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Biomedical subjects

Robert E Hill

Publications and source records attributed to Robert E Hill.

At least 19 recordsLinked to original sources
PubMed2006

Spleen versus pancreas: strict control of organ interrelationship revealed by analyses of Bapx1-/- mice.

During early stages of pancreatic development, the mesenchyme that contributes to the spleen overlies the dorsal pancreatic endoderm. Here, we show that interactions between splenic mesenchyme and pancreas proceed via a highly orchestrated morphogenetic program. Disruption of morphogenesis, as occurs in the Bapx1(Nkx3.2)(-/-) embryo, results in transformation of these tissues into well-organized, ectopic gut-like structures. Bapx1 plays a crucial organizing role effecting position and separation of the spleen and pancreas to prevent this metaplastic transformation. Similar transformations occur in organ cultures employing wild-type pancreatic endoderm and spleen mesenchyme, revealing the developmental plasticity of the pancreas and that precise spatial and temporal control of tissue interactions are required for development of both organs.

Animals↗
PubMed2005

Preaxial polydactyly: a model for defective long-range regulation in congenital abnormalities.

Point mutations in the long-range, limb-specific regulatory element of the SHH gene are responsible for the human limb abnormality called preaxial polydactyly (PPD). Disruptions of regulatory elements in developmental genes are a small but increasingly significant class of mutations responsible for congenital defects. Identifying regulatory elements that might reside hundreds of kilobases from their relevant genes is difficult but rendered possible by the emerging field of comparative genomics. Genetic analysis of PPD highlights the notion that regulatory mutations might generate phenotypes distinct from any of those identified for coding region mutations.

Animals↗
PubMed2004

The splanchnic mesodermal plate directs spleen and pancreatic laterality, and is regulated by Bapx1/Nkx3.2.

The mechanism by which left-right (LR) information is interpreted by organ primordia during asymmetric morphogenesis is largely unknown. We show that spleen and pancreatic laterality is dependent on a specialised, columnar mesodermal-derived cell layer referred to here as the splanchnic mesodermal plate (SMP). At early embryonic stages, the SMP is bilateral, surrounding the midline-located stomach and dorsal pancreatic bud. Under control of the LR asymmetry pathway, the left SMP is maintained and grows laterally. Mice carrying the dominant hemimelia (Dh) mutation lack the SMP. Significantly, the mice are asplenic and the pancreas remains positioned along the embryonic midline. In the absence of Fgf10 expression, the spleno-pancreatic mesenchyme and surrounding SMP grow laterally but contain no endodermal component, showing that leftward growth is autonomous and independent of endoderm. In the Bapx1(-/-) mutants, the SMP is defective. Normally, the SMP is a source for both Fgf9 and Fgf10; however, in the Bapx1 mutant, Fgf10 expression is downregulated and the dorsal pancreas remains at the midline. We conclude that the SMP is an organiser responsible for the leftward growth of the spleno-pancreatic region and that Bapx1 regulates SMP functions required for pancreatic laterality.

Abnormalities, Multiple↗
PubMed2004

Bapx1 regulates patterning in the middle ear: altered regulatory role in the transition from the proximal jaw during vertebrate evolution.

The middle ear apparatus is composed of three endochondrial ossicles (the stapes, incus and malleus) and two membranous bones, the tympanic ring and the gonium, which act as structural components to anchor the ossicles to the skull. Except for the stapes, these skeletal elements are unique to mammals and are derived from the first and second branchial arches. We show that, in combination with goosecoid (Gsc), the Bapx1 gene defines the structural components of the murine middle ear. During embryogenesis, Bapx1 is expressed in a discrete domain within the mandibular component of the first branchial arch and later in the primordia of middle ear-associated bones, the gonium and tympanic ring. Consistent with the expression pattern of Bapx1, mouse embryos deficient for Bapx1 lack a gonium and display hypoplasia of the anterior end of the tympanic ring. At E10.5, expression of Bapx1 partially overlaps that of Gsc and although Gsc is required for development of the entire tympanic ring, the role of Bapx1 is restricted to the specification of the gonium and the anterior tympanic ring. Thus, simple overlapping expression of these two genes appears to account for the patterning of the elements that compose the structural components of the middle ear and suggests that they act in concert. In addition, Bapx1 is expressed both within and surrounding the incus and the malleus. Examination of the malleus shows that the width, but not the length, of this ossicle is decreased in the mutant mice. In non-mammalian jawed vertebrates, the bones homologous to the mammalian middle ear ossicles compose the proximal jaw bones that form the jaw articulation (primary jaw joint). In fish, Bapx1 is responsible for the formation of the joint between the quadrate and articular (homologues of the malleus and incus, respectively) enabling an evolutionary comparison of the role of a regulatory gene in the transition of the proximal jawbones to middle ear ossicles. Contrary to expectations, murine Bapx1 does not affect the articulation of the malleus and incus. We show that this change in role of Bapx1 following the transition to the mammalian ossicle configuration is not due to a change in expression pattern but results from an inability to regulate Gdf5 and Gdf6, two genes predicted to be essential in joint formation.

Animals↗
PubMed2004

'Gotta pick a megabase or two': in silico routes to gene regulation.

A number of vertebrate genome sequences are now available in draft or high-quality form. By comparing genomes from related species, conserved elements that are located outside the coding regions can be identified, many of which represent regulatory elements. The design of the sequence comparisons, taking into account the extent of the evolutionary divergence, is crucial to the outcome. Clearly, investigations of these conserved regulatory elements are important in understanding mechanisms underlying both vertebrate evolution and human disease.

Animals↗
PubMed2004

Corneal development, limbal stem cell function, and corneal epithelial cell migration in the Pax6(+/-) mouse.

PURPOSE: To investigate the etiology of corneal dysfunction in the Pax6(+/-) mouse model of aniridia-related keratopathy. METHODS: Mosaic patterns of X-gal staining were compared in the corneal and limbal epithelia of female Pax6(+/-) and Pax6(+/+) littermates, age 3 to 28 weeks, hemizygous for an X-linked LacZ transgene, and Pax6(+/+), LacZ(-)<-->Pax6(+/+), LacZ(+) and Pax6(+/+), LacZ(-)<-->Pax6(+/-), LacZ(+) chimeras. Histologic examination of chimeric corneas was performed. RESULTS: Disrupted patterns of X-gal staining showed that heterozygosity for Pax6 perturbed clonal patterns of growth and development in the corneal and limbal epithelium. Centripetal migration of Pax6(+/-) corneal epithelial cells was diverted. Normal patterns of centripetal Pax6(+/-) cell migration and epithelial morphology were restored in Pax6(+/+)<-->Pax6(+/-) chimeras. Fewer, larger clones of limbal stem cells were present in Pax6(+/-) eyes, compared with wild-type. In the chimeras, Pax6(+/-) limbal stem cells were cell-autonomously depleted or less efficient than wild-type cells at producing progeny to populate the corneal epithelium. CONCLUSIONS: The correct Pax6 dosage is necessary for normal clonal growth during corneal development, normal limbal stem cell activity, and correct corneal epithelial cell migration. Disruption of normal cell movement in heterozygotes may be the consequence of failure of nonautonomous guidance cues. Degeneration of the corneal surface in aniridia-related keratopathy relates to both a deficiency within the limbal stem cell niche and nonautonomous diversion of corneal epithelial cell migration.

Animals↗
PubMed2004

Analysis of mouse eye development with chimeras and mosaics.

Analysis of experimental mouse chimeras (chimaeras) and mosaics provides a means of investigating patterning and differentiation within the developing mammalian eye. Chimeric and mosaic mice carry two or more genetically distinct cell populations and extend the repertoire of analytical tools available to the geneticist. Here we review the impact these techniques have had on our understanding of eye organogenesis. Chimeras and mosaics are routinely used to investigate cell lineages, patterns of growth and gene function, and provide a means to clear analytical hurdles that otherwise limit standard genetic approaches. In particular, chimeras are used to investigate the roles of genes in tissues that do not develop in conventional mutant or knock-out mice, to test whether genes act cell autonomously or non-autonomously in different tissues and to dissect tissue-tissue interactions in less tractable, complex systems. Chimeras, in which cells of different genetic composition are mixed at a fine-scale cellular level, may provide qualitatively different data from mosaic mice with conditional knockouts. The uses of chimeras, Cre-loxP mosaics and in vitro tissue recombination for study of ocular organogenesis are compared. Wider use of mosaics and chimeras should provide further insights into eye development.

Animals↗
PubMed2003

A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly.

Unequivocal identification of the full composition of a gene is made difficult by the cryptic nature of regulatory elements. Regulatory elements are notoriously difficult to locate and may reside at considerable distances from the transcription units on which they operate and, moreover, may be incorporated into the structure of neighbouring genes. The importance of regulatory mutations as the basis of human abnormalities remains obscure. Here, we show that the chromosome 7q36 associated preaxial polydactyly, a frequently observed congenital limb malformation, results from point mutations in a Shh regulatory element. Shh, normally expressed in the ZPA posteriorly in the limb bud, is expressed in an additional ectopic site at the anterior margin in mouse models of PPD. Our investigations into the basis of the ectopic Shh expression identified the enhancer element that drives normal Shh expression in the ZPA. The regulator, designated ZRS, lies within intron 5 of the Lmbr1 gene 1 Mb from the target gene Shh. The ZRS drives the early spatio-temporal expression pattern in the limb of tetrapods. Despite the morphological differences between limbs and fins, an equivalent regulatory element is found in fish. The ZRS contains point mutations that segregate with polydactyly in four unrelated families with PPD and in the Hx mouse mutant. Thus point mutations residing in long-range regulatory elements are capable of causing congenital abnormalities, and possess the capacity to modify gene activity such that a novel gamut of abnormalities is detected.

Animals↗
PubMed2003

The roles of Pax6 in the cornea, retina, and olfactory epithelium of the developing mouse embryo.

The roles of Pax6 were investigated in the murine eye and the olfactory epithelium by analysing gene expression and distribution of Pax6(-/-) cells in Pax6(+/+) <--> Pax6(-/-) chimeras. It was found that between embryonic days E10.5 and E16.5 Pax6 is autonomously required for cells to contribute fully not only to the corneal epithelium, where Pax6 is expressed at high levels, but also to the to the corneal stroma and endothelium, where the protein is detected at very low levels. Pax6(-/-) cells contributed only poorly to the neural retina, forming small clumps of cells that were normally restricted to the ganglion cell layer at E16.5. Pax6(-/-) cells in the retinal pigment epithelium could express Trp2, a component of the pigmentation pathway, at E14.5 and a small number went on to differentiate and produce pigment at E16.5. The segregation and near-exclusion of mutant cells from the nasal epithelium mirrored the behaviour of mutant cells in other developmental contexts, particularly the lens, suggesting that common primary defects may be responsible for diverse Pax6-related phenotypes.

Animals↗
PubMed2003

Pax6 regulates regional development and neuronal migration in the cerebral cortex.

Mutations in the Pax6 gene disrupt telencephalic development, resulting in a thin cortical plate, expansion of proliferative layers, and the absence of the olfactory bulb. The primary defect in the neuronal cell population of the developing cerebral cortex was analysed by using mouse chimeras containing a mixture of wild-type and Pax6-deficient cells. The chimeric analysis shows that Pax6 influences cellular activity throughout corticogenesis. At early stages, Pax6-deficient and wildtype cells segregate into exclusive patches, indicating an inability of different cell genotypes to interact. At later stages, cells are sorted further based on telencephalic domains. Pax6-deficient cells are specifically reduced in the mediocaudal domain of the dorsal telencephalon, indicating a role in regionalization. In addition, Pax6 regulates the process of radial migration of neuronal precursors. Loss of Pax6 particularly affects movement of neuronal precursors at the subventricular zone/intermediate zone boundary at a transitional migratory phase essential for entry into the intermediate zone. We suggest that the primary role of Pax6 is the continual regulation of cell surface properties responsible for both cellular identity and radial migration, defects of which cause regional cell sorting and abnormalities of migration in chimeras.

Animals↗
PubMed2003

Sonic hedgehog: restricted expression and limb dysmorphologies.

Sonic hedgehog, SHH, is required for patterning the limb. The array of skeletal elements that compose the hands and feet, and the ordered arrangement of these bones to form the pattern of fingers and toes are dependent on SHH. The mechanism of action of SHH in the limb is not fully understood; however, an aspect that appears to be important is the localized, asymmetric expression of Shh. Shh is expressed in the posterior margin of the limb bud in a region defined as the zone of polarizing activity (ZPA). Analysis of mouse mutants which have polydactyly (extra toes) shows that asymmetric expression of Shh is lost due to the appearance of an ectopic domain of expression in the anterior limb margin. One such polydactylous mouse mutant, sasquatch (Ssq), maps to the corresponding chromosomal region of the human condition pre-axial polydactyly (PPD) and thus represents a model for this condition. The mutation responsible for Ssq is located 1 Mb away from the Shh gene; however, the mutation disrupts a long-range cis-acting regulator of Shh expression. By inference, human pre-axial polydactyly results from a similar disruption of Shh expression. Other human congenital abnormalities also map near the pre-axial polydactyly locus, suggesting a major chromosomal region for limb dysmorphologies. The distinct phenotypes range from loss of all bones of the hands and feet to syndactyly of the soft tissue and fusion of the digits. We discuss the role played by Shh expression in mouse mutant phenotypes and the human limb dysmorphologies.

Embryonic Induction↗
PubMed2003

Australian tiger snake (Notechis scutatus) and mexican coral snake (Micruris species) antivenoms prevent death from United States coral snake (Micrurus fulvius fulvius) venom in a mouse model.

BACKGROUND: Wyeth-Ayerst has discontinued production of Antivenin (Micrurus fulvius). Currently, there is no other approved coral snake antivenom available in the United States. METHODS: This study was a randomized, placebo-controlled and blinded determination of the ability of a Mexican Micrurus (coral snake) antivenom and an Australian Notechis (tiger snake) antivenom to prevent lethality from a United States Micrurus fulvius fulvius venom in a mouse model. Venom dosing was based on an LD50 determined for this experiment. Our comparison groups included: (1) M. f. fulvius venom + Micrurus antivenom, (2) M. f. fulvius venom + Notechis antivenom, (3) M. f. fulvius venom + protein control, (4) 0.9% normal saline + protein control, (5) saline + Notechis antivenom, (6) saline + Micrurus antivenom. Venom dose was 5 times the determined LD50. The antivenom amounts were capable of neutralizing 10 times the venom injected (50 times the LD50). RESULTS: The LD50 of M. f. fulvius venom was determined to be 0.85 mg/kg. All mice in both antivenom test groups were protected from lethality for the entire 24-hour observation period. Six of the 7 mice in the venom test group died, with a survival time of 349 +/- 382 minutes (mean +/- s.d.) after the venom injection. All three groups of control mice survived the entire 24-hour observation period. CONCLUSIONS: Mexican Micrurus antivenom and Australian Notechis antivenom provide protection from lethality in mice envenomated with a United States M. f. filvius venom.

Animals↗
PubMed2003

Signal sequence conservation and mature peptide divergence within subgroups of the murine beta-defensin gene family.

Beta-defensins are two exon genes which encode broad spectrum antimicrobial cationic peptides. We have analyzed the largest murine cluster of these genes which localizes to chromosome 8. Using hidden Markov models, we identified six beta-defensin exon 2-like sequences and subsequently found full-length expressed transcripts for these novel genes. Expression was high in brain and reproductive tissues. Eleven beta-defensins could be grouped into two clear subgroups by virtue of their position and high signal sequence (exon 1 encoded) identity. In contrast, however, there was a very low level of sequence conservation in the exon 2 region encoding the mature antimicrobial peptide. Examination of the gene sequences of orthologs in other rodents also revealed an excess of nucleotide changes that altered amino acids in the mature peptide region. Evolutionary analysis revealed strong evidence that following gene duplication, exon 1 and surrounding noncoding DNA show little divergence within subgroups. The focus for rapid sequence divergence is localized in the DNA encoding the mature peptide and this is driven by accelerated positive selection. This mechanism of evolution is consistent with the role of this gene family as defense against bacterial pathogens and the sequence changes have implications for novel antibiotic design.

Animals↗
PubMed2003

Pax1 and Pax9 activate Bapx1 to induce chondrogenic differentiation in the sclerotome.

We have previously shown that the paired-box transcription factors Pax1 and Pax9 synergistically act in the proper formation of the vertebral column. Nevertheless, downstream events of the Pax1/Pax9 action and their target genes remain to be elucidated. We show, by analyzing Pax1;Pax9 double mutant mice, that expression of Bapx1 in the sclerotome requires the presence of Pax1 and Pax9, in a gene dose-dependent manner. By using a retroviral system to overexpress Pax1 in chick presomitic mesoderm explants, we show that Pax1 can substitute for Shh in inducing Bapx1 expression and in initiating chondrogenic differentiation. Furthermore, we demonstrate that Pax1 and Pax9 can transactivate regulatory sequences in the Bapx1 promoter and that they physically interact with the Bapx1 promoter region. These results strongly suggest that Bapx1 is a direct target of Pax1 and Pax9. Together, we conclude that Pax1 and Pax9 are required and sufficient for the chondrogenic differentiation of sclerotomal cells.

Animals↗
PubMed2002

Disruption of a long-range cis-acting regulator for Shh causes preaxial polydactyly.

Preaxial polydactyly (PPD) is a common limb malformation in human. A number of polydactylous mouse mutants indicate that misexpression of Shh is a common requirement for generating extra digits. Here we identify a translocation breakpoint in a PPD patient and a transgenic insertion site in the polydactylous mouse mutant sasquatch (Ssq). The genetic lesions in both lie within the same respective intron of the LMBR1/Lmbr1 gene, which resides approximately 1 Mb away from Shh. Genetic analysis of Ssq reveals that the Lmbr1 gene is incidental to the phenotype and that the mutation directly interrupts a cis-acting regulator of Shh. This regulator is most likely the target for generating PPD mutations in human.

Animals↗
PubMed2002

Clonal analysis of patterns of growth, stem cell activity, and cell movement during the development and maintenance of the murine corneal epithelium.

Patterns of growth and cell movement in the developing and adult corneal epithelium were investigated by analysing clonal patches of LacZ-expressing cells in chimeric and X-inactivation mosaic mice. It was found that cell proliferation throughout the basal corneal epithelium during embryogenesis and early postnatal life creates a disordered mosaic pattern of LacZ(+) clones that contrasts with patterns of proliferation and striping produced during the later embryonic stages of retinal pigmented epithelium development. The early mosaic pattern in the corneal epithelium is replaced in the first 12 postnatal weeks by an ordered pattern of radial stripes or sectors that reflects migration without mixing of the progeny of clones of limbal stem cells. In contrast to previous assumptions, it was found that maturation of the activity of limbal stem cells and the pattern of migration of their progeny are delayed for several weeks postnatally. No evidence was found for immigration of the progeny of stem cells until the 5th postnatal week. There are approximately 100 clones of limbal stem cells initially, and clones are lost during postnatal life. Our studies provide a new assay for limbal and corneal defects in mutant mice.

Animals↗