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

Notch activation is required for downregulation of HoxA3-dependent endothelial cell phenotype during blood formation.

Hemogenic endothelium (HE) undergoes endothelial-to-hematopoietic transition (EHT) to generate blood, a process that requires progressive down-regulation of endothelial genes and induction of hematopoietic ones. Previously, we have shown that the transcription factor HoxA3 prevents blood formation by inhibiting Runx1 expression, maintaining endothelial gene expression and thus blocking EHT. In the present study, we show that HoxA3 also prevents blood formation by inhibiting Notch pathway. HoxA3 induced upregulation of Jag1 ligand in endothelial cells, which led to cis-inhibition of the Notch pathway, rendering the HE nonresponsive to Notch signals. While Notch activation alone was insufficient to promote blood formation in the presence of HoxA3, activation of Notch or downregulation of Jag1 resulted in a loss of the endothelial phenotype which is a prerequisite for EHT. Taken together, these results demonstrate that Notch pathway activation is necessary to downregulate endothelial markers during EHT

Hox11 function in Region-Specific Adult Mesenchymal Stem/Stromal Cells is Required for Fracture Repari.

The mammalian skeleton boasts a remarkable capacity to completely restore the original structure and function of a bone following injury. Interestingly, the biological processes of fracture repair recapitulate many of the mechanisms of embryonic skeletal development. The Hox genes are critical regulators of skeletal development, yet the function of these genes during adult fracture repair is largely unknown. Ongoing research in the Wellik lab is focused on understanding the role(s) of these genes in this context. The Hox genes encode evolutionarily conserved transcription factors that are imperative for patterning of the axial and limb skeleton in the developing embryo. Specifically, Hox11 genes function to instruct growth and morphology of the lumbar elements of the axial skeleton and the zeugopod elements (radius/ulna and tibia/fibula) of the limbs. Previous work using a Hoxa11eGFP allele showed that Hox11 is expressed through the latest stages of embryonic development. We have now discovered that Hox11 genes continue to be expressed in the adult skeleton and are largely restricted to the previously characterized PDGFRα+/CD51+/Leptin Receptor(LepR)+ mesenchymal stem/stromal cell (MSC) population in bone marrow. These Hox11-expressing MSCs expand in response to fracture injury and are maintained throughout repair. Loss of Hox11 function results in a significantly reduced ability to generate cartilage early in repair, and at late stages, the hard callus persists and is incompletely remodeled. Together, our data suggests that Hox11 functions in MSCs at multiple stages of repair, first, for endochondral ossification and later for bone remodeling. In addition, we show more generally that the Hox expression pattern established during embryonic development is maintained in the adult skeleton. Overall, this research provides novel evidence that Hox genes have critical roles beyond embryonic patterning and that these genes are expressed and function in adult MSCs.PHDCell and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/135754/1/drrux_1.pd

Inducible cassette exchange: a rapid and efficient system enabling conditional gene expression in embryonic stem and primary cells.

Genetic modification is critically enabling for studies addressing specification and maintenance of cell fate; however, methods for engineering modifications are inefficient. We demonstrate a rapid and efficient recombination system in which an inducible, floxed cre allele replaces itself with an incoming transgene. We target this inducible cassette exchange (ICE) allele to the (HPRT) locus and demonstrate recombination in murine embryonic stem cells (ESCs) and primary cells from derivative ICE mice. Using lentivectors, we demonstrate recombination at a randomly integrated ICE locus in human ESCs. To illustrate the utility of this system, we insert the myogenic regulator, Myf5, into the ICE locus in each platform. This enables efficient directed differentiation of mouse and human ESCs into skeletal muscle and conditional myogenic transdetermination of primary cells cultured in vitro. This versatile tool is thus well suited to gain-of-function studies probing gene function in the specification and reprogramming of cell fate

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

HoxA3 Controls Notch Pathway to Repress Blood Development

Hematopoietic stem cells (HSC) are generated from a specialized subset of endothelial cells, hemogenic endothelium. Previous studies performed by our group showed that HoxA3 restrains the cell at the hemogenic endothelium stage, inhibiting further differentiation toward blood by direct repression of Runx1. Building on our previous work, we show here that overexpression of HoxA3 affects the Notch pathway. Upon HoxA3 upregulation in endothelial cells, Jag1 is induced, Mfng (Manic) and Lfng (Lunatic) fringes are downregulated, and there is a trend towards Notch target gene repression. These data suggest that in the presence of HoxA3, endothelial cells are blocked from receiving Notch signal through ligand cis-inhibition with resulting blood inhibition. In order to test this hypothesis, we evaluated the effect of activation or inhibition of the Notch pathway during blood development. We show here that the number of blood progenitors originating from the hemogenic endothelium is decreased when the Notch pathway is inhibited. Conversely, induction of the pathway by upregulation of NICD (Notch1 Intra Cellular Domain) promotes an increase in the number of blood progenitors originating from hemogenic endothelium. Furthermore, inhibition of the pathway when HoxA3 is upregulated has little or no effect in blood while induction of the pathway in HoxA3 presence in part promotes blood development. Taken together, these results demonstrate that Notch pathway is both sufficient and essential for blood development. Specifically HoxA3 inhibits Notch signal reception in two ways: 1) HoxA3 increases Jag1 ligand expression that acts in cis-inhibition; 2) represses Manic and Lunatic fringes both necessary to increase the affinity of Notch receptors for the Delta ligands. When this blockage is bypassed by NICD upregulation, blood is formed, demonstrating that HoxA3-dependent Notch inhibition results in blood suppression

Molecular diagnosis and novel genes and phenotypes in a pediatric thoracic insufficiency cohort

Abstract Thoracic insufficiency syndromes are a genetically and phenotypically heterogeneous group of disorders characterized by congenital abnormalities or progressive deformation of the chest wall and/or vertebrae that result in restrictive lung disease and compromised respiratory capacity. We performed whole exome sequencing on a cohort of 42 children with thoracic insufficiency to elucidate the underlying molecular etiologies of syndromic and non-syndromic thoracic insufficiency and predict extra-skeletal manifestations and disease progression. Molecular diagnosis was established in 24/42 probands (57%), with 18/24 (75%) probands having definitive diagnoses as defined by laboratory and clinical criteria and 6/24 (25%) probands having strong candidate genes. Gene identified in cohort patients most commonly encoded components of the primary cilium, connective tissue, and extracellular matrix. A novel association between KIF7 and USP9X variants and thoracic insufficiency was identified. We report and expand the genetic and phenotypic spectrum of a cohort of children with thoracic insufficiency, reinforce the prevalence of extra-skeletal manifestations in thoracic insufficiency syndromes, and expand the phenotype of KIF7 and USP9X-related disease to include thoracic insufficiency

Notch signaling in <i>trans</i> does not rescue HoxA3 mediated inhibition of Notch.

<p><b>A)</b> Experimental procedure <b>B)</b> Representative flow cytometric profile of endothelial surface markers Flk-1/Ve-Cadherin and hematopoietic surface markers c-Kit/CD41, and c-Kit/CD45 obtained from 200,000 EB-derived Flk1⁺/VE-cadherin⁺ cells and co-cultured on OP9 control (CON) or OP9 overexpressing Dll1 (OP9-Dll1) for 5 days in Control or HoxA3-overexpressing HE cells. <b>C)</b> Quantification of frequencies of hematopoietic surface markers (CD41, CD45) of the same cell as in <b>B</b>. <b>D)</b> Gene expression levels of the Notch pathway target genes (Hes1, Hey1, Hey2) and hematopoietic gene markers (PU.1, Runx1, Gata1) on control (white bar) or HoxA3-overexpressing (black bars) HE cells co-cultured on OP9 controls (CON) or OP9-DLL1 for 5 days (Flk1⁺/VE-cadherin⁺ and CD41⁺/c-Kit⁺ cells were pulled together). <b>E)</b> Iimmunofluorescence staining for activated Notch1 (NICD-red), VE-Cadherin (VECad-green) and Hoechst (blue) showing adherent endothelial clusters growing in Control (Con) or HoxA3 overexpression (HoxA3), derived from endothelial cells (Flk1⁺/VE-cadherin⁺) co-cultured on OP9-DLL1 cells *: p<0.05. Statistical analysis is reported on <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186818#pone.0186818.s010" target="_blank">S5 Table</a></b>.</p

Repression of EHT by HoxA3 is not affected by inhibition Notch pathway.

<p><b>A)</b> Experimental procedure, Endothelial Derived Cells (ENDO). <b>B)</b> Representative flow cytometric profile of endothelial surface markers Flk-1/Ve-Cadherin and hematopoietic surface markers c-Kit/CD41, and c-Kit/CD45 on 200,000 EB-derived Flk1⁺/VE-cadherin⁺ cells without or with HoxA3 overexpression and co-cultured on OP9 for 5 days in the presence or absence of the Notch inhibitor DAPT (20 μM). <b>C)</b> Frequency of endothelial surface markers Flk-1⁺/Ve-Cadherin⁺ in EB-derived cells. *: p<0.05; **: p<0.01. Two way ANOVA analyses of Flk-1⁺/Ve-Cadherin⁺, CD41⁺ and CD45⁺ frequencies are reported on <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186818#pone.0186818.s008" target="_blank">S3 Table</a></b>.</p