Theria-specific homeodomain and cis-regulatory element evolution of the Dlx3-4 bigene cluster in 12 different mammalian species.

The mammalian Dlx3 and Dlx4 genes are configured as a bigene cluster, and their respective expression patterns are controlled temporally and spatially by cis-elements that largely reside within the intergenic region of the cluster. Previous work revealed that there are conspicuously conserved elements within the intergenic region of the Dlx3-4 bigene clusters of mouse and human. In this paper we have extended these analyses to include 12 additional mammalian taxa (including a marsupial and a monotreme) in order to better define the nature and molecular evolutionary trends of the coding and non-coding functional elements among morphologically divergent mammals. Dlx3-4 regions were fully sequenced from 12 divergent taxa of interest. We identified three theria-specific amino acid replacements in homeodomain of Dlx4 gene that functions in placenta. Sequence analyses of constrained nucleotide sites in the intergenic non-coding region showed that many of the intergenic conserved elements are highly conserved and have evolved slowly within the mammals. In contrast, a branchial arch/craniofacial enhancer I37-2 exhibited accelerated evolution at the branch between the monotreme and therian common ancestor despite being highly conserved among therian species. Functional analysis of I37-2 in transgenic mice has shown that the equivalent region of the platypus fails to drive transcriptional activity in branchial arches. These observations, taken together with our molecular evolutionary data, suggest that theria-specific episodic changes in the I37-2 element may have contributed to craniofacial innovation at the base of the mammalian lineage.

A yeast-based recombinogenic targeting toolset for transgenic analysis of human disease genes.

Transgenic mouse models are valuable resources for analyzing functions of genes involved in human diseases. Mouse models provide critical insights into biological processes, including in vivo visualization of vasculature critical to our understanding of the immune system. Generating transgenic mice requires the capture and modification of large-insert DNAs representing genes of interest. We have developed a methodology using a yeast-bacterial shuttle vector, pClasper, that enables the capture and modification of bacterial artificial chromosomes (BAC)-sized DNA inserts. Numerous improvements and technical advances in the original pClasper vector have allowed greater flexibility and utility in this system. Examples of such pClasper mediated gene modifications include: Claspette-mediated capture of large-insert genomic fragments from BACs-human polycystic kidney disease-1 (PKD1); modification of pClasperA clones by the RareGap method-PKD1 mutations; Claspette-mediated modification of pClasper clones-mouse albumin-1 gene; and, of most relevance to our interest in lymph node vasculature-Claspimer-mediated modification of pClasper clones-high endothelial venule and lymphatic vessel genes. Mice that have been generated with these methods include mice with fluorescent high endothelial venules.

Essential functions of the Williams-Beuren syndrome-associated TFII-I genes in embryonic development.

GTF2I and GTF2IRD1 encoding the multifunctional transcription factors TFII-I and BEN are clustered at the 7q11.23 region hemizygously deleted in Williams-Beuren syndrome (WBS), a complex multisystemic neurodevelopmental disorder. Although the biochemical properties of TFII-I family transcription factors have been studied in depth, little is known about the specialized contributions of these factors in pathways required for proper embryonic development. Here, we show that homozygous loss of either Gtf2ird1 or Gtf2i function results in multiple phenotypic manifestations, including embryonic lethality; brain hemorrhage; and vasculogenic, craniofacial, and neural tube defects in mice. Further analyses suggest that embryonic lethality may be attributable to defects in yolk sac vasculogenesis and angiogenesis. Microarray data indicate that the Gtf2ird1 homozygous phenotype is mainly caused by an impairment of the genes involved in the TGFbetaRII/Alk1/Smad5 signal transduction pathway. The effect of Gtf2i inactivation on this pathway is less prominent, but downregulation of the endothelial growth factor receptor-2 gene, resulting in the deterioration of vascular signaling, most likely exacerbates the severity of the Gtf2i mutant phenotype. A subset of Gtf2ird1 and Gtf2i heterozygotes displayed microcephaly, retarded growth, and skeletal and craniofacial defects, therefore showing that haploinsufficiency of TFII-I proteins causes various developmental anomalies that are often associated with WBS.

Identification of the TFII-I family target genes in the vertebrate genome.

GTF2I and GTF2IRD1 encode members of the TFII-I transcription factor family and are prime candidates in the Williams syndrome, a complex neurodevelopmental disorder. Our previous expression microarray studies implicated TFII-I proteins in the regulation of a number of genes critical in various aspects of cell physiology. Here, we combined bioinformatics and microarray results to identify TFII-I downstream targets in the vertebrate genome. These results were validated by chromatin immunoprecipitation and siRNA analysis. The collected evidence revealed the complexity of TFII-I-mediated processes that involve distinct regulatory networks. Altogether, these results lead to a better understanding of specific molecular events, some of which may be responsible for the Williams syndrome phenotype.

Transgenic LacZ under control of Hec-6st regulatory sequences recapitulates endogenous gene expression on high endothelial venules.

Hec-6st is a highly specific high endothelial venule (HEV) gene that is crucial for regulating lymphocyte homing to lymph nodes (LN). The enzyme is also expressed in HEV-like vessels in tertiary lymphoid organs that form in chronic inflammation in autoimmunity, graft rejection, and microbial infection. Understanding the molecular nature of Hec-6st regulation is crucial for elucidating its function in development and disease. However, studies of HEV are limited because of the difficulties in isolating and maintaining the unique characteristics of these vessels in vitro. The novel pClasper yeast homologous recombination technique was used to isolate from a BAC clone a 60-kb DNA fragment that included the Hec-6st (Chst4) gene with flanking sequences. Transgenic mice were generated with the beta-galactosidase (LacZ) reporter gene inserted in-frame in the exon II of Hec-6st within the isolated BAC DNA fragment. LacZ was expressed specifically on HEV in LN, as indicated by its colocalization with peripheral node vascular addressin. LacZ was increased in nasal-associated lymphoid tissue during development and was reduced in LN and nasal-associated lymphoid tissue by LTbetaR-Ig (lymphotoxin-beta receptor human Ig fusion protein) treatment in a manner identical to the endogenous gene. The transgene was expressed at high levels in lymphoid accumulations with characteristics of tertiary lymphoid organs in the salivary glands of aged mice. Thus, the Hec-6s-LacZ construct faithfully reproduces Hec-6st tissue-specific expression and can be used in further studies to drive expression of reporter or effector genes, which could visualize or inhibit HEV in autoimmunity.

Gene expression analysis of TFII-I modulated genes in mouse embryonic fibroblasts.

TFII-I is a founding member of a family of helix-loop-helix transcription factors involved in modulation of genes through interaction with various nuclear factors and chromatin remodeling complexes. Recent studies indicate that TFII-I performs important function in cell physiology and mouse embryogenesis. In order to understand its molecular role, TFII-I was overexpressed in primary mouse embryonic fibroblasts (MEFs) and alterations in gene expression were monitored with a mouse 16 K oligonucleotide microarray. These studies allowed us to identify genes that lie downstream of TFII-I-dependent pathways. Among the modulated candidates were genes involved in the immunity response, catalytic activity, signaling pathways and transcriptional regulation. Expression of several candidates including those for the interferon-stimulated protein (G1p2), small inducible cytokine A7 (Ccl7), ubiquitin-conjugating enzyme 8 (Ube2l6), cysteine-rich protein (Csrp2) and Drosophila delta-like 1 homolog (Dlk1) were confirmed by real-time PCR. The obtained results suggest that TFII-I participates in multiple signaling and regulatory pathways in MEFs.

Mouse naked cuticle 2 (mNkd2) as a direct transcriptional target of Hoxc8 in vivo.

Mouse naked cuticle 2 (mNkd2), the mammalian homolog of the Drosophila segment polarity gene naked cuticle (nkd), encodes an EF hand protein that regulates early Wg activity by acting as an inducible antagonist. The transcription factor, Hoxc8, a member of the homeobox gene family, is vital for growth and differentiation. Chromatin immunoprecipitation (ChIP) assay, an electrophoretic mobility shift assay (EMSA), and a reporter assay demonstrated that endogenous Hoxc8 protein binds directly to the enhancer region of the mNkd2 gene, implying a Hoxc8-dependent transcriptional activity. Introduction of exogenous Hoxc8 into NIH3T3 cell lines lacking wild-type Hoxc8 dramatically reduced expression of mNkd2 mRNA. If, as the results suggest, mNkd2 is a direct target of Hoxc8, it represents a novel mechanism by which Hoxc8 might cross-talk with the Wnt signaling pathway by regulating mNkd2.

Expression profiling of BEN regulated genes in mouse embryonic fibroblasts.

BEN is a member of the TFII-I family of helix-loop-helix transcription factors. Both TFII-I and BEN are involved in gene regulation through interactions with tissue-specific transcription factors and chromatin remodeling complexes. Identification of the downstream target genes of TFII-I proteins is critical in delineating the regulatory effects of these proteins. In this study, we conducted a microarray analysis to determine gene expression alterations following the overexpression of BEN in primary mouse embryonic fibroblasts (MEFs). We found the BEN-dependent modulation in the expression of large groups of genes representing a wide variety of functional categories including genes important in the immune response, cell cycle, transcriptional regulation and cell signaling. A set of genes identified by the microarray analysis was validated by independent real-time PCR analysis. Among upregulated genes were Shrm, Tgfb2, Ube2l6, G1p2, Ccl7 while downregulated genes were Folr1, Tgfbr2, Csrp2, and Dlk1. These results support a versatile function of TFII-I proteins in vertebrate physiology and lead to an increased understanding of the BEN-dependent molecular events.

Laser-assisted microdissection (LAM) in developmental biology.

The analysis of gene expression in developing organs is a valuable tool for the assessment of genetic fingerprints during the various stages of differentiation. Complex processes in developing tissues are particularly difficult to understand in terms of biochemical phenomena. Laser-assisted microdissection (LAM) allows the efficient and precise capture of cells or groups of cells from developing tissues in sufficient quantities and within the context of time and space to permit the subsequent molecular characterization of the targeted tissue. The technique development has dramatically increased the ease of isolating specific cells which, together with progress in tissue preparation and microextraction protocols, allows for broad-range down-stream applications in the fields of genomics, transcriptomics and proteomics. This review gives an overview of the LAM technology and its application in developmental biology.