Lin28a/let-7 pathway modulates the Hox code via Polycomb regulation during axial patterning in vertebrates.

The body plan along the anteroposterior axis and regional identities are specified by the spatiotemporal expression of Hox genes. Multistep controls are required for their unique expression patterns; however, the molecular mechanisms behind the tight control of Hox genes are not fully understood. In this study, we demonstrated that the Lin28a/let-7 pathway is critical for axial elongation. Lin28a-/- mice exhibited axial shortening with mild skeletal transformations of vertebrae, which were consistent with results in mice with tail bud-specific mutants of Lin28a. The accumulation of let-7 in Lin28a-/- mice resulted in the reduction of PRC1 occupancy at the Hox cluster loci by targeting Cbx2. Consistently, Lin28a loss in embryonic stem-like cells led to aberrant induction of posterior Hox genes, which was rescued by the knockdown of let-7. These results suggest that the Lin28/let-7 pathway is involved in the modulation of the 'Hox code' via Polycomb regulation during axial patterning.

The Mohawk homeobox gene is a critical regulator of tendon differentiation.

Mohawk (Mkx) is a member of the Three Amino acid Loop Extension superclass of atypical homeobox genes that is expressed in developing tendons. To investigate the in vivo functions of Mkx, we generated Mkx(-/-) mice. These mice had hypoplastic tendons throughout the body. Despite the reduction in tendon mass, the cell number in tail tendon fiber bundles was similar between wild-type and Mkx(-/-) mice. We also observed small collagen fibril diameters and a down-regulation of type I collagen in Mkx(-/-) tendons. These data indicate that Mkx plays a critical role in tendon differentiation by regulating type I collagen production in tendon cells.

Sox9 directly promotes Bapx1 gene expression to repress Runx2 in chondrocytes.

The transcription factor, Sry-related High Mobility Group (HMG) box containing gene 9 (Sox9), plays a critical role in cartilage development by initiating chondrogenesis and preventing the subsequent maturation process called chondrocyte hypertrophy. This suppression mechanism by Sox9 on late-stage chondrogenesis partially results from the inhibition of Runt-related transcription factor 2 (Runx2), the main activator of hypertrophic chondrocyte differentiation. However, the precise mechanism by which Sox9 regulates late chondrogenesis is poorly understood. In the present study, the transcriptional repressor vertebrate homolog of Drosophila bagpipe (Bapx1) was found to be a direct target of Sox9 for repression of Runx2 expression in chondrocytes. We identified a critical Sox9 responsive region in the Bapx1 promoter via a luciferase reporter assay. Analysis by chromatin immunoprecipitation and electrophoretic mobility shift assays indicated that Sox9 physically bound to this region of the Bapx1 promoter. Consistent with the notion that Bapx1 and Sox9 act as negative regulators of chondrocyte hypertrophy by regulating Runx2 expression, transient knockdown of Sox9 or Bapx1 expression by shRNA in chondrocytes increased Runx2 expression, as well as expression of the late chondrogenesis marker, Col10a1. Furthermore, while over-expression of Sox9 decreased Runx2 and Col10a1 expressions, simultaneous transient knockdown of Bapx1 diminished that Sox9 over-expressing effect. Our findings reveal that the molecular pathway modulated by Bapx1 links two major regulators in chondrogenesis, Sox9 and Runx2, to coordinate skeletal formation.

A systems approach reveals that the myogenesis genome network is regulated by the transcriptional repressor RP58.

We created a whole-mount in situ hybridization (WISH) database, termed EMBRYS, containing expression data of 1520 transcription factors and cofactors expressed in E9.5, E10.5, and E11.5 mouse embryos--a highly dynamic stage of skeletal myogenesis. This approach implicated 43 genes in regulation of embryonic myogenesis, including a transcriptional repressor, the zinc-finger protein RP58 (also known as Zfp238). Knockout and knockdown approaches confirmed an essential role for RP58 in skeletal myogenesis. Cell-based high-throughput transfection screening revealed that RP58 is a direct MyoD target. Microarray analysis identified two inhibitors of skeletal myogenesis, Id2 and Id3, as targets for RP58-mediated repression. Consistently, MyoD-dependent activation of the myogenic program is impaired in RP58 null fibroblasts and downregulation of Id2 and Id3 rescues MyoD's ability to promote myogenesis in these cells. Our combined, multi-system approach reveals a MyoD-activated regulatory loop relying on RP58-mediated repression of muscle regulatory factor (MRF) inhibitors.

Loss of short dystrophin isoform Dp71 in olfactory ensheathing cells causes vomeronasal nerve defasciculation in mouse olfactory system.

The Duchenne muscular dystrophy (DMD) gene encodes dystrophin, which is a protein defective in DMD patients, as well as a number of shorter isoforms, which have been shown to be expressed in various non-muscle, primarily neural, tissues. As of yet, the physiological function of the various dystrophin isoforms is not fully understood. In the present study, we investigated the neurological phenotype that arises in the DMD-null mice, where expression of all dystrophin isoforms had been disrupted. We demonstrate that vomeronasal axons in the DMD-null mice are defasciculated, and some of the defasciculated vomeronasal axons aberrantly entered into the main olfactory bulb, which indicates that the product(s) of the DMD gene plays an important role in vomeronasal nerve organization. Through western blot and immunofluorescence analyses, we determined that the dystrophin isoform Dp71 was exclusively expressed in the mouse olfactory system: mainly in the olfactory ensheathing cells (OECs), an olfactory system-specific glia cell that ensheaths fascicles of the olfactory nerve. In the OECs, Dp71 was co-localized with beta-dystroglycan, utrophin, laminin, and perlecan. Since beta-dystroglycan and perlecan expression was decreased in the OECs of DMD-null mice, we hypothesize that Dp71 expressed in the OECs participates in fasciculation of the vomeronasal nerve, most likely through interactions with extracellular matrix.

Dynamic gene expression of Lin-28 during embryonic development in mouse and chicken.

The Caenorhabditis elegans heterochronic gene lin-28 regulates developmental timing in the nematode trunk. We report the dynamic expression patterns of Lin-28 homologues in mouse and chick embryos. Whole mount in situ hybridization revealed specific and intriguing expression patterns of Lin-28 in the developing mouse and chick limb bud. Mouse Lin-28 expression was detected in both the forelimb and hindlimb at E9.5, but disappeared from the forelimb at E10.5, and finally from the forelimb and hindlimb at E11.5. Chicken Lin-28, which was first detected in the limb primordium at stage 15/16, was also downregulated as the stage proceeded. The amino acid sequences of mouse and chicken Lin-28 genes are highly conserved and the similar expression patterns of Lin-28 during limb development in mouse and chicken suggest that this heterochronic gene is also conserved during vertebrate limb development.

A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins.

A major challenge of the post-genomic era is the functional characterization of anonymous open reading frames (ORFs) identified by the Human Genome Project. In this context, there is a strong requirement for the development of technologies that enhance our ability to analyze gene functions at the level of the whole organism. Here, we describe a rapid and efficient procedure to generate transgenic chimaeric mice that continuously secrete a foreign protein into the systemic circulation. The transgene units were inserted into the genomic site adjacent to the endogenous immunoglobulin (Ig) kappa locus by homologous recombination, using a modified mouse embryonic stem (ES) cell line that exhibits a high frequency of homologous recombination at the Igkappa region. The resultant ES clones were injected into embryos derived from a B-cell-deficient host strain, thus producing chimaerism-independent, B-cell-specific transgene expression. This feature of the system eliminates the time-consuming breeding typically implemented in standard transgenic strategies and allows for evaluating the effect of ectopic transgene expression directly in the resulting chimaeric mice. To demonstrate the utility of this system we showed high-level protein expression in the sera and severe phenotypes in human EPO (hEPO) and murine thrombopoietin (mTPO) transgenic chimaeras.

A new model mouse for Duchenne muscular dystrophy produced by 2.4 Mb deletion of dystrophin gene using Cre-loxP recombination system.

Duchenne muscular dystrophy (DMD) is caused by mutation in the 2.4-Mb dystrophin (DMD) gene . This gene encodes a number of tissue-specific isoforms of dystrophin generated by transcription from at least seven promoters and also by alternative splicing. We deleted entire genomic region of the DMD gene on mouse chromosome X using a Cre-loxP recombination system. Introduction of a loxP site in dystrophin's first and last exon by homologous recombination in mouse embryonic stem (ES) cells generated "DMD-floxed" (flanked by loxP sites) ES cells, which we subjected to Cre-mediated excision leading to establishment of "DMD-null" ES cell lines. The DMD-null mice produced from the DMD-null ES cells were viable but displayed severe muscular hypertrophy and dystrophy. In addition to the muscular impairment, the DMD-null mouse exhibited some behavioral abnormality and male sterility. The DMD-floxed mice produced from the DMD-floxed ES cells were viable, phenotypically normal, and were born with the expected Mendelian frequency, despite the absence of brain (cortical)-type dystrophin (Dp427c) expression. Since production of multiple dystrophin isoforms due to alternative splicing or exon skipping is totally prevented in the DMD-null mouse, these new mutants will provide an improved model system for functional studies of dystrophin and its isoforms.