The H2.0-like homeobox transcription factor modulates yolk sac vascular remodeling in mouse embryos.
OBJECTIVE
The H2.0-like homeobox transcription factor (HLX) plays an essential role in visceral organogenesis in mice and has been shown to regulate angiogenic sprouting in vitro and in zebrafish embryos. We therefore examined the role of HLX in vascular development in mouse and avian embryos.


RESULTS
In situ hybridization showed that Hlx is expressed in a subset of sprouting blood vessels in postnatal mouse retinas and embryos. Hlx expression was conserved in quail embryos and upregulated in blood vessels at the onset of circulation. In vitro assays showed that Hlx is dynamically regulated by growth factors and shear stress alterations. Proangiogenic vascular endothelial growth factor induces Hlx expression in cultured endothelial cells, whereas signals that induce stalk cell identity lead to a reduction in Hlx expression. HLX was also downregulated in embryos in which flow was ablated, whereas injection of a starch solution, which increases blood viscosity and therefore shear stress, causes an upregulation in HLX. HLX knockdown in vitro resulted in a reduction in tip cell marker expression and in reduced angiogenic sprouting, but Hlx(-/-) embryos showed no defect in vascular sprouting at E8.5, E9.5, or E11.5 in vivo. Vascular remodeling of the capillary plexus was altered in Hlx(-/-) embryos, with a modestly enlarged venous plexus and reduction of the arterial plexus.


CONCLUSIONS
Our findings indicate not only that Hlx regulates sprouting in vitro, but that its role in sprouting is nonessential in vivo. We find HLX is regulated by shear stress and a subtle defect in vascular remodeling is present in knockout embryos.
From the Department of Internal Medicine, Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT (C.P., F.M., B.L., F.Z., A.E.); Lady Davis Institute, Jewish General Hospital (B.K., E.D.J., D.V., H.C.Z., E.A.V.J.) and Departments of Experimental Medicine (B.K.), Biomedical Engineering, (E.D.J.), and Chemical Engineering (E.A.V.J.), McGill University, Montreal, Quebec, Canada; CIRB Collège de France, INSERM U1050/CNRS UMR7241, Paris, France (L.P., L.P.-F., A.E.); Department of Molecular and Cellular Physiology, Yale University Medical School, New Haven, CT (A.E.); and Department of Cardiovascular Science, KU Leuven, Leuven, Belgium (B.K., E.A.V.J).
Claudia
C
Prahst
From the Department of Internal Medicine, Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT (C.P., F.M., B.L., F.Z., A.E.); Lady Davis Institute, Jewish General Hospital (B.K., E.D.J., D.V., H.C.Z., E.A.V.J.) and Departments of Experimental Medicine (B.K.), Biomedical Engineering, (E.D.J.), and Chemical Engineering (E.A.V.J.), McGill University, Montreal, Quebec, Canada; CIRB Collège de France, INSERM U1050/CNRS UMR7241, Paris, France (L.P., L.P.-F., A.E.); Department of Molecular and Cellular Physiology, Yale University Medical School, New Haven, CT (A.E.); and Department of Cardiovascular Science, KU Leuven, Leuven, Belgium (B.K., E.A.V.J).
Bahar
B
Kasaai
From the Department of Internal Medicine, Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT (C.P., F.M., B.L., F.Z., A.E.); Lady Davis Institute, Jewish General Hospital (B.K., E.D.J., D.V., H.C.Z., E.A.V.J.) and Departments of Experimental Medicine (B.K.), Biomedical Engineering, (E.D.J.), and Chemical Engineering (E.A.V.J.), McGill University, Montreal, Quebec, Canada; CIRB Collège de France, INSERM U1050/CNRS UMR7241, Paris, France (L.P., L.P.-F., A.E.); Department of Molecular and Cellular Physiology, Yale University Medical School, New Haven, CT (A.E.); and Department of Cardiovascular Science, KU Leuven, Leuven, Belgium (B.K., E.A.V.J).
Filipa
F
Moraes
From the Department of Internal Medicine, Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT (C.P., F.M., B.L., F.Z., A.E.); Lady Davis Institute, Jewish General Hospital (B.K., E.D.J., D.V., H.C.Z., E.A.V.J.) and Departments of Experimental Medicine (B.K.), Biomedical Engineering, (E.D.J.), and Chemical Engineering (E.A.V.J.), McGill University, Montreal, Quebec, Canada; CIRB Collège de France, INSERM U1050/CNRS UMR7241, Paris, France (L.P., L.P.-F., A.E.); Department of Molecular and Cellular Physiology, Yale University Medical School, New Haven, CT (A.E.); and Department of Cardiovascular Science, KU Leuven, Leuven, Belgium (B.K., E.A.V.J).
Espen D
ED
Jahnsen
From the Department of Internal Medicine, Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT (C.P., F.M., B.L., F.Z., A.E.); Lady Davis Institute, Jewish General Hospital (B.K., E.D.J., D.V., H.C.Z., E.A.V.J.) and Departments of Experimental Medicine (B.K.), Biomedical Engineering, (E.D.J.), and Chemical Engineering (E.A.V.J.), McGill University, Montreal, Quebec, Canada; CIRB Collège de France, INSERM U1050/CNRS UMR7241, Paris, France (L.P., L.P.-F., A.E.); Department of Molecular and Cellular Physiology, Yale University Medical School, New Haven, CT (A.E.); and Department of Cardiovascular Science, KU Leuven, Leuven, Belgium (B.K., E.A.V.J).
Bruno
B
Larrivee
From the Department of Internal Medicine, Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT (C.P., F.M., B.L., F.Z., A.E.); Lady Davis Institute, Jewish General Hospital (B.K., E.D.J., D.V., H.C.Z., E.A.V.J.) and Departments of Experimental Medicine (B.K.), Biomedical Engineering, (E.D.J.), and Chemical Engineering (E.A.V.J.), McGill University, Montreal, Quebec, Canada; CIRB Collège de France, INSERM U1050/CNRS UMR7241, Paris, France (L.P., L.P.-F., A.E.); Department of Molecular and Cellular Physiology, Yale University Medical School, New Haven, CT (A.E.); and Department of Cardiovascular Science, KU Leuven, Leuven, Belgium (B.K., E.A.V.J).
David
D
Villegas
From the Department of Internal Medicine, Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT (C.P., F.M., B.L., F.Z., A.E.); Lady Davis Institute, Jewish General Hospital (B.K., E.D.J., D.V., H.C.Z., E.A.V.J.) and Departments of Experimental Medicine (B.K.), Biomedical Engineering, (E.D.J.), and Chemical Engineering (E.A.V.J.), McGill University, Montreal, Quebec, Canada; CIRB Collège de France, INSERM U1050/CNRS UMR7241, Paris, France (L.P., L.P.-F., A.E.); Department of Molecular and Cellular Physiology, Yale University Medical School, New Haven, CT (A.E.); and Department of Cardiovascular Science, KU Leuven, Leuven, Belgium (B.K., E.A.V.J).
Luc
L
Pardanaud
From the Department of Internal Medicine, Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT (C.P., F.M., B.L., F.Z., A.E.); Lady Davis Institute, Jewish General Hospital (B.K., E.D.J., D.V., H.C.Z., E.A.V.J.) and Departments of Experimental Medicine (B.K.), Biomedical Engineering, (E.D.J.), and Chemical Engineering (E.A.V.J.), McGill University, Montreal, Quebec, Canada; CIRB Collège de France, INSERM U1050/CNRS UMR7241, Paris, France (L.P., L.P.-F., A.E.); Department of Molecular and Cellular Physiology, Yale University Medical School, New Haven, CT (A.E.); and Department of Cardiovascular Science, KU Leuven, Leuven, Belgium (B.K., E.A.V.J).
Laurence
L
Pibouin-Fragner
From the Department of Internal Medicine, Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT (C.P., F.M., B.L., F.Z., A.E.); Lady Davis Institute, Jewish General Hospital (B.K., E.D.J., D.V., H.C.Z., E.A.V.J.) and Departments of Experimental Medicine (B.K.), Biomedical Engineering, (E.D.J.), and Chemical Engineering (E.A.V.J.), McGill University, Montreal, Quebec, Canada; CIRB Collège de France, INSERM U1050/CNRS UMR7241, Paris, France (L.P., L.P.-F., A.E.); Department of Molecular and Cellular Physiology, Yale University Medical School, New Haven, CT (A.E.); and Department of Cardiovascular Science, KU Leuven, Leuven, Belgium (B.K., E.A.V.J).
Feng
F
Zhang
From the Department of Internal Medicine, Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT (C.P., F.M., B.L., F.Z., A.E.); Lady Davis Institute, Jewish General Hospital (B.K., E.D.J., D.V., H.C.Z., E.A.V.J.) and Departments of Experimental Medicine (B.K.), Biomedical Engineering, (E.D.J.), and Chemical Engineering (E.A.V.J.), McGill University, Montreal, Quebec, Canada; CIRB Collège de France, INSERM U1050/CNRS UMR7241, Paris, France (L.P., L.P.-F., A.E.); Department of Molecular and Cellular Physiology, Yale University Medical School, New Haven, CT (A.E.); and Department of Cardiovascular Science, KU Leuven, Leuven, Belgium (B.K., E.A.V.J).
Hans C
HC
Zaun
From the Department of Internal Medicine, Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT (C.P., F.M., B.L., F.Z., A.E.); Lady Davis Institute, Jewish General Hospital (B.K., E.D.J., D.V., H.C.Z., E.A.V.J.) and Departments of Experimental Medicine (B.K.), Biomedical Engineering, (E.D.J.), and Chemical Engineering (E.A.V.J.), McGill University, Montreal, Quebec, Canada; CIRB Collège de France, INSERM U1050/CNRS UMR7241, Paris, France (L.P., L.P.-F., A.E.); Department of Molecular and Cellular Physiology, Yale University Medical School, New Haven, CT (A.E.); and Department of Cardiovascular Science, KU Leuven, Leuven, Belgium (B.K., E.A.V.J).
Anne
A
Eichmann
From the Department of Internal Medicine, Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT (C.P., F.M., B.L., F.Z., A.E.); Lady Davis Institute, Jewish General Hospital (B.K., E.D.J., D.V., H.C.Z., E.A.V.J.) and Departments of Experimental Medicine (B.K.), Biomedical Engineering, (E.D.J.), and Chemical Engineering (E.A.V.J.), McGill University, Montreal, Quebec, Canada; CIRB Collège de France, INSERM U1050/CNRS UMR7241, Paris, France (L.P., L.P.-F., A.E.); Department of Molecular and Cellular Physiology, Yale University Medical School, New Haven, CT (A.E.); and Department of Cardiovascular Science, KU Leuven, Leuven, Belgium (B.K., E.A.V.J). liz.jones@med.kuleuven.be.
Elizabeth A V
EA
Jones
D000818:Animals; D001808:Blood Vessels / Q000196:embryology / Q000378:metabolism*; D001809:Blood Viscosity; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000098:blood supply; D004625:Embryo, Nonmammalian; D018507:Gene Expression Regulation, Developmental; D005865:Gestational Age; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D040542:Mechanotransduction, Cellular; D051379:Mice; D018345:Mice, Knockout; D018919:Neovascularization, Physiologic; D011784:Quail; D034622:RNA Interference; D012039:Regional Blood Flow; D013314:Stress, Mechanical; D014157:Transcription Factors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D015017:Yolk Sac / Q000098:blood supply*
C084058:HLX protein, human; C489999:Hlx protein, mouse; D018398:Homeodomain Proteins; D014157:Transcription Factors
angiogenesis modulators; genes, homeobox; mechanosensory transduction, post-implantation embryonic development