC 6803.

The translation start codon for psbC, the gene encoding CP43, a chlorophyll-binding protein of photosystem II, has been identified for the cyanobacterium Synechosystis sp. PCC 6803 using site-directed mutagenesis. An AUG codon, about 50 bases upstream from the end of psbD-I had previously been assumed to be the translation start site of psbC. However, the fact that the AUG codon is not present in psbC from several other organisms, whereas a GUG codon 14 bases upstream from the end of psbD-I is strictly conserved suggests that CP43 translation starts at the latter codon. Mutation of GUG, but not of AUG, led to a loss of CP43 and photoautotrophic growth, indicating that the GUG codon is the sole initiation site for translation of the CP43 protein in Synechocystis sp. PCC 6803.

Primary structure of the glycoprotein E2 of coronavirus MHV-A59 and identification of the trypsin cleavage site.

The nucleotide sequence of the peplomer (E2) gene of MHV-A59 was determined from a set of overlapping cDNA clones. The E2 gene encodes a protein of 1324 amino acids including a hydrophobic signal peptide. A second large hydrophobic domain is found near the COOH terminus and probably represents the membrane anchor. Twenty glycosylation sites are predicted. Cleavage of the E2 protein results in two different 90K species, 90A and 90B (L.S. Sturman, C. S. Ricard, and K. V. Holmes (1985) J. Virol. 56, 904-911), and activates cell fusion. Protein sequencing of the trypsin-generated N-terminus revealed the position of the cleavage site. 90A and 90B could be identified as the C-terminal and the N-terminal parts, respectively. Amino acid sequence comparison of the A59 and JHM E2 proteins showed extensive homology and revealed a stretch of 89 amino acids in the 90B region of the A59 E2 protein that is absent in JHM.