HoxA3 is an apical regulator of haemogenic endothelium.

During development, haemogenesis occurs invariably at sites of vasculogenesis. Between embryonic day (E) 9.5 and E10.5 in mice, endothelial cells in the caudal part of the dorsal aorta generate haematopoietic stem cells and are referred to as haemogenic endothelium. The mechanisms by which haematopoiesis is restricted to this domain, and how the morphological transformation from endothelial to haematopoietic is controlled are unknown. We show here that HoxA3, a gene uniquely expressed in the embryonic but not yolk sac vasculature, restrains haematopoietic differentiation of the earliest endothelial progenitors, and induces reversion of the earliest haematopoietic progenitors into CD41-negative endothelial cells. This reversible modulation of endothelial-haematopoietic state is accomplished by targeting key haematopoietic transcription factors for downregulation, including Runx1, Gata1, Gfi1B, Ikaros, and PU.1. Through loss-of-function, and gain-of-function epistasis experiments, and the identification of antipodally regulated targets, we show that among these factors, Runx1 is uniquely able to erase the endothelial program set up by HoxA3. These results suggest both why a frank endothelium does not precede haematopoiesis in the yolk sac, and why haematopoietic stem cell generation requires Runx1 expression only in endothelial cells

Transformation, Somatic Embryogenesis and Whole Plant Regeneration Method for Glycine Species

A method for somatic embryogenesis of soybean, (Glycine max), Glycine soja and other Glycine species is provided using immature cotyledon tissue, preferably with the embryonic axis removed, comprising culturing said tissue on a medium containing auxin, preferably NAA at a concentration of at least about 15 mg/l. A further method for such somatic embryogenesis is provided wherein the culture medium contains a synergistically acting lowered carbohydrate and auxin concentration. Particularly embryogenic cells of such tissue are identified and improved maceration methods for contacting such cells with regeneration and transformation media are disclosed. Methods for transforming somatic tissue from soybean and other Glycine species are also provided. Whole, fertile, transformed plants are obtained

Dominant lethal pathologies in male mice engineered to contain an X-linked DUX4 transgene

Facioscapulohumeral muscular dystrophy (FSHD) is an enigmatic disease associated with epigenetic alterations in the subtelomeric heterochromatin of the D4Z4 macrosatellite repeat. Each repeat unit encodes DUX4, a gene that is normally silent in most tissues. Besides muscular loss, most patients suffer retinal vascular telangiectasias. To generate an animal model, we introduced a doxycycline-inducible transgene encoding DUX4 and 3' genomic DNA into a euchromatic region of the mouse X chromosome. Without induction, DUX4 RNA was expressed at low levels in many tissues and animals displayed a variety of unexpected dominant leaky phenotypes, including male-specific lethality. Remarkably, rare live-born males expressed DUX4 RNA in the retina and presented a retinal vascular telangiectasia. By using doxycycline to induce DUX4 expression in satellite cells, we observed impaired myogenesis in vitro and in vivo. This mouse model, which shows pathologies due to FSHD-related D4Z4 sequences, is likely to be useful for testing anti-DUX4 therapies in FSHD

Excess protein O-GlcNAcylation links metabolic derangements to right ventricular dysfunction in pulmonary arterial hypertension

The hexosamine biosynthetic pathway (HBP) converts glucose to uridine-diphosphate-N-acetylglucosamine, which, when added to serines or threonines, modulates protein function through protein O-GlcNAcylation. Glutamine-fructose-6-phosphate amidotransferase (GFAT) regulates HBP flux, and AMP-kinase phosphorylation of GFAT blunts GFAT activity and O-GlcNAcylation. While numerous studies demonstrate increased right ventricle (RV) glucose uptake in pulmonary arterial hypertension (PAH), the relationship between O-GlcNAcylation and RV function in PAH is unexplored. Therefore, we examined how colchicine-mediated AMP-kinase activation altered HBP intermediates, O-GlcNAcylation, mitochondrial function, and RV function in pulmonary artery-banded (PAB) and monocrotaline (MCT) rats. AMPK activation induced GFAT phosphorylation and reduced HBP intermediates and O-GlcNAcylation in MCT but not PAB rats. Reduced O-GlcNAcylation partially restored the RV metabolic signature and improved RV function in MCT rats. Proteomics revealed elevated expression of O-GlcNAcylated mitochondrial proteins in MCT RVs, which fractionation studies corroborated. Seahorse micropolarimetry analysis of H9c2 cardiomyocytes demonstrated colchicine improved mitochondrial function and reduced O-GlcNAcylation. Presence of diabetes in PAH, a condition of excess O-GlcNAcylation, reduced RV contractility when compared to nondiabetics. Furthermore, there was an inverse relationship between RV contractility and HgbA1C. Finally, RV biopsy specimens from PAH patients displayed increased O-GlcNAcylation. Thus, excess O-GlcNAcylation may contribute to metabolic derangements and RV dysfunction in PAH

Expression of the Human FSHD-Linked DUX4 Gene Induces Neurogenesis During Differentiation of Murine Embryonic Stem Cells

Misexpression of the double homeodomain protein DUX4 in muscle is believed to cause facioscapulohumeral muscular dystrophy (FSHD). Although strategies are being devised to inhibit DUX4 activity in FSHD, there is little known about the normal function of this protein. Expression of DUX4 has been reported in pluripotent cells and testis. To test the idea that DUX4 may be involved in initiating a germ lineage program in pluripotent cells, we interrogated the effect of expressing the human DUX4 gene at different stages during in vitro differentiation of murine embryonic stem (ES) cells. We find that expression of even low levels of DUX4 is incompatible with pluripotency: DUX4-expressing ES cells downregulate pluripotency markers and rapidly differentiate even in the presence of leukemia inhibitory factor (LIF) and bone morphogenetic protein 4 (BMP4). Transcriptional profiling revealed unexpectedly that DUX4 induced a neurectodermal program. Embryoid bodies exposed to a pulse of DUX4 expression displayed severely inhibited mesodermal differentiation, but acquired neurogenic potential. In a serum-containing medium in which neurogenic differentiation is minimal, DUX4 expression served as a neural-inducing factor, enabling the differentiation of Tuj1+ neurites. These data suggest that besides effects in muscle and germ cells, the involvement of DUX4 in neurogenesis should be considered as anti-DUX4 therapies are developed

New cellular and animal models for FSHD

Darko Bosnakovski1,2, Lynn M. Hartweck1, Abhijit Dandapat1, John Day3, Ramiro Nandez1, Radbod Darabi4, Rita R. Perlingeiro4, Lauren Snider5, Stephen J. Tapscott5, Janet Sowden6, Rabi Tawil6 and Michael Kyba1 1Lillehei Heart Institute and Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455 2Faculty of Technology and Technical Science‐Veles, University St. Kliment Ohridski‐Bitola, Skopje, R. Macedonia 3Paul and Sheila Wellstone Center for Muscular Dystrophy and Department of Neurology, University of Minnesota, Minneapolis, MN 55455 4Lillehei Heart Institute and Department of Medicine, University of Minnesota, Minneapolis, MN 55455 5Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109 6Fields Center for FSHD and Neuromuscular Research and Department of Neurology, University of Rochester, Rochester, NY 14642 There is currently no animal model bearing the actual FSHD mutation (D4Z4 contraction), and the lack of a suitable model system to study the effects of this mutation has severely hampered progress in understanding FSHD. We present data from two approaches to model FSHD: the generation of a mouse bearing a doxycycline‐inducible Dux4 gene, and the differentiation of iPS cells derived from myoblasts cultures established from FSHD and control biopsies. We have inserted a construct starting at the MAL initiation of Dux4 and continuing to the end of the EcoRI site, therefore containing the pLAM polyA signal from the FSHD A haplotype, into constitutively open chromatin 5’ of the HPRT gene. Even without induction we observed that males are almost never born and carrier females present several pathologies, although not in muscle. Rare males are severely affected, and do not survive past about 1 month of age. In addition to the phenotypes observed in the females, males also present testis abnormalities, including a defect in spermatogenesis. We are currently investigating vascular pathologies, which are especially evident in embryos, and severely enhanced when doxycycline is applied to induce expression of the transgene. Although muscle is not dystrophic, these are the first animals presenting a phenotype due to the presence of human D4Z4 DNA. In a second strategy, we have derived iPS cells from myoblast cultures taken from FSHD patients and controls. We present preliminary data on the ability of these cells to differentiate into muscle in vitro, and on the chromatin state and transcription of DUX4/D4Z4 at different stages of development

Mouse model to understand the role of Dux4 in FSHD

FSHD is an autosomal dominant disease that affects 1:20000 individuals. Mapping studies have associated the disease with a reduced number (1-10) of the D4Z4 macrosatellite repeats from the usual ~100. These repeats lie adjacent to the telomeres and are usually present in a highly silenced epigenetic state. It is not clear which genes are affected or how DNA methylation patterns affect the disease. Within the D4Z4 repeat is an ORF encoding a putative transcription factor named Dux4, containing two homeodomains. Although the function of Dux4 is unknown, the Dux4 homeodomains are similar to those of Pax7, a protein known to be involved in muscle development, proliferation and differentiation. We have previously reported that Dux4 is toxic when misexpressed at high levels in many cell types, and blocks differentiation of myoblasts when expressed at low levels, and competes with Pax7 for regulation of myogenic target genes. To model Dux4 function in vivo, we made a Dux4-inducible mES cell line by inserting a doxycycline-inducible Dux4 allele (iDux4+3’UTR) onto the X chromosome at a euchromatic region (HPRT). High-level induction of the Dux4 was toxic to mES cells but low-level Dux4 resulted in altered differentiation. When iDux4+3’UTR mice where generated and bred, this allele demonstrated leaky phenotypes in females, and male-specific lethality. Rare live-born males were small and underdeveloped with abnormal skin and defective sperm development and showed changes to muscle fibers, but no overt muscle degeneration. However, mice died within 1 month, well before the stage degeneration usually begins in FSHD. Dux4 protein could be induced and observed in cultured primary cells, and we are evaluatingpups and embryos for Dux4 expression in vivo. Dux4 carrier females were smaller and displayed the skin phenotype in transverse stripes. We hypothesize that the 3’ UTR contains an enhancer which drives leaky expression in some embryonic cell types and that X chromosome inactivation combined with selective survival of XDUX4-inactive cells protects the females from the lethality. To test for selectively biased X inactivation, we crossed Dux4 carrier females with XGFP males. Upon FACS analysis of the GFP+ cells in XGFP/ X+ vs. XGFP/ XDux4 female progeny, we found that the latter had an elevated frequency of GFP positive cells in most tissues, including the satellite cell compartment of skeletal muscle, confirming our hypothesis of selective XDUX4 inactivation. This mouse model suggests that Dux4 is a dominant lethal gene even when expressed at very low levels and can cause a variety of developmental defects in EBs and in embryo development