Biomedical subjects

A Awgulewitsch

Publications and source records attributed to A Awgulewitsch.

At least 19 recordsLinked to original sources

PubMed2001

Overexpression of Hoxc13 in differentiating keratinocytes results in downregulation of a novel hair keratin gene cluster and alopecia.

Studying the roles of Hox genes in normal and pathological development of skin and hair requires identification of downstream target genes in genetically defined animal models. We show that transgenic mice overexpressing Hoxc13 in differentiating keratinocytes of hair follicles develop alopecia, accompanied by a progressive pathological skin condition that resembles ichthyosis. Large-scale analysis of differential gene expression in postnatal skin of these mice identified 16 previously unknown and 13 known genes as presumptive Hoxc13 targets. The majority of these targets are downregulated and belong to a subgroup of genes that encode hair-specific keratin-associated proteins (KAPs). Genomic mapping using a mouse hamster radiation hybrid panel showed these genes to reside in a novel KAP gene cluster on mouse chromosome 16 in a region of conserved linkage with human chromosome 21q22.11. Furthermore, data obtained by Hoxc13/lacZ reporter gene analysis in mice that overexpress Hoxc13 suggest negative autoregulatory feedback control of Hoxc13 expression levels, thus providing an entry point for elucidating currently unknown mechanisms that are required for regulating quantitative levels of Hox gene expression. Combined, these results provide a framework for understanding molecular mechanisms of Hoxc13 function in hair growth and development.

Alopecia↗

PubMed2000

Loss of fibula in mice overexpressing Hoxc11.

This study demonstrates severe malformations of the appendicular skeleton in mice overexpressing Hoxc11. Consistent with the endogenous expression pattern, the most conspicuous defect in Hoxc11 overexpressing neonates is aplasia/hypoplasia of the fibula. This is preceded at day 15.5 of embryonic development by marked reduction of chondrocyte proliferation, lack of PTHR expressing prehypertrophic cells, and the absence of hypertrophic and calcifying chondrocytes. Combined with the lack of an overt phenotype in the majority of Hoxc11 overexpressing embryos at day 13.5, the data suggest inhibition of chondrocyte differentiation during the elongation phase of the fibula bone as a primary effect of elevated Hoxc11 expression. This interpretation is further corroborated by Hoxc11 reporter gene expression in the joint areas at embryonic day 15.5, suggesting an involvement of the periarticular perichondrium in generating the mutant phenotype.

Animals↗

PubMed1998

Murine Hoxc-9 gene contains a structurally and functionally conserved enhancer.

Reporter gene analysis of the Hoxc-9 genomic region in transgenic mice allowed us to identify a positional enhancer in the Hoxc-9 intron that drives expression in the posterior neural tube of midgestation mouse embryos in a Hoxc-9-related manner. Sequence comparison to the chicken Choxc-9 intron revealed the existence of two highly conserved sequence elements (CSEs) in a similar spatial arrangement. These structural similarities in the mammalian and avian lineage are mirrored by conserved function of the chicken Choxc-9 intron in transgenic mice. Deletion analysis of the two introns suggests that full activity of both enhancers depends on cooperation between the two CSEs located close to the respective 5' and 3' splice sites. Following the paradigm of phylogenetically conserved developmental control mechanisms, the Hoxc-9 intragenic enhancer was tested in Drosophila. Our data show that the mouse Hoxc-9 enhancer acts in a conserved fashion in transgenic flies, conferring posteriorly restricted reporter gene expression to the developing central nervous system in third instar larvae. This finding indicates that the Hoxc-9 intragenic enhancer is involved in transcriptional regulatory circuits conserved between vertebrates and arthropods.

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PubMed1994

The murine Hoxc cluster contains five neighboring AbdB-related Hox genes that show unique spatially coordinated expression in posterior embryonic subregions.

A common feature of the murine Abdominal B (AbdB) -related Hox genes, located in the 5' regions of the four Hox clusters, appears to be a function in patterning the developing limb. As a prerequisite for studying the role of the AbdB-related Hoxc genes during limb development, we have isolated and mapped the three predicted AbdB-related Hoxc-11, -12, and -13 loci, thus defining the 5' end of the Hoxc cluster. Sequence comparisons based on the homeobox sequences of presumably all murine AbdB-related Hox genes strongly support the concept of a two step process in their evolution. As expected, Hoxc-11, -12 and -13 exhibit nested and extremely posteriorly restricted expression domains, whose anterior boundaries reflect their map positions, in accordance with the colinearity rule. A limited comparison of the primary expression domains of all five AbdB-related Hoxc genes in the developing hindlimb revealed nested and increasingly restricted domains of expression in the mesenchyme for only Hoxc-9, -10 and -11. However, separate localized expression was detected for Hoxc-9, -10, -11, -12 and possibly -13 in distal epidermal regions of the developing hind- and forelimb, whereas no expression of any of the five genes was observed in mesenchymal tissues of the developing forelimb. These data suggest a specific role for the AbdB-related Hoxc genes in patterning the hindlimb and pelvic girdle, which is separate from a second role relevant for both hind- and forelimb development.

Amino Acid Sequence↗

PubMed1992

Deformed autoregulatory element from Drosophila functions in a conserved manner in transgenic mice.

The striking similarities in the structure, organization and anterior-posterior expression patterns between the murine Hox gene system and the Drosophila homeotic gene complexes, called HOM-C (ref. 3), may point to highly conserved mechanisms for specifying positional identities (reviewed in ref. 4). Strong support for this concept lies in the observation of conserved colinearity between the genomic order of the Hox/HOM genes and their unique successive expression domains along the anterior-posterior axes of both mouse and fly embryos. These unique and precise expression patterns appear to be facilitated by multiple cis-regulatory elements (reviewed in ref. 5). One of the few elements characterized in detail is the autoregulatory enhancer of the homeotic gene Deformed (Dfd), which supports expression in subregions of posterior head segments of Drosophila embryos. Here we present evidence that this enhancer is capable of conferring reporter gene expression to a discrete subregion of the hindbrain in transgenic mouse embryos. Remarkably, this anterior-posterior subregion lies within the common anterior expression domain of the Dfd cognate Hox genes in the postotic hindbrain. Our results indicate that the Dfd autoregulatory enhancer is part of a highly conserved mechanism for establishing region-specific gene expression along the anterior-posterior axis of the embryo.

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PubMed1992

Clonal proliferation of murine lymphohemopoietic progenitors in culture.

We have used a two-step clonal culture system to unequivocally demonstrate that individual primitive lymphohemopoietic progenitor cells have the capacity for differentiation along either the myeloid or the B-lymphoid lineage. Highly enriched murine marrow cells were plated individually in culture by micromanipulation in the presence of pokeweed mitogen-stimulated spleen cell conditioned medium, erythropoietin, steel factor (SF), and interleukin (IL) 7. Forty-five percent of the single cells formed primary colonies expressing multiple hemopoietic lineages. When aliquots from individual colonies were replated in secondary methyl cellulose culture containing SF and IL-7, 41% of the primary colonies gave rise to lymphocyte colonies. Cells of the lymphocyte colonies were blast-like and B220+, sIg-, Mac-1-, Gr-1-, Ly-1-, L3T4-, Ly-2-, and CD3-. Thirty to 70% of the cells were Thy-1+. mu-chain mRNA was detected in most of the cells by in situ hybridization with an antisense RNA probe. When lymphocyte colonies derived from a single cell were pooled and individually injected into scid mice, donor-type IgM was measurable in the serum of mice and spleens contained donor-type B cells. We then carried out initial screening of growth factors to identify growth factors that might replace pokeweed mitogen-stimulated spleen cell conditioned medium in the primary culture. Combinations of two factors that included SF plus IL-6, IL-11, or granulocyte colony-stimulating factor were all effective in the primary culture in the maintenance of the B-lymphoid potential. Interestingly, IL-3 could neither replace nor act synergistically with SF to support the lymphoid potential of the primary cultures. Our observations demonstrate that many primitive progenitors previously believed to be myeloid-committed also possess B-lymphoid potential. This culture system should prove valuable for elucidation of the mechanisms regulating early stages of lymphohemopoiesis.

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PubMed1992

Hox-3.6: isolation and characterization of a new murine homeobox gene located in the 5' region of the Hox-3 cluster.

Most members of the murine Hox gene system can be grouped into two subclasses based on their structural similarity to either one of the Drosophila homeotic genes Antennapedia (Antp) or Abdominal B (AbdB). All the AbdB-like genes reported thus far are located in the 5' region of their respective cluster. We describe here the isolation, structural characterization and spatio-temporal expression pattern of a new AbdB-like homeobox gene designated Hox-3.6 that is located in the 5' region of the Hox-3 cluster. Hox-3.6 has an extreme posterior expression domain in embryos of 12.5 days of gestation, a feature that has thus far only been observed for the 5' most genes of the Hox-4 cluster. Like the other members of the AbdB subfamily, Hox-3.6 exhibits spatially restricted expression in the hindlimb bud, but the expression domain is antero-proximal in contrast to the postero-distal domain reported for its cognate gene Hox-4.5. Structural analysis of the 5' region revealed the presence of a 35 bp sequence which shares homology and relative 5' position with an upstream sequence present in its two nearest downstream neighbors, Hox-3.2 and -3.1.

Amino Acid Sequence↗

PubMed1990

Structural analysis of the Hox-3.1 transcription unit and the Hox-3.2--Hox-3.1 intergenic region.

The mouse Hox gene family is a set of mammalian homeobox genes that may represent developmental control genes. Complete information about the primary structure of these genes is a prerequisite for a systematic analysis of the mechanisms that determine their complex tempero-spatial expression patterns. In this report we describe the complete sequence of the Hox-3.1 locus and provide evidence for several closely spaced transcriptional start sites. Sequence analysis of the 5' region of the Hox-3.1 gene extending to its nearest upstream neighbor, Hox-3.2, allowed us to identify sequences known to be capable of interactions with transcription factors. Several of these sequence motifs are similar to cis-regulatory elements found in the regulatory regions of other known developmentally regulated genes.

Amino Acid Sequence↗

PubMed1990

Evidence for positive and negative regulation of the Hox-3.1 gene.

The region-specific patterns of expression of mouse homeobox genes are considered important for establishing the embryonic body plan. A 5-kilobase (kb) DNA fragment from the Hox-3.1 locus that is sufficient to confer region-specific expression to a beta-galactosidase reporter gene in transgenic mouse embryos has been defined. The observed reporter gene expression pattern closely parallels endogenous Hox-3.1 expression in 8- to 9.5-day postcoitum (p.c.) embryos. At 10.5 days p.c. and later, the pattern of beta-galactosidase activity diverges from the Hox-3.1 pattern, and an inappropriately high level of reporter gene expression is observed in posterior spinal ganglia. Inclusion of an additional 2 kb of upstream sequences is sufficient to suppress this aberrant expression in the developing spinal ganglia. Together, these results show that the control of early Hox-3.1 expression is complex, involving at least one positively acting and one negatively acting element.

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PubMed1990

Differential expression of Hox 3.1 protein in subregions of the embryonic and adult spinal cord.

Synthetic oligopeptides derived from the predicted Hox 3.1 protein coding sequence were used for the production of antibodies (anti-aa2) that specifically recognize Hox 3.1 protein in tissue sections. These antibodies were applied in immunohistochemical studies to monitor the expression of Hox 3.1 protein within the central nervous system (CNS) of embryonic and adult mice. We demonstrate congruency between the distinct Hox 3.1 RNA and protein expression patterns in the developing spinal cord by direct comparison of in situ hybridization and immunohistochemical staining in frozen sagittal sections from embryos of 12.5 days of gestation. A distinct pattern of spatially restricted expression of Hox 3.1 protein within the spinal cord was first detected at around 10.5 days of embryonic development. Within certain anteroposterior limits the geometries of this expression pattern change drastically during subsequent embryonic stages, concomitant with important cytoarchitectural changes in the developing spinal cord. Analyses on subcellular levels indicate predominant accumulation of Hox 3.1 protein within nuclei of neuronal cells. In addition to the nuclear localization in subsets of embryonic cells, persistent accumulation of Hox 3.1 protein was shown in nuclei of fully differentiated and mature neuronal cells of the adult CNS.

Animals↗

PubMed1989

The developmental expression pattern of a new murine homeo box gene: Hox-2.5.

To examine the possible role of homeo box genes in murine development we have studied the structure and expression pattern of Hox-2.5, a newly isolated homeo box gene that maps to the left end of the mouse Hox-2 locus on chromosome 11. The sequence of the Hox-2.5 homeo box has been determined. It is highly homologous to Hox-1.7 and Hox-3.2, demonstrating extended conservation among three homeo box complexes in the mouse. Northern and in situ hybridization analyses of Hox-2.5 demonstrate a novel, regionally restricted pattern of expression in developing mesoderm and neurectoderm. We detect localized Hox-2.5 transcripts as early as 8.5 days postcoitum. The expression pattern of Hox-2.5 was analyzed over the next 3 days of ontogeny, as well as in later embryonic, newborn, and adult stages. Three-dimensional reconstruction of Hox-2.5 transcript localization within the central nervous system of early embryos clearly illustrates the neural expression domain. Although the Hox-2.5 expression pattern is regionally restricted during all of these stages of development, the pattern changes along the anteroposterior and dorsoventral axes of the CNS as the embryo undergoes complex morphogenetic movements and cytodifferentiation.

Amino Acid Sequence↗

PubMed1988

Structure and function of Y chromosomal DNA. I. Sequence organization and localization of four families of repetitive DNA on the Y chromosome of Drosophila hydei.

The sequence organization of four different families of Y chromosomal repetitive DNA is characterized at three levels of spatial extension along the Y chromosome of Drosophila hydei. At the lowest level of resolution, DNA blot analysis of Y chromosomal fragments of different lengths and in situ hybridization experiments on metaphase chromosomes demonstrate the clustering of each particular sequence family within one defined region of the chromosome. At a higher level of resolution, family specific repeats can be detected within these clusters by crosshybridization within 10-20 kb long continuous stretches of cloned DNA in EMBL3 phages. At the highest level of resolution, detailed sequence analysis of representative subclones about 1 kb in length reveals a satellite-like head to tail arrangement of family specific degenerated subrepeats as the building scheme common to all four families. Our results provide the first comparative sequence analysis of three novel families of repetitive DNA on the long arm of the Y chromosome of D. hydei. Additional data are presented which support the existence of two related subfamilies of repetitive DNA on the short arm of the Y chromosome.

Animals↗

PubMed1988

Structure and expression of Hox-2.2, a murine homeobox-containing gene.

The Hox-2.2 gene is one of a cluster of homeobox-containing genes on mouse chromosome 11. A cDNA clone containing the Hox-2.2 homeobox has been isolated from an adult spinal cord library. Our analysis of the Hox-2.2 cDNA and genomic clones indicates that there are at least two oxons and one intron. The largest open reading frame includes the homeobox and codes for a 224 amino acid protein of molecular weight 25,312. Comparisons of the predicted Hox-2.2 protein with other homeodomain-containing proteins revealed four regions of sequence similiarity: an N-terminal octapeptide, a hexapeptide upstream of the homeodomain, the homeodomain, and a glutamic acid-rich region at the C terminus. Possible functions of these regions are discussed. The Hox-2.2 gene is expressed in 13.5-day embryos in the developing hindbrain and spinal cord. The expression patterns of Hox-2.2 and Hox-2.1 in 13.5-day embryos are compared.

Amino Acid Sequence↗

PubMed1987

Region-specific expression of two mouse homeo box genes.

Mammalian homeo box genes have been identified on the basis of sequence homology to Drosophila homeotic and segmentation genes. These studies examine the distribution of transcripts from two mouse homeo box genes, Hox-2.1 and Hox-3.1, throughout the latter third of prenatal development. Transcripts from these genes are regionally localized along the rostro-caudal axis of the developing central nervous system, yielding expression patterns very similar to patterns of Drosophila homeotic gene expression.

Animals↗

PubMed1987

Expression of the murine homeo box gene Hox 1.5 during embryogenesis.

The spatial pattern of expression of the murine homeo box-containing gene Hox 1.5 was studied during embryogenesis. In situ hybridization of single-stranded RNA probes to mouse embryo sections revealed a specific spatial distribution of the Hox 1.5 transcripts in mouse embryos 8.5 to 12.5 days postcoitum (p.c.). Analysis of mouse embryos 8.5 days p.c. showed that the gene is expressed in a spatially restricted manner. Expression appears to be limited to the central nervous system with an anterior boundary in the hindbrain and extending posteriorly through caudal regions of the spinal cord. The same spatial pattern of expression was observed in embryos 9.5 to 12.5 days p.c. These results show that the murine Hox 1.5 gene is expressed in a spatially restricted manner during embryonic development similar to the patterns observed in Drosophila homeotic genes.

Animals↗

PubMed1987

Homeo box genes in murine development.

Considerable information has accumulated on mouse homeo box gene organization and expression. Homeo box genes are expressed in a wide variety of tissues, developmental stages, and cell lines. How can this be interpreted in view of the relationship of these genes to Drosophila morphogenetic loci? One view is that homeo box genes control determinative decisions by modulating transcription of as yet unidentified target genes. Proponents of this view are faced with two tasks: to identify developmental processes that are controlled by homeo box genes, and to identify the target genes that mediate this control. Such target genes might be identified on the basis of in vitro homeo domain-DNA interactions. Candidate morphogenetic processes might be identified on the basis of the observed patterns of homeo box gene expression. It must be stressed that finding expression in a given tissue in no way demonstrates that the expression is necessary for the determination of that tissue. The role of Drosophila homeo box genes in determinative decisions is based upon analysis of mutants to demonstrate that the pattern of homeo box gene expression determines the morphogenetic outcome. To test whether the expression of a mouse homeo box gene is involved in a determinative decision, one must disrupt the normal pattern of expression of that gene and observe the resulting morphogenetic effect. In mouse this can be approached by looking for allelism with known morphogenetic loci, by isolating mutants in homeo box genes through large-scale mutagenesis screens, or by introducing altered homeo box genes into transgenic mice. One of the most intriguing possibilities is that homeo box genes are involved in regional specification along the anteroposterior axis. In situ hybridization and Northern blot analysis have demonstrated that at least four different homeo box genes display distinct regional patterns of expression along the anteroposterior axis of the developing CNS. The expression of each of these genes has a unique anterior boundary from which expression extends posteriorly within the CNS. Hox 1.5 expression has an anterior boundary within the hindbrain just posterior to the pontine flexure. The anterior boundary of Hox 2.1 expression lies more posteriorly within the medulla of the hindbrain. Weak expression of Hox 2.5 is detected in the spinal cord just posterior to the first cervical vertebra, and maximal expression is found posterior to the second cervical vertebra.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1986

Isolation of Y-chromosomal repetitive DNA sequences of Drosophila hydei via enrichment of chromosome-specific sequences by heterogeneous hybridization between female and male DNA.

Male or female DNA of Drosophila hydei was sheared by sonication, denatured, reannealed to different C0t-values and fractionated by hydroxyapatite. The highly repetitive, moderately repetitive and unique fractions of female DNA were denatured again and coupled via diazotization or cyanogen bromide to macroporous Sephacryl S-500. Enrichment of Y-chromosomal sequences was achieved by cycling each of the different fractions of male DNA under optimized hybridization conditions over a column with a manifold excess of immobilized female DNA of the corresponding complexity. Thereby, Y-chromosomal sequences of D. hydei could be enriched about 100-fold for highly and moderately repetitive DNA and about 10-fold for unique DNA. When a library of male D. hydei DNA was screened with Y-enriched highly repetitive DNA, more than 98% of all hybridizing phages contained inserts of repetitive Y-chromosomal DNA of at least four different sequence families.

Animals↗

PubMed1985

Homeo box gene complex on mouse chromosome 11: molecular cloning, expression in embryogenesis, and homology to a human homeo box locus.

The homeo box is a 180 bp protein-coding domain found within homeotic genes of Drosophila and conserved in a variety of invertebrate and vertebrate species. It has been suggested that the mammalian homeo box sequences may play a role in controlling pattern formation during embryogenesis. We report findings that support this hypothesis. We have cloned three overlapping recombinant phage clones that cover a region of mouse chromosome 11 that contains a cluster of four homeo boxes (the Hox-2 locus). This locus encodes multiple transcripts that are expressed during embryogenesis. Forty kilobases of the Hox-2 region is devoid of repetitive elements and shows extensive homology with the human Hox-2 locus. These results provide direct evidence for genetic expression during embryonic development, a conserved organization in comparison to the cognate human locus, and a complexity of organization and transcript expression similar to that found in Drosophila.

Animals↗