Cooduvalli S. Shashikant

Functional and molecular features of the Id4+ germline stem cell population in mouse testes

The maintenance of cycling cell lineages relies on undifferentiated subpopulations consisting of stem and progenitor pools. Features that delineate these cell types are undefined for many lineages, including spermatogenesis, which is supported by an undifferentiated spermatogonial population. Here, we generated a transgenic mouse line in which spermatogonial stem cells are marked by expression of an inhibitor of differentiation 4 (Id4)-green fluorescent protein (Gfp) transgene. We found that Id4-Gfp(+) cells exist primarily as a subset of the type A(single) pool, and their frequency is greatest in neonatal development and then decreases in proportion during establishment of the spermatogenic lineage, eventually comprising ∼ 2% of the undifferentiated spermatogonial population in adulthood. RNA sequencing analysis revealed that expression of 11 and 25 genes is unique for the Id4-Gfp(+)/stem cell and Id4-Gfp(-)/progenitor fractions, respectively. Collectively, these findings provide the first definitive evidence that stem cells exist as a rare subset of the A(single) pool and reveal transcriptome features distinguishing stem cell and progenitor states within the mammalian male germline.

Loss of Glutamatergic Pyramidal Neurons in Frontal and Temporal Cortex Resulting from Attenuation of FGFR1 Signaling Is Associated with Spontaneous Hyperactivity in Mice

Fibroblast growth factor receptor (FGFR) gene products (Fgfr1, Fgfr2, Fgfr3) are widely expressed by embryonic neural progenitor cells throughout the CNS, yet their functional role in cerebral cortical development is still unclear. To understand whether the FGF pathways play a role in cortical development, we attenuated FGFR signaling by expressing a tyrosine kinase domain-deficient Fgfr1 (tFgfr1) gene construct during embryonic brain development. Mice carrying the tFgfr1 transgene under the control of the Otx1 gene promoter have decreased thickness of the cerebral cortex in frontal and temporal areas because of decreased number of pyramidal neurons and disorganization of pyramidal cell dendritic architecture. These alterations may be, in part, attributable to decreased genesis of T-Brain-1-positive early glutamatergic neurons and, in part, to a failure to maintain radial glia fibers in medial prefrontal and temporal areas of the cortical plate. No changes were detected in cortical GABAergic interneurons, including Cajal-Retzius cells or in the basal ganglia. Behaviorally, tFgfr1 transgenic mice displayed spontaneous and persistent locomotor hyperactivity that apparently was not attributable to alterations in subcortical monoaminergic systems, because transgenic animals responded to both amphetamine and guanfacine, an α2A adrenergic receptor agonist. We conclude that FGF tyrosine kinase signaling may be required for the genesis and growth of pyramidal neurons in frontal and temporal cortical areas, and that alterations in cortical development attributable to disrupted FGF signaling are critical for the inhibitory regulation of motor behavior.

Genomic structure and functional control of the Dlx3-7 bigene cluster

The Dlx genes are involved in early vertebrate morphogenesis, notably of the head. The six Dlx genes of mammals are arranged in three convergently transcribed bigene clusters. In this study, we examine the regulation of the Dlx3-7 cluster of the mouse. We obtained and sequenced human and mouse P1 clones covering the entire Dlx3-7 cluster. Comparative analysis of the human and mouse sequences revealed several highly conserved noncoding regions within 30 kb of the Dlx3-7-coding regions. These conserved elements were located both 5′ of the coding exons of each gene and in the intergenic region 3′ of the exons, suggesting that some enhancers might be shared between genes. We also found that the protein sequence of Dlx7 is evolving more rapidly than that of Dlx3. We conducted a functional study of the 79-kb mouse genomic clone to locate cis-element activity able to reproduce the endogenous expression pattern by using transgenic mice. We inserted a lacZ reporter gene into the first exon of the Dlx3 gene by using homologous recombination in yeast. Strong lacZ expression in embryonic (E) stage E9.5 and E10.5 mouse embryos was found in the limb buds and first and second visceral arches, consistent with the endogenous Dlx3 expression pattern. This result shows that the 79-kb region contains the major cis-elements required to direct the endogenous expression of Dlx3 at stage E10.5. To test for enhancer location, we divided the construct in the mid-intergenic region and injected the Dlx3 gene portion. This shortened fragment lacking Dlx7-flanking sequences is able to drive expression in the limb buds but not in the visceral arches. This observation is consistent with a cis-regulatory enhancer-sharing model within the Dlx bigene cluster.

Ksp-cadherin gene promoter. II. Kidney-specific activity in transgenic mice

Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a tissue-specific member of the cadherin superfamily that is expressed exclusively in the basolateral membrane of tubular epithelial cells in the kidney. To determine the basis for tissue-specific expression of Ksp-cadherin in vivo, we evaluated the activity of the promoter in transgenic mice. Transgenic mice containing 3.3 kb of the mouse Ksp-cadherin promoter and an Escherichia coli lacZ reporter gene were generated by pronuclear microinjection. Assays of β-galactosidase enzyme activity showed that the transgene was expressed exclusively in the kidney in both adult and developing mice. Within the kidney, the transgene was expressed in a subset of renal tubular epithelial cells that endogenously expressed Ksp-cadherin and that were identified as collecting ducts by colabeling with Dolichos biflorus agglutinin. In the developing metanephros, expression of the transgene in the branching ureteric bud correlated with the developmental expression of Ksp-cadherin. Identical patterns of expression were observed in multiple founder mice, indicating that kidney specificity was independent of transgene integration site. However, heterocellular expression was observed consistent with repeat-induced gene silencing. We conclude that the Ksp-cadherin gene promoter directs kidney-specific expression in vivo. Regulatory elements that are sufficient to recapitulate the tissue- and differentiation-specific expression of Ksp-cadherin in the renal collecting duct are located within 3.3 kb upstream to the transcriptional start site.Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a tissue-specific member of the cadherin superfamily that is expressed exclusively in the basolateral membrane of tubular epithelial cells in the kidney. To determine the basis for tissue-specific expression of Ksp-cadherin in vivo, we evaluated the activity of the promoter in transgenic mice. Transgenic mice containing 3.3 kb of the mouse Ksp-cadherin promoter and an Escherichia coli lacZ reporter gene were generated by pronuclear microinjection. Assays of beta-galactosidase enzyme activity showed that the transgene was expressed exclusively in the kidney in both adult and developing mice. Within the kidney, the transgene was expressed in a subset of renal tubular epithelial cells that endogenously expressed Ksp-cadherin and that were identified as collecting ducts by colabeling with Dolichos biflorus agglutinin. In the developing metanephros, expression of the transgene in the branching ureteric bud correlated with the developmental expression of Ksp-cadherin. Identical patterns of expression were observed in multiple founder mice, indicating that kidney specificity was independent of transgene integration site. However, heterocellular expression was observed consistent with repeat-induced gene silencing. We conclude that the Ksp-cadherin gene promoter directs kidney-specific expression in vivo. Regulatory elements that are sufficient to recapitulate the tissue- and differentiation-specific expression of Ksp-cadherin in the renal collecting duct are located within 3.3 kb upstream to the transcriptional start site.

Evolution of Chordate Hox Gene Clustersa

ABSTRACT: In this article, we consider the role of the Hox genes in chordate and vertebrate evolution from the viewpoints of molecular and developmental evolution. Models of Hox cluster duplication are considered with emphasis on a threefold duplication model. We also show that cluster duplication is consistent with a semiconservative model of duplication, where following duplication, one daughter cluster remains unmodified, while the other diverges and assumes a new architecture and presumably new functions. Evidence is reviewed, suggesting that Hox gene enhancers have played an important role in body plan evolution. Finally, we contrast the invertebrates and vertebrates in terms of genome and Hox cluster duplication which are present in the latter, but not the former. We question whether gene duplication has been important in vertebrates for the introduction of novel features such as limbs, a urogenital system, and specialized neuromuscular interactions.

Multiple phases of expression and regulation of mouse Hoxc8 during early embryogenesis

Hox genes are expressed in dynamic patterns during embryogenesis consistent with their role in axial specifications. To study the distribution of mouse Hoxc8, a homeodomain containing protein, we raised monoclonal antibodies against the least conserved portion of Hoxc8. Using these antibodies, we have examined early and mid-gestation embryos for the distribution of the protein. At the end of gastrulation Hoxc8 is expressed in the caudal portion of the embryo. In the neural tube, an early phase when all cells express Hoxc8 is distinguished from a late phase with predominant expression in differentiating neurons. A comparison of this expression pattern with that of a reporter gene under the control of the early Hoxc8 enhancer demarcates three separate regulatory components: (1) initiation and establishment; (2) maintenance; and (3) downregulation. We propose that Hoxc8 expression during embryogenesis is established in multiple phases. Possible regulatory mechanisms involved in generating such a complex domain of Hox gene expression are discussed.

Transcriptional regulation of human MAP2 gene in melanoma: role of neuronal bHLH factors and Notch1 signaling

Microtubule-associated protein 2 (MAP2), a neuron-specific protein, stabilizes microtubules and is critical for neurite outgrowth and dendrite development. Although MAP2 is widely used as a marker of neuronal differentiation, regulation of its transcription has not been investigated. We showed that MAP2 is frequently activated in human cutaneous melanoma. Here, we identified a 2.2 kb region that is sufficient for neuronal-specific expression in vitro and in vivo. Comparative analysis of the mouse, rat and human MAP2 promoter sequences showed the presence of a conserved bHLH factor binding sites. Electrophoretic mobility shift analysis, promoter mutagenesis and co-transfection experiments showed that NeuroD, a pro-neuronal differentiation factor, and Hairy and Enhancer of Split (HES1), a transcription repressor, are involved in the regulation of MAP2 promoter activity. Melanoma cells express both NeuroD and HES1. Chromatin immunoprecipitation showed that in metastatic melanoma cells N-box region of the MAP2 promoter is occupied by endogenous HES1. We show that the inhibition of Notch signaling, a regulator of HES1 gene expression, and/or shRNA knockdown of HES1 results in the upregulation of MAP2 promoter activity. Thus, our data suggest that Notch signaling, which is implicated in melanoma progression, and HES1 play a role in MAP2 gene regulation during melanoma progression.

RECOMBINOGENIC TARGETING : A NEW APPROACH TO GENOMIC ANALYSIS : A REVIEW

Currently, recombinational cloning procedures based upon methods developed for yeast, Saccharomyces cerevisiae, are being exploited for targeted cloning and in-vivo modification of genomic clones. In this review, we will discuss the development of large-insert vectors, homologous recombination-based techniques for cloning and modification, and their application towards functional analysis of genes using transgenic mouse model systems.

Divergence of Hoxc8 early enhancer parallels diverged axial morphologies between mammals and fishes

There is considerable interest in understanding how cis-regulatory modifications drive morphological changes across species. Because developmental regulatory genes, including Hox genes, are remarkably conserved, their noncoding regulatory regions are likely sources for variations. Modifications of Hox cis-regulatory elements have potential to alter Hox gene expression and, hence, axial morphologies. In vertebrates, differences in the axial levels of Hox gene expression correlate with differences in the number and relative position of thoracic vertebrae. Variation in cis-regulatory elements of Hox genes can be identified by comparative sequence and reporter gene analyses in transgenic mouse embryos. Using these approaches, we show a remarkable divergence of the Hoxc8 early enhancers between mammals and fishes representing diverse axial morphologies. Extensive restructuring of the Hoxc8 early enhancer including nucleotide substitutions, inversion, and divergence result in distinct patterns of reporter gene expression along the embryonic axis. Our results provide an evolutionary perspective on how the enhancer elements are engineered and support the hypothesis that remodeling of Hox regulatory elements in different species has played a significant role in generating morphological diversity.

Molecular evolution of Hox gene regulation: cloning and transgenic analysis of the lamprey HoxQ8 gene.

The mammalian Hox clusters arose by duplication of a primordial cluster. The duplication of Hox clusters created redundancy within cognate groups, allowing for change in function over time. The lamprey, Petromyzon marinus, occupies an intermediate position within the chordates, both in terms of morphologic complexity and possibly cluster number. To determine the extent of divergence among Hox genes after duplication events within vertebrates, we analyzed Hox genes belonging to cognate group 8. Here we report characterization of the HoxQ8 gene, which shows conservation with mammalian genes in its amino-terminal, homeobox and hexapeptide sequences, and in the position of its splice sites. A beta-galactosidase reporter gene was introduced in the HoxQ8 genomic region by targeted recombinational cloning using a yeast-bacteria shuttle vector, pClasper. These reporter gene constructs were tested for their ability to direct region-specific expression patterns in transgenic mouse embryos. Lamprey enhancers direct expression to posterior neural tube but not to mesoderm, suggesting conservation of neuronal enhancers. In the presence of the mouse heat shock promoter, lamprey enhancers could also direct expression to the posterior mesoderm suggesting that there has been some divergence in promoter function. Our results suggest that comparative studies on Hox gene structure and analysis of regulatory elements may provide insights into changes concomitant with Hox cluster duplications in the chordates.