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W de Graaff

Publications and source records attributed to W de Graaff.

9 recordsLinked to original sources
PubMed2001

Axial skeletal patterning in mice lacking all paralogous group 8 Hox genes.

We present a detailed study of the genetic basis of mesodermal axial patterning by paralogous group 8 Hox genes in the mouse. The phenotype of Hoxd8 loss-of-function mutants is presented, and compared with that of Hoxb8- and Hoxc8-null mice. Our analysis of single mutants reveals common features for the Hoxc8 and Hoxd8 genes in patterning lower thoracic and lumbar vertebrae. In the Hoxb8 mutant, more anterior axial regions are affected. The three paralogous Hox genes are expressed up to similar rostral boundaries in the mesoderm, but at levels that strongly vary with the axial position. We find that the axial region affected in each of the single mutants mostly corresponds to the area with the highest level of gene expression. However, analysis of double and triple mutants reveals that lower expression of the other two paralogous genes also plays a patterning role when the mainly expressed gene is defective. We therefore conclude that paralogous group 8 Hox genes are involved in patterning quite an extensive anteroposterior (AP) axial region. Phenotypes of double and triple mutants reveal that Hoxb8, Hoxc8 and Hoxd8 have redundant functions at upper thoracic and sacral levels, including positioning of the hindlimbs. Interestingly, loss of functional Hoxb8 alleles partially rescues the phenotype of Hoxc8- and Hoxc8/Hoxd8-null mutants at lower thoracic and lumbar levels. This suggests that Hoxb8 affects patterning at these axial positions differently from the other paralogous gene products. We conclude that paralogous Hox genes can have a unique role in patterning specific axial regions in addition to their redundant function at other AP levels.

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PubMed1998

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.

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PubMed1997

A 3' remote control region is a candidate to modulate Hoxb-8 expression boundaries.

Hox genes have been shown to play a key role in the acquisition of positional identity by precursors of embryonic axial, paraxial and limb structures. This function is thought to depend on the sequential, concerted expression of these genes in time and space. However the underlying molecular mechanisms of this collinear expression are still largely unknown. So far we had identified proximal regulatory elements driving expression of Hoxb-8/LacZ transgenes in Hox-like expression patterns with rostral boundaries more posterior than those of the endogenous gene. In this work we have analyzed 30 kb of 3' genomic sequences for Hoxb-8 regulatory activity in transgenic mice. We have identified a control region in the Hoxb-5/b-4 intergenic region that rostrally extends the Hoxb-8/LacZ expression domain into the posterior hindbrain. In combination with the Hoxb-8 minimal promoter, the 3' control region drives transgene expression with boundaries more anterior than those of Hoxb-8 in the neural tube. When combined with a 4.5 kb Hoxb-8 upstream sequence, where essential proximal regulatory sequences are located, the 3' control region drives transgene expression in a domain which seems to correspond to that of the endogenous Hoxb-8. By deletion analysis we have narrowed down to 550bp the regulatory activity interacting with the Hoxb-8 minimal promoter. We discuss the possibility that this remote 3' enhancer, which is the closest regulatory region found in the cluster to rostrally extend Hoxb-8/LacZ expression, could be involved in the regulation of Hoxb-8 and interact with the proximal control elements.

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PubMed1995

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.

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PubMed1995

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.

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PubMed1994

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.

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PubMed1993

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.

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PubMed1990

Expression of the murine homeobox-containing gene Hox-2.3 suggests multiple time-dependent and tissue-specific roles during development.

This study reports the expression pattern of the murine homeobox-containing gene Hox-2.3 during development. Using in situ hybridization, we first detect Hox-2.3 transcripts in the allantois primordium at 7.5 days post coitum (p.c.). One day later transcripts are found in embryonic ectoderm and mesoderm. In 9.5- and 10.5- day embryos Hox-2.3 expression is observed in the central nervous system (CNS) from a rostral boundary in the upper spinal cord to the caudal end. Within this anteroposterior domain, Hox-2.3 expression is also found in the peripheral nervous system, in the mesoderm and in the hindgut epithelium. The rostral boundary in the mesoderm is located at the level of the 11th somite and thus shifted posteriorwards compared to the rostral boundary in the neural tube. During subsequent development, the initially broad expression pattern in the somitic, lateral plate and intermediate mesoderm becomes restricted to structures in the urogenital system. In adults, the spinal cord and the derivatives of the Wolffian and Müllerian ducts continue to express the gene at a high level. The described temporal and tissue-specific changes in expression of Hox-2.3 are suggestive of several levels of regulation as reported for Drosophila homeotic genes and argue for more than one role of the gene during development and in adults.

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