Barriga EH , Maxwell PH , Reyes AE , Mayor R .

Biotechnology and Biological Sciences Research Council , Medical Research Council , Wellcome Trust , MR/J000655/1 Medical Research Council , MRC_MR/J000655/1 Medical Research Council

	Figure 1. Hif-1α is required for neural crest migration. (A–Q) Xenopus (A–H) and zebrafish (I–Q) embryos were injected with antisense MOs as indicated in the figure, and the expression of the neural crest markers Snail2 (A–H) or crestin (I–M) was analyzed. Asterisks show the injected side. (A–D) No effect of ATGMOhif-1α on neural crest induction. (E–H) Strong effect of ATGMOhif-1α on neural crest migration. (I–J′) Dorsal (I and J) or lateral (I′ and J′) view of zebrafish embryos. (J) Note that in ATGMOhif-1α–injected embryos, neural crests are present around to the dorsal midline. (L) Percentage of embryos with the described phenotype. (M) The number of neural crest streams was counted by analyzing the expression of crestin for each treatment. (K, K′, and M) Note that the number of streams is reduced with the ATGMOhif-1α but rescued by the coinjection of Hif-1α mRNA. (N–Q) Analysis of cartilage development in zebrafish embryos. The bars in D, H, L, M, and Q histograms represent the standard deviations of three independent experiments, with ∼40 embryos used in each case. som, somite.
	Figure 2. Hif-1α is required cell autonomously for neural crest migration. (A–H) Frames of a time-lapse video (Video 1) of zebrafish embryos Tg(sox10:mRFP). Leader cells are shown pseudocolored in red for embryos injected with a control MO (A–C) or an ATGMOhif-1α (E–G). (D and H) Centered tracks of leader cells. (I and J) Analysis of cell directionality and velocity of leader cells under indicated conditions. Errors bars represent standard deviation of three independent experiments, with a minimal of 20 embryos in each case. ***, P < 0.005. (K–O) Xenopus graft experiments. (K) FDX/MOs were injected into the ectoderm of blastula embryos. At early neurula stages (St), the injected neural crest was grafted into noninjected embryos, and neural crest migration was analyzed in vivo. (L and N) Normal neural crest migration (n = 10, 80% migration). Arrows show migrating neural crest. (M and O) Inhibition of neural crest migration after ATGMOhif-1α injection (n = 10, 20% migration). (P) Western blot of whole neurula Xenopus embryos, under the indicated conditions. (Q) Western blot of cephalic Xenopus regions enriched in neural crest cells versus tail of young tadpoles that contain very few neural crest cells.
	Figure 3. Twist and Cxcr4, but not Snail1 or Snail2, are Hif-1α targets in the neural crest. (A–L) Xenopus embryos. In situ hybridization for Snail1, Snail2, Twist, and Cxcr4 after ATGMOhif-1α injection. Asterisks show the injected side. (A–F) Snail1 and Snail2 expression. Note that neither Snail1 nor Snail2 are affected by inhibition of Hif-1α, but a clear inhibition of neural crest migration is observed. (G–I) Twist expression is lost after ATGMOhif-1α injection (arrows). (J–L) Cxcr4 expression. (J) Note that strong expression in the neural plate is not affected, whereas the neural crest expression is visible only in the right (noninjected) side as three masses next to the neural plate. Arrows indicate absence of gene expression in the neural crest at the injected side. (M and N) Quantification of results shown in A–L. Bars represent the standard deviation from three independent experiments, a minimal of 40 embryos was analyzed for each case. (O and P) Quantitative PCR (O) and RT-PCR (P) of neural crest dissected from embryos injected with a control MO or with ATGMOhif-1α genes analyzed as indicated. EF-1α was used as a loading control. Bars represent the standard deviation from three independent experiments. A minimal of 30 embryos was analyzed for each case. ***, P < 0.005.
	Figure 4. Hif-1α controls chemotaxis in a Cxcr4-dependent manner. Chemotaxis toward a localized source of SDF-1, which is located to the right of each panel, for each treatment as indicated at the top of the figure. (A–C) Time 0. (D–I) 3 h after culture. (A–I) Fluorescent (A–F) and bright-field (G–I) images. (J–L) Tracks of leader migrating cells. Chemotactic cells should move toward the right side of the panel, where SDF-1 is higher. (M and N) Angles of migration for individual cells. (O) Chemotaxis index for leader cells. (P) Persistence. (Q) Leader cells (green) are more efficiently attracted than trailing cells (red). (R) Chemotaxis of leader (green) and trailing (orange) cells under the indicated conditions. Bars represent the standard deviation from three independent experiments, with a minimal of 60 cells analyzed for each treatment. ***, P < 0.005. Ctrl, control.
	Figure 5. Hif-1α gain of function induces cell dispersion and inhibits chemotaxis. Chemotaxis was analyzed as described in Fig. 4 for each treatment as indicated at the top of the figure. (A–E) Time 0. (F–J) 3 h after culture. (K–O) Tracks of migrating cells. (P–T) Angles of migration for individual cells. (U) Cell velocity. (V) Persistence. (W) Chemotaxis index. Each experiment was performed at least three times, and ∼60 cells were analyzed for each treatment. ***, P < 0.005. Errors bars represent standard deviation.
	Figure 6. Hif-1α promotes neural crest dispersion. Neural crest cells labeled with nuclear-RFP and membrane-GFP were cultured in vitro for 3 h, and cells dispersion was analyzed. (A–E) Nuclear labeling is shown at time 0 and after 3 h. (F–J) Delaunay triangulation shown for a 3-h time to analyze the distance between neighbor cells. Color coded according to the area of the triangles. (K–O) Bright-field images after the indicate treatments. (P–T) Membrane-GFP merged with nuclear-RFP to observe cell morphology, after the indicated treatments. (P) Quantification of Delaunay triangulation for all the conditions analyzed. ***, P < 0.005. Errors bars represent standard deviation, calculated from three independent experiments, with a minimal of 60 cells for each condition.
	Figure 7. Hif-1α controls cell dispersion in a Twist-dependent manner. (A–D) In situ hybridization against the neural crest marker Snail2. (A) Injection of ATGMOTwist into the prospective neural crest at the 16- and 32-cell stage embryo does not affect neural crest induction. Asterisk shows the injected side. (B–D) Injection of ATGMOTwist blocks neural crest migration. (E–S) Analysis of dispersion of cultured neural crest cells as described in Fig 6. (E–J) Nuclear labeling is shown at time 0 and after 3 h. (K–M) Delaunay triangulation shown for 3 h time to analyze the distance between neighbor cells. Color coded according to the area of the triangles after the indicate treatments. (N–P) Bright-field images after the indicated treatments. (Q–S) Membrane-GFP merged with nuclear-RFP to observe cell morphology. (T) Quantification of Delaunay triangulation for all the conditions analyzed. ***, P < 0.005. Errors bars represent standard deviation of three independent experiments, with a minimal of 60 cells analyzed for each condition. (U) Percentage of explants exhibiting cell dispersion.
	Figure 8. Hif-1α/Twist are repressors of E-cadherin in the neural crest. (A–L) Immunofluorescence against E-cadherin performed in neural crest cultured in vitro. (M–S) Histological sections of immunofluorescence against E-cadherin performed in whole embryos. The diagram shows the orientation of the sections. White outlines show epidermis and placodes; green outlines show three streams of cephalic neural crest; e, eye. Red staining shows E-cadherin; blue staining shows DAPI; purple staining in P and S shows cells injected with ATGMOhif-1α. Arrows show neural crest. Note that some of the neural crests injected with the MO show E-cadherin staining (arrows in Q). (T and U) Temporal analysis of E-cadherin expression. Prospective neural crest (stage 13) and premigratory neural crest (stage 16) were dissected, and quantitative PCR was performed for E-cadherin and EF-1α (loading control). Bars represent the standard deviation from three independent experiments. ***, P < 0.005. NC, neural crest; qPCR, quantitative PCR; St, stage.
	Figure 9. Model of Hif-1 controlling neural crest migration. Neural crest, shown as blue cells, delaminates from the neural tube (NT). Prospective neural crest expresses E-cadherin (E-cad; red), just before EMT Hif-1α activates Twist, which in turn represses E-cadherin expression, allowing neural crest EMT. In addition, Hif-1α activates Cxcr4, which is required for chemotaxis toward SDF-1.

Akimenko, Combinatorial expression of three zebrafish genes related to distal-less: part of a homeobox gene code for the head. 1994, Pubmed

Akimenko, Combinatorial expression of three zebrafish genes related to distal-less: part of a homeobox gene code for the head. 1994, Pubmed
Alfandari, Integrin alpha5beta1 supports the migration of Xenopus cranial neural crest on fibronectin. 2003, Pubmed , Xenbase
Aybar, Snail precedes slug in the genetic cascade required for the specification and migration of the Xenopus neural crest. 2003, Pubmed , Xenbase
Barrallo-Gimeno, Neural crest survival and differentiation in zebrafish depends on mont blanc/tfap2a gene function. 2004, Pubmed
Barrallo-Gimeno, The Snail genes as inducers of cell movement and survival: implications in development and cancer. 2005, Pubmed
Betancur, Assembling neural crest regulatory circuits into a gene regulatory network. 2010, Pubmed
Bildsoe, Requirement for Twist1 in frontonasal and skull vault development in the mouse embryo. 2009, Pubmed
Breau, A nonneural epithelial domain of embryonic cranial neural folds gives rise to ectomesenchyme. 2008, Pubmed
Cacheux, Loss-of-function mutations in SIP1 Smad interacting protein 1 result in a syndromic Hirschsprung disease. 2001, Pubmed
Cairns, Regulation of cancer cell metabolism. 2011, Pubmed
Carmona-Fontaine, Complement fragment C3a controls mutual cell attraction during collective cell migration. 2011, Pubmed , Xenbase
Carmona-Fontaine, Contact inhibition of locomotion in vivo controls neural crest directional migration. 2008, Pubmed , Xenbase
Chang, Hypoxia predicts aggressive growth and spontaneous metastasis formation from orthotopically grown primary xenografts of human pancreatic cancer. 2011, Pubmed
Chen, twist is required in head mesenchyme for cranial neural tube morphogenesis. 1995, Pubmed
Chong, Expression pattern of two zebrafish genes, cxcr4a and cxcr4b. 2001, Pubmed
Cockman, Hypoxia inducible factor-alpha binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein. 2000, Pubmed
Compernolle, Cardia bifida, defective heart development and abnormal neural crest migration in embryos lacking hypoxia-inducible factor-1alpha. 2003, Pubmed
Dady, Timing and kinetics of E- to N-cadherin switch during neurulation in the avian embryo. 2012, Pubmed
De Calisto, Essential role of non-canonical Wnt signalling in neural crest migration. 2005, Pubmed , Xenbase
DeSimone, The Xenopus embryo as a model system for studies of cell migration. 2005, Pubmed , Xenbase
Erler, Hypoxia-mediated down-regulation of Bid and Bax in tumors occurs via hypoxia-inducible factor 1-dependent and -independent mechanisms and contributes to drug resistance. 2004, Pubmed
Evans, VHL promotes E2 box-dependent E-cadherin transcription by HIF-mediated regulation of SIP1 and snail. 2007, Pubmed
Gort, The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2alpha. 2008, Pubmed
Graeber, Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. 1996, Pubmed
Gray, HIF-1alpha, STAT3, CBP/p300 and Ref-1/APE are components of a transcriptional complex that regulates Src-dependent hypoxia-induced expression of VEGF in pancreatic and prostate carcinomas. 2005, Pubmed
Gupta, Cancer metastasis: building a framework. 2006, Pubmed
Gómez-Skarmeta, Xiro, a Xenopus homolog of the Drosophila Iroquois complex genes, controls development at the neural plate. 1998, Pubmed , Xenbase
Harland, In situ hybridization: an improved whole-mount method for Xenopus embryos. 1991, Pubmed , Xenbase
Harland, Translation of mRNA injected into Xenopus oocytes is specifically inhibited by antisense RNA. 1985, Pubmed , Xenbase
Harris, Hypoxia--a key regulatory factor in tumour growth. 2002, Pubmed
Hendrix, Reprogramming metastatic tumour cells with embryonic microenvironments. 2007, Pubmed
Hill, Cancer stem cells, hypoxia and metastasis. 2009, Pubmed
Hong, The activity of Pax3 and Zic1 regulates three distinct cell fates at the neural plate border. 2007, Pubmed , Xenbase
Hopwood, A Xenopus mRNA related to Drosophila twist is expressed in response to induction in the mesoderm and the neural crest. 1989, Pubmed , Xenbase
Ishii, Msx2 and Twist cooperatively control the development of the neural crest-derived skeletogenic mesenchyme of the murine skull vault. 2003, Pubmed
Kamura, Activation of HIF1alpha ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex. 2000, Pubmed
Kelsh, Expression of zebrafish fkd6 in neural crest-derived glia. 2000, Pubmed
Kimmel, Stages of embryonic development of the zebrafish. 1995, Pubmed , Xenbase
Kioi, Inhibition of vasculogenesis, but not angiogenesis, prevents the recurrence of glioblastoma after irradiation in mice. 2010, Pubmed
Kirby, In vivo time-lapse imaging shows dynamic oligodendrocyte progenitor behavior during zebrafish development. 2006, Pubmed
Knight, lockjaw encodes a zebrafish tfap2a required for early neural crest development. 2003, Pubmed
Koh, HAF : the new player in oxygen-independent HIF-1alpha degradation. 2009, Pubmed
Kucia, Trafficking of normal stem cells and metastasis of cancer stem cells involve similar mechanisms: pivotal role of the SDF-1-CXCR4 axis. 2005, Pubmed
Kulesa, Reprogramming metastatic melanoma cells to assume a neural crest cell-like phenotype in an embryonic microenvironment. 2006, Pubmed
Kuriyama, Molecular analysis of neural crest migration. 2008, Pubmed , Xenbase
Linker, Relationship between gene expression domains of Xsnail, Xslug, and Xtwist and cell movement in the prospective neural crest of Xenopus. 2000, Pubmed , Xenbase
Locascio, Modularity and reshuffling of Snail and Slug expression during vertebrate evolution. 2002, Pubmed
Luo, Specific pan-neural crest expression of zebrafish Crestin throughout embryonic development. 2001, Pubmed
Mancilla, Neural crest formation in Xenopus laevis: mechanisms of Xslug induction. 1996, Pubmed , Xenbase
Matthews, Directional migration of neural crest cells in vivo is regulated by Syndecan-4/Rac1 and non-canonical Wnt signaling/RhoA. 2008, Pubmed , Xenbase
Mayor, Induction of the prospective neural crest of Xenopus. 1995, Pubmed , Xenbase
Moepps, Characterization of a Xenopus laevis CXC chemokine receptor 4: implications for hematopoietic cell development in the vertebrate embryo. 2000, Pubmed , Xenbase
Morrison, Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells. 2000, Pubmed
Nagao, HIF-1alpha signaling upstream of NKX2.5 is required for cardiac development in Xenopus. 2008, Pubmed , Xenbase
Nandadasa, N- and E-cadherins in Xenopus are specifically required in the neural and non-neural ectoderm, respectively, for F-actin assembly and morphogenetic movements. 2009, Pubmed , Xenbase
Ocaña, Metastatic colonization requires the repression of the epithelial-mesenchymal transition inducer Prrx1. 2012, Pubmed
Ohh, Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein. 2000, Pubmed
Olesnicky Killian, A role for chemokine signaling in neural crest cell migration and craniofacial development. 2009, Pubmed
Ota, Twist is required for patterning the cranial nerves and maintaining the viability of mesodermal cells. 2004, Pubmed
Peinado, Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? 2007, Pubmed
Pennacchietti, Hypoxia promotes invasive growth by transcriptional activation of the met protooncogene. 2003, Pubmed
Rojas, Cloning of hif-1alpha and hif-2alpha and mRNA expression pattern during development in zebrafish. 2007, Pubmed
Rouschop, The unfolded protein response protects human tumor cells during hypoxia through regulation of the autophagy genes MAP1LC3B and ATG5. 2010, Pubmed
Schulte-Merker, The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo. 1992, Pubmed
Sefton, Conserved and divergent roles for members of the Snail family of transcription factors in the chick and mouse embryo. 1998, Pubmed
Semenza, Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics. 2010, Pubmed
Semenza, Hypoxia and cancer. 2007, Pubmed
Semenza, Hypoxia-inducible nuclear factors bind to an enhancer element located 3' to the human erythropoietin gene. 1991, Pubmed
Semenza, HIF-1: using two hands to flip the angiogenic switch. 2000, Pubmed
Semenza, HIF-1 and tumor progression: pathophysiology and therapeutics. 2002, Pubmed
Soo, Twist function is required for the morphogenesis of the cephalic neural tube and the differentiation of the cranial neural crest cells in the mouse embryo. 2002, Pubmed
Stachel, Lithium perturbation and goosecoid expression identify a dorsal specification pathway in the pregastrula zebrafish. 1993, Pubmed
Staller, Chemokine receptor CXCR4 downregulated by von Hippel-Lindau tumour suppressor pVHL. 2003, Pubmed
Steventon, Genetic network during neural crest induction: from cell specification to cell survival. 2005, Pubmed
Stoetzel, Dorso-ventral and rostro-caudal sequential expression of M-twist in the postimplantation murine embryo. 1995, Pubmed
Studer, Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen. 2000, Pubmed
Tanimoto, Mechanism of regulation of the hypoxia-inducible factor-1 alpha by the von Hippel-Lindau tumor suppressor protein. 2000, Pubmed
Theveneau, Collective cell migration of the cephalic neural crest: the art of integrating information. 2011, Pubmed
Theveneau, Integrating chemotaxis and contact-inhibition during collective cell migration: Small GTPases at work. 2010, Pubmed
Theveneau, Collective chemotaxis requires contact-dependent cell polarity. 2010, Pubmed , Xenbase
Thiery, Epithelial-mesenchymal transitions in development and disease. 2009, Pubmed
Tsai, Spatiotemporal regulation of epithelial-mesenchymal transition is essential for squamous cell carcinoma metastasis. 2012, Pubmed
Wang, Oxygen-mediated endocytosis in cancer. 2010, Pubmed
Weinberg, Developmental regulation of zebrafish MyoD in wild-type, no tail and spadetail embryos. 1996, Pubmed
Yang, Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. 2004, Pubmed
Yang, Direct regulation of TWIST by HIF-1alpha promotes metastasis. 2008, Pubmed
Zhang, Unexpected functional redundancy between Twist and Slug (Snail2) and their feedback regulation of NF-kappaB via Nodal and Cerberus. 2009, Pubmed , Xenbase
Zhong, Modulation of hypoxia-inducible factor 1alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. 2000, Pubmed
Zhou, Cytokines and hormones in the regulation of hypoxia inducible factor-1alpha (HIF-1alpha). 2006, Pubmed
de Beaucourt, Molecular cloning and characterization of the Xenopus hypoxia-inducible factor 1alpha (xHIF1alpha). 2007, Pubmed , Xenbase