An oxygen-insensitive Hif-3α isoform inhibits Wnt signaling by destabilizing the nuclear β-catenin complex
Hypoxia-inducible factors ( HIF ) are evolutionarily conserved transcriptional regulators that play key roles in coordinating the cellular response to hypoxia ( Semenza , 2012 ) . HIFs are heterodimers , consisting of an oxygen-regulated α subunit and a stable β subunit . All three HIFα proteins contain a bHLH ( basic helix-loop-helix ) domain , two PAS ( Per-Arnt-Sim ) domains , a PAC ( PAS associated C-terminal ) domain , and an ODD ( oxygen-dependent degradation ) domain . HIF-1α and HIF-2α have two TADs ( transactivation domains ) ( Pugh et al . , 1997; Tian et al . , 1997 ) . HIF-3α has only one TAD but it has a unique LZIP ( leucine zipper ) domain in the C-terminal region ( Gu et al . , 1998; Hara et al . , 2001 ) . Under normal oxygen tension , HIFα proteins are hydroxylated at conserved proline residues in their ODD domains by PHDs ( prolyl hydroxlase domain proteins ) . The prolyl hydroxylated HIFα proteins are recognized by the pVHL ( von Hippel-Lindau protein ) . pVHL polyubiqutinates HIFα proteins and targets them to the proteasome for degradation ( Huang et al . , 2002; Kageyama et al . , 2004 ) . Under hypoxia , prolyl hydroxylation is decreased and HIFα proteins are stabilized . They then dimerize with HIFβ , translocate into the nucleus , bind to the hypoxia response elements ( HREs ) in the promoter regions of their target genes , and up-regulate their expression ( Semenza , 2012; Simon and Keith , 2008 ) . These HIF target genes participate in many adaptive and pathological processes such as erythropoiesis , angiogenesis , metabolic reprograming , cell-cycle regulation , and tumorigenesis ( Semenza , 2012; Simon and Keith , 2008 ) . In addition to their well-known functions in coordinating the transcriptional responses to hypoxia , HIFs have been shown to have oxygen-independent roles in early development in mammals , frogs , fish , and invertebrates ( Barriga et al . , 2013; Dunwoodie , 2009; Simon and Keith , 2008 ) . The mechanisms underlying these oxygen-independent roles of HIFs , however , are not well understood . The HIFα genes are subjected to sophisticated post-transcriptional regulation with that of HIF-3α being the most complicated ( Prabhakar and Semenza , 2012 ) . The human HIF-3α gene , for example , has 19 predicted variants that result from the use of different promoters , different transcription initiation sites , and alternative splicing ( Duan , 2015 ) . Eight of them have been experimentally shown to encode proteins ( Heikkila et al . , 2011; Maynard et al . , 2003; Pasanen et al . , 2010 ) . The mouse Hif-3α locus also gives rise to several different variants , resulting in the full-length HIF-3α , NEPAS ( neonatal and embryonic PAS ) , IPAS ( inhibitory PAS ) and possibly others ( Gu et al . , 1998; Makino et al . , 2001; Yamashita et al . , 2008 ) . These isoforms are often expressed in different tissues , at different developmental stages , and are differentially regulated . They have distinct or even opposite functions when tested by overexpression approaches ( Duan , 2015 ) . For instance , while human HIF-3α1 , the full-length human HIF-3α , can stimulate HRE-dependent reporter construct activity and up-regulate unique target genes ( Gu et al . , 1998; Zhang et al . , 2014 ) , human HIF-3α4 isoform , a shorter isoform that lacks the TAD domain , inhibits the activity of HIF-1α and HIF-2α ( Maynard et al . , 2005; Maynard et al . , 2007 ) . Similarly , IPAS was shown to inhibit HIF-1α activity ( Makino et al . , 2001 ) , while NEPAS has weak transcriptional activity and is thought to inhibit HIF-1/2α activity by competing for the common HIFβ in cells with limited amounts of HIFβ ( Yamashita et al . , 2008 ) . The existence of such a large array of HIF-3α variants has posed enormous challenges to studying HIF-3 biology . While the conventional gene knockout technology has been used to knockout the NEPAS/HIF-3α/IPAS in mice ( Yamashita et al . , 2008 ) , the interpretation of the results is not straightforward because multiple isoforms are deleted . The new CRISPR/Cas9 genome editing technology makes it possible to address this problem . Wnts are secreted glycoproteins that play crucial roles in cell fate specification , body axis determination , cell proliferation , and cell migration during embryogenesis ( Clevers and Nusse , 2012; MacDonald et al . , 2009 ) . The Wnt signaling pathway also regulates stem cell renewal and adult tissue homeostasis . Aberrant expression and/or activation in Wnt signaling leads to many human diseases such as birth defects , cancer , and degenerative disorders ( Clevers and Nusse , 2012; MacDonald et al . , 2009 ) . In the absence of Wnt ligands , the transcriptional co-activator β-catenin is phosphorylated in the cytoplasm by a protein complex consisting of APC , CK1 , Axin , and GSK3β . This leads to β-catenin recognition by the ubiquitin ligase β-TrCP . β-TrCP binds to the N-terminal region of β-catenin in a phosphorylation-dependent manner and promotes β-catenin degradation . The binding of a Wnt ligand to Frizzled and co-receptors inhibits the phosphorylation and degradation of β-catenin . The stabilized β-catenin accumulates and translocates into the nucleus to form complexes with TCF/LEF , and thereby activates target gene expression ( Clevers and Nusse , 2012; MacDonald et al . , 2009 ) . In addition to the canonical pathway , Wnt also regulates planar cell polarity and Akt/mTOR through non-canonical pathways ( Clevers and Nusse , 2012; MacDonald et al . , 2009 ) We have recently shown that the full-length zebrafish Hif-3α is an oxygen-dependent transcription factor and that it activates a transcriptional program distinct from that of Hif-1α in zebrafish embryos under hypoxia ( Zhang et al . , 2014 ) . In this study , we have identified a novel zebrafish Hif-3α spliced variant , termed Hif-3α isoform 2 ( Hif-3α2 ) . Hif-3α2 is an oxygen-insensitive nuclear protein . Despite its lack of the bHLH and PAS domains , Hif-3α2 has HRE-dependent transcriptional activity . We investigated the in vivo role of Hif-3α2 using transgenesis and CRISPR/Cas9-mediated gene editing . Our results suggest that Hif-3α2 inhibits canonical Wnt signaling by binding to β-catenin and destabilizing the nuclear β-catenin complex . This action is independent of its HRE-dependent transcriptional activity and is evolutionarily conserved .
RT-PCR analysis of zebrafish embryo RNA detected two major transcripts ( Figure 1A ) . In addition to the previously reported full-length hif-3α transcript ( Zhang et al . , 2012 ) , there is a short transcript . These transcripts are referred to as hif-3α isoforms 1 and 2 , respectively ( hif-3α1 and -3α2 ) hereafter . The complete sequence of the hif-3α2 transcript ( 893 bp ) was determined by 5' and 3' RACE ( GenBank access number KR338972 ) . It lacks exons 3–10 and a portion of exon 2 ( Figure 1B ) . The predicted ORF is 513 bp , encoding a protein of 170 amino acids with a predicted size of 19 kDa . The encoded protein contains the TAD and LZIP domains but lacks the bHLH , PAS , PAC , and ODD domains ( Figure 1B ) . During early development , hif-3α2 mRNA levels peaked at 9–16 hr post fertilization ( hpf ) , decreased after 20 hpf , and increased again in the larval stage ( Figure 1C ) . In the adult stage , the highest levels of hif-3α2 mRNA were found in the kidney , followed by ovary , spleen , testis , intestine , brain , and heart . The lowest levels were detected in the liver ( Figure 1D ) . Its levels were lower compared with those of hif-3α1 mRNA ( Figure 1—figure supplemental 1A ) . 10 . 7554/eLife . 08996 . 003Figure 1 . Hif-3α2 is an oxygen-insensitive Hif-3α isoform resulting from alternative splicing . ( A ) Hif-3α isoform 1 ( Hif-3α1 ) and isoform 2 ( Hif-3α2 ) mRNA expression . RNA isolated from zebrafish embryos was analyzed by RT-PCR . ( B ) Schematic illustration of the hif-3α gene ( top ) , Hif-3α1 and Hif-3α2 mRNAs ( middle ) , and proteins ( bottom ) . ( C , D ) Hif-3α2 expression in early development ( C ) and in adult tissues ( D ) . The Hif-3α2 mRNA levels were measured by qRT-PCR and normalized by β-actin levels . Values are means +S . E . ( n = 3 ) . ( E , F ) Hif-3α1-EGFP but not Hif-3α2-EGFP is degraded under normoxia in vivo . Capped mRNA encoding EGFP , Hif-3α1-EGFP , and Hif-3α2-EGFP was injected into zebrafish embryos . The embryos were raised to 6 hpf under normoxia and observed under fluorescence microscopy ( E ) or analyzed by Western blotting using an anti-GFP antibody ( F ) . ( G ) 6-hpf wild-type embryos were transferred to hypoxic ( Hyp ) or normoxic water ( Nor ) for 24 hr and analyzed by Western blotting using a specific Hif-3α antibody . ( H ) Adult fish were subjected to hypoxia treatments for the indicated time period and analyzed by Western blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 00310 . 7554/eLife . 08996 . 004Figure 1—figure supplement 1 . Hif-3α mRNA expression in adult and antibody validation . ( A ) Relative Hif-3α1 and Hif-3α2 mRNA levels in the indicated adult tissues . ( B ) The Hif-3α antibody recognizes both Hif-3α1 and Hif-3α2 . HEK293T cells were transfected with pCS2-Hif-3α1-EGFP or pCS2-Hif-3α2-EGFP . Twenty-four hours after the transfection , cells were lysed and subjected to Western blotting analysis using a GFP antibody ( left ) or the Hif-3α antibody ( right ) . Note: The Hif-3α1-EGFP levels were much lower due to its oxygen-dependent degradation . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 004 Because Hif-3α2 lacks a complete ODD domain , we speculated that its protein stability might not be regulated by oxygen tension . To test this idea , capped mRNA encoding GFP , Hif-3α1-GFP , and Hif-3α2-GFP was injected into 3 separate groups of zebrafish embryos . The embryos were raised to 6 hpf under normoxia . As previously reported , Hif-3α1-GFP was rapidly degraded under normoxia and no GFP signal was observed in this group ( Zhang et al . , 2014 ) . In contrast , strong signal was observed in Hif-3α2-GFP and GFP mRNA injected embryos ( Figure 1E ) . The Hif-3α2-GFP signal was observed in the nucleus , while the GFP protein was present in the cytoplasm and nucleus ( Figure 1E ) . Western blotting confirmed these results ( Figure 1F ) . The levels of endogenous Hif-3α2 protein were examined using a validated Hif-3α antibody ( Zhang et al . , 2012 ) . This antibody detected both Hif-3α1 and Hif-3α2 in HEK293T cells transfected with a GFP tagged construct ( Figure 1—figure supplement 1B ) . Western blotting results showed comparable levels of Hif-3α2 in zebrafish embryos raised in normoxic and hypoxic water ( Figure 1G ) , while hypoxia induced a robust increase in Hif-3α1 levels ( Figure 1G ) . Likewise , 6 hr , 12 hr , and 24 hr hypoxia treatments did not change Hif-3α2 levels in adult zebrafish ( Figure 1H ) . These results suggest that unlike Hif-3α1 , Hif-3α2 expression is not regulated by oxygen availability . Co-expression of Hif-3α2 with p2 . 1 , a HRE reporter construct ( Semenza et al . , 1996 ) , resulted in a highly significant , 11 . 3-fold increase in the reporter activity in HEK293 cells ( Figure 2A ) . This activity was abolished when the HRE was mutated ( i . e . , p2 . 4 in Figure 2A ) . In comparison , zebrafish Hif-3α1 and Hif-1α caused a 27 . 4-fold and 14 . 5-fold increase , respectively ( Figure 2A ) . Similar results were obtained in HeLa cells ( Figure 2—figure supplemental 1B ) . When tagged with GFP and transfected into human cells , Hif-3α2-GFP signal was seen in the nucleus in cultured cells and in zebrafish embryos ( Figure 2C and E ) . Deletion of the TAD abolished the transcriptional activity , while it only slightly reduced the nuclear GFP signal intensity ( Figure 2B–C ) . Deletion of the LZIP domain had no effect on the nuclear localization but increased the transcriptional activity ( Figure 2B–D ) . The expression levels of the ΔLZIP mutant were higher than that of Hif-3α and ΔTAD ( Figure 2—figure supplemental 1A ) . Overexpression of Hif-3α2 in zebrafish embryos resulted in significant increases in the mRNA levels of igfbp-1a , mlp3c , and redd1 ( Figure 2F–H ) . These genes are known Hif-1α and Hif-3α target genes in zebrafish ( Feng et al . , 2012; Kajimura et al . , 2006; Kamei et al . , 2008 ) . The ΔTAD mutant had no such effect ( Figure 2F–H ) . The magnitude of induction by Hif-3α2 , however , was much lower compared to that of Hif-3α1 or Hif-1α ( Figure 2F–H ) . While Hif-1α increased the expression of its target gene vegfAb , Hif-3α2 had no such effect ( Figure 2—figure supplemental 1C ) . Likewise , Hif-3α1 but not Hif-3α2 increased the mRNA levels of zp3v2 and sqrdl ( Figure 2—figure supplemental 1D–E ) . These data suggest that Hif-3α2 is a nuclear protein capable of activating HRE-dependent gene expression in vitro and in vivo . They also suggest that Hif-3α2 either does not activate the target genes to the same degree or has target genes differ from that of full-length Hif-3α1 . 10 . 7554/eLife . 08996 . 005Figure 2 . Hif-3α2 is a nuclear protein and has HRE-dependent transcriptional activity . ( A ) HRE-dependent transcriptional activity . HEK293 cells were transfected with the indicated plasmid together with 100 ng p2 . 1 ( red ) or p2 . 4 plasmid ( black ) . The results are normalized and expressed as fold change over the GFP p2 . 4 group . Values are means + S . E . ( n = 3 ) . ***p < 0 . 001 . ( B ) Schematic diagram of the Hif-3α2 truncation mutants tested . ( C ) U2OS cells were transfected with the constructs shown in ( B ) . The GFP signal was visualized 24 hr after transfection ( left panels ) . Cells were counterstained with DAPI ( middle panels ) . Merged views are shown in the right panels . Scale bar = 50 μm . ( D ) HRE-dependent transcriptional activity of Hif-3α2 mutants . Values are means + S . E . ( n = 3 ) . Groups labeled with different letters are significantly different from each other ( P < 0 . 05 ) . ( E ) Nuclear localization of Hif-3α2-EGFP in zebrafish embryo cells . Embryos injected with EGFP ( 600 pg ) or Hif-3α2-EGFP capped mRNA ( 600 pg ) were raised to 6 hpf under normoxia . The cells were dispersed and observed under bright field ( BF ) and GFP fluorescence microscopy . Scale bar = 50 μm . ( F–H ) Effects of Hif-3α2 ( 600 pg ) , Hif-3α1’ ( stabilized Hif-3α1 , 800 pg ) , and Hif-1α’ ( stabilized Hif-1α , 800 pg ) on endogenous gene expression . Embryos injected with the indicated capped mRNA were raised to 12 hpf under normoxia . The mRNA levels of the indicated genes were determined by qRT-PCR and normalized by the β-actin levels . Values are means + S . E . ( n = 3 ) . Groups labeled with different letters are significantly different from each other ( P < 0 . 05 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 00510 . 7554/eLife . 08996 . 006Figure 2—figure supplement 1 . Hif-3α2's transcriptional activity differs from that of Hif-1α and Hif-3α1 . ( A ) Western blotting results showing the successful expression of Hif-3α2 and its mutants . ( B ) HeLa cells were transfected with the indicated plasmid together with 100 ng p2 . 1 ( red ) or p2 . 4 ( black ) . The results are normalized and expressed as fold changes over the GFP p2 . 4 group . n = 3 . ***p < 0 . 001 , ns , not significant . ( C–E ) Embryos injected with the indicated capped mRNA were raised to 12 hpf under normoxia . The mRNA levels of vegfAb ( C ) , zp3v2 ( D ) , and sqrdl ( E ) mRNA were determined and presented as described in Figure 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 006 The discovery of Hif-3α2 has raised the possibility that it may play a role in early development in an O2-independent manner . Overexpression of Hif-3α2 resulted in reduced body growth and developmental timing in zebrafish embryos ( Figure 3—figure supplemental 1A–B ) . This effect is similar to what has been reported for the full-length Hif-3α1 ( Zhang et al . , 2014 ) . This action of Hif-3α2 appears to require its transcriptional activity because overexpression of the ΔTAD mutant did not change body size or somite number , while the ΔLZIP mutant did ( Figure 3—figure supplemental 1A–B ) . While GFP mRNA-injected or ΔTAD mRNA-injected embryos were mostly morphologically normal , many Hif-3α2- and ΔLZIP-expressing embryos exhibited morphological abnormality , ( Figure 3—figure supplemental 1C–D ) . Approximately 84% of the Hif-3α2 mRNA-injected embryos had partial or complete loss of myod1-mRNA expressing somites on one or both sides of the body ( Figure 3A ) . Similar results were found with myog mRNA expression ( Figure 3B ) . The expression patterns of two asymmetric genes , spaw and lefty2 ( lft2 ) ( Amack et al . , 2007 ) , were examined . While the spaw expression was detected on the left side of the body plan in all GFP mRNA-injected embryos , its expression was detected on the right side , bilaterally , or was completely lost in ~58% of the Hif-3α2-expressing embryos ( Figure 3C ) . Similar changes were found with lft2 expression ( Figure 3D ) . During zebrafish development , the shape of the heart changes from a tube-like structure in the middle to a left looped structure ( Stainier , 2001 ) . The cardiac tube remained in the middle or even looped to the right side in half of the Hif-3α2 expressing embryos ( Figure 3E ) . No such phenotype was observed in Hif-3α1 mRNA-injected embryos ( Figure 3E ) . The effect of Hif-3α2 expression was further examined using LiPan fish , a transgenic zebrafish line that has liver-specific expression of DsRed RFP and pancreas-specific expression of GFP ( Korzh et al . , 2008 ) . In the control embryos , the liver was located on the left and pancreas on the right side of the body plan in all embryos ( Figure 3F ) . In the Hif-3α2 group , the liver was found on the right side or in the midline in many embryos ( 28 . 5% ) ( Figure 3F ) . These data suggest that overexpression of Hif-3α2 but not the full-length Hif-3α1 impairs LR axis development . 10 . 7554/eLife . 08996 . 007Figure 3 . Forced expression of Hif-3α2 causes LR asymmetry defects . Embryos injected with GFP or Hif-3α2 capped mRNA ( 600 pg ) were raised and analyzed by in situ hybridization . ( A , B ) Somite development was visualized by myod1 ( A ) and myog ( B ) mRNA expression at 12–14 hpf . The embryos were scored based on the criteria shown in the left panel . The quantification results are shown in the right . The total embryo number is shown on the top of each column . ( C ) Altered expression of spaw ( C ) mRNA at 18 hpf and lft2 mRNA ( D ) at 20 hpf . ( E ) The cardiac tube looping was visualized by cmlc2 mRNA expression at 48 hpf . Representative views are shown in the left panel . The quantification results are shown in the right panel . Hif-3α1 ( stabilized Hif-3α1 , 800 pg ) injected embryos were used as controls . ( F ) Changes in liver and pancreas location . Hif-3α2 capped mRNA was injected into LiPan transgenic embryos . Liver ( red ) and pancreas ( green ) location was examined at 96 hpf . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 00710 . 7554/eLife . 08996 . 008Figure 3—figure supplement 1 . Forced expression of Hif-3α2 slows down embryonic growth and developmental timing and causes morphological abnormality . Body length ( A ) and somite number ( B ) of the embryos injected with the indicated capped mRNA ( 600 pg ) were measured at 24 hpf and are shown . The total embryo number is shown at the top of each column . ( C , D ) Embryos were scored based on the morphological criteria shown in ( C ) . The percentage of embryos in each category is shown in ( D ) . The total number of embryos analyzed is shown at the top of each column . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 008 Kupffer's vesicle ( KV ) , a transient embryonic organ , plays a key role in establishing the LR asymmetry axis in zebrafish ( Essner et al . , 2005 ) . The rotating cilia of KV establish a counterclockwise fluid flow and promote intracellular Ca2+ elevation in cells localized on the left side of KV , which in turn stimulates the processing and/or secretion of the Nodal-related ligand gene spaw on the left side of KV ( Husken and Carl , 2013 ) . KV is derived from the dorsal forerunner cells ( DFCs ) . DFCs emerge at mid-gastrulation and migrate collectively to the vegetal pole . At the tail bud , they form a rosette structure . The lumen and cilia are then developed to form KV ( Matsui and Bessho , 2012 ) . We investigated whether Hif-3α2 overexpression alters LR asymmetry axis by affecting DFCs and/or KV development . In situ hybridization and qRT-PCR analyses using the DFC marker gene sox17 indicated that Hif-3α2 overexpression caused a modest increase in DFC cluster size and total mRNA levels , but these increases were not statistically significant ( Figure 4A and C ) . There was a slight decrease in the number of migrating DFCs ( Figure 4B ) . We analyzed KV forming using charon , which is expressed in KV lumen epithelia and surrounding cells ( Hashimoto et al . , 2004 ) . While wild-type embryos had normally formed KVs , many Hif-3α2 mRNA-injected embryos had a partially formed KVs ( 48% ) or no KV ( 20% ) ( Figure 4D ) . This action is specific to Hif-3α2 because Hif-3α1 overexpression had no such effect ( Figure 4D ) . Taken together , the results suggest that Hif-3α2 overexpression causes LR asymmetry defects by impairing KV formation . 10 . 7554/eLife . 08996 . 009Figure 4 . Forced expression of Hif-3α2 impairs Kupffer's vesicle ( KV ) development and inhibits Wnt/β-catenin signaling . ( A , B ) Lack of effect on dorsal forerunner cell ( DFC ) development . The DFC cluster size ( A ) was determined by measuring sox17 mRNA expression domain in the Hif-3α2 mRNA injected embryos at 8 hpf ( n = 22 ) using ImageJ . The values were normalized by those of the GFP mRNA injected embryos ( n = 19 ) . The number of migrating DFCs were quantified and shown in ( B ) . ( C ) The sox17 mRNA levels were determined by qRT-PCR and normalized by the β-actin levels . Values are means +S . E . ( n = 3 ) . No significant difference is detected . ( D ) Effects on KV development . KV was visualized by charon mRNA expression at 12 hpf . The embryos were scored based on the criteria shown in the left panel . The quantification results are shown in the right . ( E , F ) Effects on Wnt target gene expression . Embryos injected with the indicated mRNA were raised to 9 hpf and vent ( E ) and vox ( F ) mRNA levels were determined by qRT-PCR and normalized to the β-actin levels . Values are means + S . E . ( n = 3 ) . ***p < 0 . 001 . ( G , H ) Inhibition of Wnt3a ( G ) and β-cateninΔN ( H ) activity . Embryos injected with Topflash plasmid DNA ( 90 pg ) together with the 30 pg Wnt3a or 100 pg β-cateninΔN and 600 pg Hif-3α2 capped mRNA were raised to 9 hpf and luciferase activity was measured . Values are means + S . E . ( n = 3 ) . Groups labeled with different letters are significantly different from each other ( P < 0 . 05 ) . ( I , J ) Inhibition of β-cateninΔN-induced cav1 . 2 ( I ) and foxj1a ( J ) expression . qRT-PCR was performed and analyzed as described above . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 00910 . 7554/eLife . 08996 . 010Figure 4—figure supplement 1 . Forced expression of Hif-3α2 does not change Fgf , Hedgehog , Notch , Akt , and Erk signaling . ( A-C ) Embryos injected with the indicated capped mRNA ( 600 pg ) were raised to 12 hpf in normoxic water . The mRNA levels of the Fgf target gene dusp6 ( A ) , Hedgehog target gene gli1 ( B ) , and Notch target gene her4 ( C ) were determined by qRT-PCR and normalized by the β-actin levels . n = 3 . ( D , E ) . Some of the embryos were subjected to Western blotting using antibodies for phospho- and total Akt ( D ) and Erk ( E ) . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 01010 . 7554/eLife . 08996 . 011Figure 4—figure supplement 2 . Effects of Hif-3α2 , Hif-3α1' ( a stabilized Hif-3α1 ) , and Hif-1α' ( a stabilized Hif-1α ) on Wnt3a- ( A ) and β-cateninΔN-induced ( B ) Topflash activity . Embryos injected with 90 pg Topflash plasmid DNA together with the indicated capped mRNA ( 600 or 800 pg ) were raised to 9 hpf in normoxic water and luciferase activity was measured . Values are means +S . E . ( n = 3 ) . * , ** , and ***p < 0 . 05 , 0 . 01 and 0 . 001 . ns , not significant . ( C ) Hif-3α2 overexpression antagonizes Wnt3a and β-cateninΔN actions . Zebrafish embryos were injected with the indicated capped mRNA . They were raised and photographed at 12 hpf . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 011 Several signaling pathways , including Wnt/β-catenin , Fgf , Notch , Hedgehog , and Akt , have been implicated in KV organogenesis ( Husken and Carl , 2013; Matsui and Bessho , 2012; Roussigne et al . , 2012 ) . qRT-PCR assays showed that Hif-3α2 overexpression resulted in a significant reduction in the mRNA expression of vent and vox ( Figure 4E , F ) , two genes acting downstream of the canonical Wnt/β-catenin pathway . No significant changes were detected in the mRNA expression of the Fgf target gene dusp6 , the Hedgehog target gene gli1 , or the Notch target gene her4 ( Figure 4—figure supplemental 1A–C ) . No marked changes were observed in the phospho-Akt and phospho-Erk levels ( Figure 4—figure supplemental 1D–E ) . Topflash , a Luciferase reporter construct containing TCF binding sites was used to directly test whether Hif-3α2 inhibits Wnt/β-catenin signaling ( Flowers et al . , 2012 ) . Injection of Wnt3a mRNA into zebrafish embryos resulted in a robust induction in Topflash reporter activity ( Figure 4G ) . This induction was abolished by Hif-3α2 co-injection ( Figure 4G ) . Co-injection of Hif-3α2 also abolished the induction of Topflash activity by β-cateninΔN ( Figure 4H ) . β-cateninΔN is a constitutively active β-catenin mutant lacking part of the N-terminal sequence ( Xiong et al . , 2006 ) . This action is specific to Hif-3α2 because the full-length Hif-3α1 did not inhibit Wnt3a- or β-cateninΔN-induced Topflash activity ( Figure 4—figure supplemental 2A–B ) . Hif-1α had a modest but statistically significant effect on Wnt3a-induced Topflash activity ( Figure 4—figure supplemental 2A–B ) . Co-injection of Hif-3α2 also abolished the Wnt3a- and β-cateninΔN-induced dorsalization phenotype ( Figure 4—figure supplemental 2C ) . Wnt/β-catenin signaling has been shown to regulate KV development and LR asymmetry by stimulating the expression of the ciliogenic transcription factor foxj1a ( Caron et al . , 2012 ) and the calcium channel cav1 . 2 ( Muntean et al . , 2014 ) . If Hif-3α2 impairs KV development via inhibition of Wnt/β-catenin signaling , then expression of Hif-3α2 should block Wnt/β-catenin-induced foxj1a and cav1 . 2 gene expression . Indeed , co-injection of Hif-3α2 mRNA abolished the β-cateninΔN induction of foxj1a and cav1 . 2 gene expression ( Figure 4I–J ) . These results suggest that forced expression of Hif-3α2 but not the full-length Hif-3α1 inhibits Wnt/β-catenin signaling and impairs Kupffer's vesicle development . To determine whether endogenous Hif-3α2 functions as a negative regulator of the Wnt/β-catenin signaling pathway , we generated hif-3α-/- mutants using a CRISPR/Cas9-mediated approach targeting the Hif-3α2 ATG site in exon 11 ( Figure 5A ) . We identified several alleles . The hif-3αΔ42 allele has a 42 bp deletion surrounding the Hif-3α2 ATG site ( Figure 5A–B ) . RT-PCR assays showed that the expression of Hif-3α1 mRNA was not affected in the mutant embryos ( Figure 5C ) . The successful ablation of Hif-3α2 protein was confirmed by Western blotting ( Figure 5D ) . We next cloned and sequenced the Hif-3α1 cDNA from the mutant embryos . The Hif-3α1Δ42 has a 14 aa deletion ( residues 456 to 469 ) located in the N-terminal end of the TAD ( Figure 5—figure supplemental 1A ) . When its transcriptional activity was compared with that of the wild type Hif-3α1 , Hif-3α1Δ42 had comparable activity ( Figure 5—figure supplemental 1B ) . These data suggest that the expression and functionality of Hif-3α1 protein remain largely unchanged in the hif-3αΔ42 line . 10 . 7554/eLife . 08996 . 012Figure 5 . CRISPR/Cas9-mediated deletion of Hif-3α2 increases Wnt/β-catenin signaling . ( A ) Top: Design of the gRNA used to target the Hif-3α2 ATG site ( bold letters ) in exon 11 . The gRNA targeting sequence is underlined ( green ) . The PAM motif is labeled in red , and the NcoI digestion site is indicated in italic letters . Bottom: Sequencing results of the hif-3α locus in the wild type and homozygous mutant fish . ( B ) Top: Location of the 42 bp deletion and PCR primers used for genotyping . Middle and Bottom: Genotyping results using two PCR-based assays . Water was used as negative control ( NC ) . ( C ) The expression of Hif-3α1 mRNA expression is not affected . Top: PCR primers used for detecting Hif-3α2 and Hif-3α1 mRNAs . Bottom left panel: RT-PCR results . This primer set amplified Hif-3α2 and Hif-3α1 mRNAs in the wild type ( +/+ ) and Hif-3α1 mRNA ( albeit smaller size ) in the hif-3αΔ42 mutant ( -/- ) embryos . Cloned Hif-3α1 , Hif-3α2 , and Hif-3α1Δ42 DNA were used as controls . ( D ) Western blotting results indicating the ablation of Hif-3α2 protein in the hif-3αΔ42 mutant embryos . 24 hpf hif-3αΔ42 mutant embryos and wild-type siblings were analyzed . ( E ) Elevated Wnt/β-catenin signaling activity . Wild-type and hif-3αΔ42 mutant embryos were injected with 90 pg Topflash plasmid DNA and luciferase activity was measured at 9 hpf . ( F , G ) The mRNA levels of vent ( F ) vox ( G ) were measured in mutant embryos at 9 hpf and presented as relative values to the wild type controls . Values are means +S . E . ( n = 3 ) . * and **p < 0 . 05 and 0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 01210 . 7554/eLife . 08996 . 013Figure 5—figure supplement 1 . The 14-amino acid deletion does not alter HIF-3α1 transcriptional activity . ( A ) hif-3αΔ42 mutation results in a 14 amino acid deletion in Hif-3α1 . The Hif-3α1 cDNA was cloned from hif-3αΔ42 mutant embryos and sequenced . The location of the deletion is shown . ( B ) HEK293 cells were transfected with the indicated plasmid together with p2 . 1 . The results are normalized and expressed as fold change over the GFP group . No significant difference was detected . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 01310 . 7554/eLife . 08996 . 014Figure 5—figure supplement 2 . CRISPR/Cas9-mediated mutation of Hif-3α1/2 increases Wnt/β-catenin signaling . ( A ) Top: Design of the gRNA . Bottom: Sequencing results of the hif-3α locus in the wild type and homozygous mutant fish . ( B ) Hif-3α1Δ20 protein structure . ( C ) Wild-type and hif-3αΔ20 mutant embryos were injected with 90 pg Topflash plasmid DNA and luciferase activity was measured at 9 hpf . *p < 0 . 05 . ( D ) Mortality of wild-type and hif-3αΔ20 mutant embryos . The number of dead embryo was determined at 9 hpf and is shown as percentage of total embryos . The total embryo number is shown on the top of each column . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 014 We next investigated whether loss of Hif-3α2 alters Wnt/β-catenin signaling activity . Compared with the wild-type embryos , the Topflash reporter activity was significantly elevated in the hif-3αΔ42 mutant embryos at 9 hpf ( Figure 5E ) . Likewise , the mRNA levels of vent and vox were elevated in hif-3αΔ42 embryos at 9 hpf ( Figure 5F–G ) . Similar results were obtained using the hif-3αΔ20 allele , which has a 20 bp deletion covering the Hif-3α2 ATG site . It is predicted to be a hif-3α2 null mutant and encode a truncated Hif-3α1 protein lacking the TAD and LZIP domains ( Figure 5—figure supplemental 2A and B ) . The truncated protein also contains 8 incorrect aa ( residues 455-462 ) . Compared to wild type embryos , a significant increase in Topflash reporter activity was found in the hif-3αΔ20 mutant line ( Figure 5—figure supplemental 2C ) . These results indicate that endogenous Hif-3α2 negatively regulates the Wnt/β-catenin signaling . A most notable phenotype observed in the hif-3αΔ42 mutant line was elevated embryo mortality ( Figure 6A ) . Many hif-3αΔ42 embryos died before 6–9 hpf . The mortality rate , although varied considerably among different pairs of mutant fish and between different breeding cycles in the same pair ( Figure 6—figure supplement 1 ) , was significantly higher than that of the wild-type controls ( Figure 6A ) . The hif-3αΔ20 mutant line also had significantly elevated mortality rate ( Figure 5—figure supplemental 2D ) . Although two out of 105 of the surviving hif-3αΔ42 embryos had reduced forebrain and smaller eyes , most of the surviving hif-3αΔ42 mutant embryos looked morphologically normal and continued to develop ( Figure 6B ) . No difference was detected in the somite development and cardiac looping between the mutant and wild-type embryos ( Figure 6C–D ) . Expression of flh , a mid/caudal diencephalon marker gene , increased at 6 hpf but returned to the control levels at 10 hpf in the hif-3αΔ42 mutant embryos ( Figure 6E–F ) . Meanwhile , no difference in pax2a expression was observed between the mutant and the wild type embryos at 6 and 10 hpf ( Figure 6G–H ) . 10 . 7554/eLife . 08996 . 015Figure 6 . Genetic deletion of Hif-3α2 increases embryo mortality and transient Wnt/β-catenin signaling phenotype . ( A ) Increased embryo mortality . Wild-type and F3 homozygous hif-3αΔ42 mutant embryos were raised under normoxia . The number of dead embryo was determined at 9 hpf and is shown as percentage of total embryos . The total embryo number is shown on the top of each column . ( B ) Morphology of the surviving embryos at 24 hpf . The total embryo number is shown on the top of each column . ( C , D ) The cardiac tube looping ( C ) and somite development ( D ) were examined and quantified at 48 and 12–14 hpf . ( E-H ) Expression of flh and pax2 . a mRNA in wide type and hif-3αΔ42 mutant embryos at 6 hpf ( E , G ) and 10 hpf ( F , H ) . The frequency of embryos with the indicated expression patterns is shown at the bottom of each panel . Scale bar = 200 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 01510 . 7554/eLife . 08996 . 016Figure 6—figure supplement 1 . The hif-3αΔ42 line had higher mortality rate . ( A ) Embryo mortality rates . Six pairs of hif-3αΔ42 mutant fish were bred 4 times and the number of embryos and the mortality rate ( % ) were determined at 9 hpf and are shown . ( B ) Genotyping results of the 6 pairs of mutant fish used in ( A ) . Wild type ( +/+ ) and heterozygous fish ( +/- ) were used as controls . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 016 The lack of major phenotypes in body patterning was surprising . Excessive Wnt/β-catenin activity has been linked to major phenotypic changes in zebrafish . For example , the masterblind ( mbl ) mutant fish had no eyes , reduced telencephalon , and expanded diencephalon ( Heisenberg et al . , 2001 ) . Likewise , treatment of zebrafish embryos with Lithium and BIO , which increase β-catenin signaling by inhibiting GSK3β , results in the absence of eyes or reduced eyes ( Kim et al . , 2002; Nishiya et al . , 2014 ) . A recent study has shown that zebrafish can activate compensation mechanisms to buffer against deleterious genetic mutations ( Rossi et al . , 2015 ) . For instance , while knockdown of egfl7 resulted in major phenotypic changes , no phenotype was found in the egfl7-/- genetic mutants ( Rossi et al . , 2015 ) . To test this possibility , we designed a morpholino ( MO ) targeting the Hif-3α2 ATG site . Injection of the Hif-3α2 MO but not the control MO reduced the Hif-3α2 protein levels ( Figure 7A—figure supplement 1A ) . Injection of Hif-3α2 MO did not change the levels of Hif-3α1 mRNA ( Figure 7—figure supplement 1B ) . Because the full-length Hif-3α1 protein is rapidly degraded under normoxia ( Zhang et al . , 2012 ) , it could not be detected nor affected by the Hif-3α2 MO . Topflash reporter assay and qRT-PCR measurement showed that knockdown of Hif-3α2 significantly increased Wnt/β-catenin activity at 9 hpf ( Figure 7B–D ) . Therefore , the Wnt/β-catenin activity is elevated in the morphants . Many of the morphants had phenotypes resembling those of mbl mutant embryos ( Heisenberg et al . , 2001 ) , including the absent or reduced eyes and reduced forebrain ( Figure 7E ) . These phenotypes were unlikely caused by MO toxicity because the Hif-3α2 targeting MO did not increase embryo mortality nor did it increase p53 mRNA expression at the injected dose ( 3 ng ) ( Figure 7—figure supplement 1C ) . Furthermore , the hif-3αΔ42 embryos were significantly less sensitive to the Hif-3α2 MO ( Figure 7F ) . qRT-PCR results showed that the expression of boz , a maternal Wnt signaling target gene ( Leung et al . , 2003 ) , increased at 6 hpf but returned to the control levels at 9 and 24 hpf in the hif-3αΔ42 mutants ( Figure 8A ) . No change was detected in boz expression in the morphants at any of these stages ( Figure 8D ) . The vent and vox mRNA levels increased significantly at 6 and 9 hpf but these increases disappeared at 24 hpf in the hif-3αΔ42 mutant embryos ( Figure 8B–C ) . In contrast , knockdown of Hif-3α2 resulted in elevated vent and vox mRNA expression at 6 , 9 , as well as 24 hpf ( Figure 8E–F ) . A reciprocal decrease was observed with tp63 expression; tp63 is a direct transcriptional target of Bmp signaling in zebrafish ( Bakkers et al . , 2002 ) ( Figure 8G–H ) . These results support the notion that the hif-3αΔ42 mutant embryos have activated certain compensatory mechanisms . Next , we subjected the hif-3αΔ42 mutant embryos to Lithium and BIO . We reasoned that the hif-3αΔ42 mutant embryos should be more sensitive to these Wnt activating agents even if they can survive by activating as-yet-unidentified compensatory mechanisms . Indeed , when treated with Lithium and BIO , a significantly greater portion of the hif-3αΔ42 embryos exhibited the no eye or small eye phenotype compared to the wild type controls ( Figure 8I–J ) . 10 . 7554/eLife . 08996 . 017Figure 7 . Hif-3α2 knockdown leads to Wnt/β-catenin signaling phenotypes . ( A ) Wild type ( WT ) , control MO ( cMO ) , and Hif-3α2 targeting MO ( Hif-3α2 MO ) -injected embryos were raised to 24 hpf in normoxic water . They were lysed and subjected to Western blotting . ( B ) Wild-type embryos injected with Topflash plasmid DNA together with the indicated capped mRNA or MO were raised to 9 hpf and the luciferase activity was measured . Values are means + S . E . ( n = 3 ) . * and **p < 0 . 05 and 0 . 01 . ( C , D ) Embryos injected with the indicated MOs were raised to 9 hpf and vent ( C ) and vox ( D ) mRNA levels were determined and presented as described above . ( E ) Phenotypes . The frequency of embryos with the indicated phenotypes is shown at the bottom of each panel . Scale bar = 200 μm . ( F ) hif-3αΔ42 mutant embryos are less sensitive to Hif-3α2 MO . Control MO or Hif-3α2 MO were injected into wild type ( +/+ ) or F3 homozygous hif-3αΔ42 mutant embryos ( -/- ) . The frequency of embryos with the abnormal phenotype ( E ) at 9 hpf was determined and shown . Total embryo number is shown on the top of each column . P value of Chi analysis is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 01710 . 7554/eLife . 08996 . 018Figure 7—figure supplement 1 . Hif-3α2 targeting MO design and verification . ( A ) Design of the Hif-3α2 targeting MO . ( B ) Injection of Hif-3α2 MO does not alter Hif-3α1 mRNA expression . ( C ) Injection of Hif-3α2 MO does not significantly affect p53 mRNA expression at 3 ng per embryo but it does so at higher doses . p53 mRNA levels were measured in 24 hpf were determined and presented as described above . Values are means +S . E . ( n = 3 ) . Groups labeled with different letters are significantly different from each other ( P < 0 . 05 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 01810 . 7554/eLife . 08996 . 019Figure 8 . Genetic deletion but not knockdown of Hif-3α2 result in dynamic and compensatory changes in the expression of Wnt/β-catenin and BMP target genes . ( A-C ) Expression of boz ( A ) , vent ( B ) , and vox ( C ) in hif-3αΔ42 mutant embryos at the indicated stages . ( D-F ) Expression of boz ( D ) , vent ( E ) , and vox ( F ) in morphants at the indicated stages . ( G , H ) Expression of tp63 in hif-3αΔ42 mutant embryos ( G ) and morphants ( H ) at the indicated stages . In all above , the mRNA levels of the indicated genes were measure and presented as described above . Values are means +S . E . ( n = 3 ) . * and **p < 0 . 05 and P < 0 . 01 . ( I , J ) Treatment of wild type and hif-3αΔ42 mutant embryos with LiCl ( 0 . 3 mM ) and BIO ( 4 μM ) results in no eyes or small eyes phenotypes . Representative views are shown in the left panel and quantification results are shown in the right panel . The total embryo number is shown on the top . P value of Chi analysis is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 019 To investigate whether this action of Hif-3α2 is evolutionarily conserved and determine the underlying biochemical mechanisms , we tested the effects of Hif-3α2 in cultured HEK293T cells . Overexpression of Hif-3α2 abolished Wnt3a-induced Topflash reporter activity in these human cells ( Figure 9—figure supplemental 1A ) . In contrast , Hif-3α2 had no effect on Wnt5a-induced non-canonical Ap1 reporter activity ( Figure 9—figure supplemental 1B ) , suggesting that Hif-3α2 only inhibits the canonical Wnt signaling pathway . We engineered a truncated human HIF-3α9 by deleting its N-terminal 452 residues ( Figure 9—figure supplemental 1C ) . This truncated HIF-3α9 , like zebrafish Hif-3α2 , had significant HRE-dependent transcriptional activity . It also inhibited Wnt3a- and β-catenin-induced Topflash activity ( Figure 9—figure supplemental 1D–E ) . These data suggest that both activities are conserved in human HIF-3α . Next , Flag-tagged β-catenin and GFP-tagged Hif-3α2 were co-transfected into HEK293T cells . Reciprocal co-IP experiments detected β-catenin and Hif-3α2 in the same complex ( Figure 9A ) . A direct protein-protein interaction between β-catenin and Hif-3α2 was confirmed by GST pull-down assays ( Figure 9—figure supplement 2A ) . No direct interaction was detected between Lef1 and Tcf4 and GST-Hif-3α2 ( Figure 9—figure supplement 2B–C ) . To test whether the Hif-3α2 can compete for β-catenin binding with its nuclear partners , Flag-β-catenin and Myc-Lef1 were co-expressed with increasing levels of Hif-3α2-GFP . Co-expression of Hif-3α2-GFP decreased the levels of both β-catenin and Lef1 in the complex ( Figure 9B ) . Likewise , overexpression of Hif-3α2-GFP also reduced the β-catenin and Tcf4 levels in the IP complex ( Figure 9C ) . Analysis of the Western blotting results of the cell lysates showed that the levels of total β-catenin , Lef1 , and Tcf4 were all decreased by Hif-3α2 in a dose-dependent manner ( Figure 9B–C ) . Overexpression of Hif-3α2 had no effect on the levels of Tcf3 levels and Hif-3α2-GST failed to pull down Tcf3 ( Figure 9—figure supplement 3A–B ) . We postulated that Hif-3α2 inhibits β-catenin by destabilizing β-catenin and its nuclear co-activators . To test this idea , the proteasome inhibitor MG132 was added . While Hif-3α2 expression resulted in a marked reduction in β-catenin levels , this reduction was blocked by MG132 treatment ( Figure 9D ) . Likewise , MG132 treatment also partially inhibited the Hif-3α2-induced reduction in Lef1 levels ( Figure 9E ) . MG132 treatment increased Hif-3α2 levels ( Figure 9D–E ) . Next , HEK293T cells co-transfected with β-catenin and Hif-3α2-GFP were fractionated into nuclear and cytoplasmic fractions and analyzed . The expression of Hif-3α2 greatly reduced the levels of nuclear β-catenin , while it had a minimal effect on the levels of cytoplasmic β-catenin ( Figure 9F ) . Addition of BIO and Lithium , two chemical inhibitors of GSKβ , did not affect the Hif-3α2-induced β-catenin degradation ( Figure 9G ) . Taken together , these data indicate that Hif-3α2 binds to β-catenin and destabilizes the active β-catenin complex through a mechanism that is independent of GSKβ/β-TrCP-mediated degradation . 10 . 7554/eLife . 08996 . 020Figure 9 . Hif-3α2 binds to β-catenin and destabilizes the nuclear β-catenin complex . ( A ) Co-IP experiment . HEK 293T cells were co-transfected with the indicated plasmids . 24 hr later , the cells were lysed and subjected to IP with an anti-GFP ( left panel ) or anti-Flag antibody ( right panel ) followed by immunoblotting using the indicated antibodies . ( B , C ) Dose-dependent effects of Hif-3α2 in reducing the β-catenin-Left1 complex ( B ) and β-catenin-Tcf4 complex ( C ) . HEK 293T cells were co-transfected with the indicated plasmids . + and +++ indicate 1 and 3 μg DNA . 24 hr later , the cells were lysed and subjected to IP followed by Western blotting . ( D ) Hif-3α2 promotes β-catenin degradation . HEK 293T cells were co-transfected with the indicated plasmids . 24 hr after transfection , the cells were treated with MG132 ( 10 μM ) for 12 hr and analyzed by Western blotting . ( E ) Hif-3α2 increases Lef1 degradation . HEK 293T cells transfected with the indicated plasmids . 24 hr after transfection , the cells were treated with MG132 ( 10 μM ) for 12 hr and analyzed by Western blotting . ( F ) Hif-3α2 decreases nuclear β-catenin levels . HEK 293T cells transfected with the indicated plasmids were fractionated into nuclear and cytosolic fractions and analyzed by Western blotting . + and ++ , indicate 1 and 2 μg DNA used . α-Tubulin and Histone H3 were measured as cytosolic and nuclear protein markers . ( G ) Lack of effects of GSKβ inhibitors . HEK293T cells were co-transfected with the indicated plasmids . 24 hr after transfection , the cells were treated with 6-bromoindirubin-3'-xime ( BIO , 100 nM ) , LiCl ( 10 mM ) , or MG132 ( 10 μM ) for 12 hr and lysed . The cell lysates were analyzed . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 02010 . 7554/eLife . 08996 . 021Figure 9—figure supplement 1 . The HER-dependent transcriptional activity and Wnt/β-catenin inhibitory activity are conserved in human cells . ( A , B ) Hif-3α2 inhibits the canonical but not non-canonical Wnt signaling pathway in human cells . HEK293T cells were transfected with 100 ng Topflash reporter ( A ) or Ap1 reporter ( B ) plasmid together with the indicated plasmids . 24 hr after the transfection , luciferase activity was measured and the results are normalized and expressed as fold change over the control group . Values are means +S . E . ( n = 3 ) . ** and ***p < 0 . 01 and 0 . 001 . ( C ) Sequence alignment of zebrafish Hif-3α2 and the human HIF-3α9 C-terminal region . ( D , E ) The HRE-dependent transcriptional activity ( D ) and β-catenin inhibitory activities ( E ) of the human HIF-3α9Δ1–452 . In ( D ) , HEK293T cells were transfected with the indicated plasmid together with 100 ng p2 . 1 DNA . In ( E ) , HEK293T cells were transfected with 100 ng Topflash DNA together with the indicated plasmids . 24 hr after the transfection , luciferase activity was measured and the results are normalized and expressed as fold change over the GFP group . Values are means +S . E . ( n = 3 ) . ***p < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 02110 . 7554/eLife . 08996 . 022Figure 9—figure supplement 2 . Hif-3α2 binds to β-catenin but not Lef1 or Tcf4 directly . Purified GST or GST-Hif-3α2 bound to glutathione-Sepharose beads was incubated with lysates of HEK293T cells transfected with β-catenin ( A ) , Lef1 ( B ) , and Tcf4 ( C ) . The beads were washed and the eluent was analyzed by Western blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 02210 . 7554/eLife . 08996 . 023Figure 9—figure supplement 3 . Hif-3α2 dose not affect Tcf3 stability . ( A ) HEK 293T cells were co-transfected with the indicated plasmids at the indicated levels . + , ++ , and +++ indicate 1 , 2 , and 3 μg DNA . 24 hr after transfection , the cell lysates were analyzed by Western blotting . ( C ) Purified GST or GST-Hif-3α2 bound to glutathione-SepharoseTM beads was incubated with HEK293T cell lysates transfected with Tcf3 . The beads were washed and the eluent was analyzed by Western blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 023 Next , the molecular mechanisms underlying Hif-3α2's actions were investigated . While deletion of the TAD completely abolished HRE-dependent transcriptional activity ( see Figure 2D ) , it only partially reduced the β-catenin inhibiting activity ( Figure 10A ) . The ΔLZIP mutant had greater transcriptional activity ( see Figure 2D ) and full β-catenin inhibitory activity ( Figure 10A ) . Next , several mutants were engineered to target several motifs conserved in the HIF/Hif-1α and -3α TAD ( Figure 10—figure supplement 1 ) . Changing L30D31L32 into A ( i . e . , M1 mutant ) did not affect the β-catenin inhibitory activity but reduced the HRE-dependent transcriptional activity by half ( Figure 10A–B ) . Mutant M2 ( changing D44F45Q46 into A ) had partial β-catenin inhibitory activity ( Figure 10A ) , while it retained half of its HRE-dependent transcriptional activity ( Figure 10B ) . The A36G mutant had full β-catenin inhibitory activity ( Figure 10A ) , but completely lost its transcriptional activity ( Figure 10B ) . Changing P37 to A , however , eliminated both activities ( Figure 10A , B ) . The expression levels of these proteins were comparable ( Figure 10C ) . These data suggest that Hif-3α2 inhibits . 10 . 7554/eLife . 08996 . 024Figure 10 . Hif-3α2 regulates LR asymmetry by destabilizing β-catenin independently of its transcriptional activity . ( A ) Inhibition of β-catenin-induced Topflash reporter activity . Zebrafish embryos were injected with 90 pg Topflash plasmid DNA together with capped mRNA ( 600 pg ) of the indicated Hif-3α2 mutants ( Figure 10—figure supplement 1 for mutant details ) . Luciferase activity was measured at 9 hpf . Values are means +S . E . ( n = 3 ) . Groups labeled with different letters are significantly different from each other ( P < 0 . 05 ) . ( B ) HRE-dependent transcriptional activities of the indicated Hif-3α2 mutants . HEK293 cells were transfected with the indicated plasmid together with p2 . 1 . The results are normalized and expressed as fold change over the GFP group and presented as described above . ( C ) Effects of Hif-3α2 mutants on β-catenin stability . HEK 293T cells were co-transfected with Flag-tagged β-catenin and the indicated Hif-3α2 mutants . The cell lysates were analyzed by Western blotting . ( D , E ) Effects of the indicated Hif-3α2 mutants on heart looping ( D ) and somite development ( E ) . Embryos injected with the indicated capped mRNA were analyzed and scored as described in Figure 3 . ( F ) Oxygen-dependent and -independent action of Hif-3α . The hif-3α gene gives rise to Hif-3α1 ( the full-length protein ) and Hif-3α2 ( a short spliced variant ) . Hif-3α1 is an oxygen-dependent transcription factor . It is rapidly degraded under normoxia . Under hypoxia , Hif-3α1 is stabilized and regulates the expression of many target genes , which in turn slow down growth and developmental timing ( Zhang et al . , 2014 ) . Hif-3α2 is insensitive to oxygen tension . Hif-3α2 regulates target gene expression in an HRE-dependent manner under both normoxia and hypoxia . It is unclear whether Hif-3α2 can form a dimer with HIFβ . Hif-3α2 also binds to β-catenin and destabilizes the nuclear β-catenin complex . This Wnt signaling inhibitory action is independent of its HRE-dependent transcriptional activity . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 02410 . 7554/eLife . 08996 . 025Figure 10—figure supplement 1 . Sequence comparison of the TAD in the indicated human and zebrafish HIF/Hif proteins . The Hif-3α2 point mutants engineered are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 02510 . 7554/eLife . 08996 . 026Figure 10—figure supplement 2 . Effects of full-length human HIF-3α9 , its N-terminal deletion mutant , zebrafish Hif-3α1 , and Hif-3α2 on β-catenin stability . HEK293T cells were co-transfected with Flag-tagged β-catenin and the indicated plasmid . The cell lysates were analyzed by Western blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 08996 . 026 The effects of these Hif-3α2 mutants in promoting β-catenin degradation were studied next . All three mutants possessing full β-catenin inhibitory activity , i . e . , ΔLZIP , M1 , and A36G , strongly induced β-catenin degradation ( Figure 10C ) . P37A , which had no Figure 10C ) . M2 and ΔTAD , which had partial β-catenin inhibitory activity , had a modest impact on β-catenin stability ( Figure 10C ) . Human HIF-3α9Δ 1–452 had similar activity in destabilizing β-catenin ( Figure 10—figure supplement 2 ) . Finally , the effects of the A36G and P37A mutants on the LR asymmetry development were determined . Overexpression of A36G and Hif-3α2 resulted in heart randomization and abnormal somite development ( Figure 10D , E ) . In contrast , overexpression of P37A , which lacks the ability to induce β-catenin degradation , had little effect ( Figure 10D–E ) . These findings suggest that Hif-3α2 alters LR asymmetryby destabilizing β-catenin and this action is independent of its HRE-dependent transcriptional activity ( Figure 10F ) .
In this study , we have identified a novel and oxygen-insensitive Hif-3α variant , Hif-3α2 . To date , a number of HIF-3α protein-coding variants have been reported , including: a ) the full-length protein; b ) a HIF-3α variant similar to the full-length protein; c ) HIF-3α variants lacking the LZIP domain; d ) HIF-3α variants lacking both bHLH and LZIP domains; e ) Short HIF-3α variants lacking the C-terminal half region; f ) Truncated HIF-3α variants lacking the bHLH , ODD/N-TAD , and LZIP domains; and g ) HIF-3α variants lacking the bHLH domain ( Duan , 2015 ) . Zebrafish Hif-3α2 has a structure distinct from those reported previously . Hif-3α2 lacks the entire N-terminal region and only contains the N-TAD and LZIP domains . Hif-3α2 is expressed in all embryonic stages and in many adult tissues examined . Although the Hif-3α2 mRNA levels were lower compared to those of the full-length Hif-3α1 , the Hif-3α2 protein was abundantly expressed in embryonic and adult tissues under both normoxia and hypoxia . Functional analysis results show that Hif-3α2 has significant HRE-dependent transcriptional activity . This activity depends on its TAD . Although Hif-3α2 has the ability to induce HRE-dependent gene expression , its action is not identical to that of the full-length Hif-3α1 . The transcriptional activity of Hif-3α2 is weaker compared to that of Hif-3α1 . Among the five Hif-31α target genes examined , Hif-3α2 increases the expression of igfbp-1a , mlp3c and redd1 but not that of zp3v2 and sqrdl . We observed two major phenotypes resulting from Hif-3α2 overexpression . One is the global growth retardation and developmental timing delay . This is similar to what has been reported for Hif-3α1 ( Zhang et al . , 2014 ) . This action apparently requires the transcriptional activity because it was abolished by deletion the TAD but not LZIP domain . Another phenotype is the defective LR asymmetry . Forced expression of Hif-3α2 altered the expression of several asymmetry genes and resulted in the randomization of laterality of the heart , liver , and pancreas . This action is unique to Hif-3α2 because no such phenotype was observed in the Hif-3α1 expressing embryos . Importantly , the A36G mutant , which had no HRE-dependent transcriptional activity , caused a similar heart randomization , suggesting that this action does not required its HRE transcriptional activity . The LR axis development in zebrafish is divided into several phases , beginning from the symmetry breaking , and continuing with DFC appearance and clustering , KV organogenesis , lateral plate mesoderm asymmetry , and left- or right-specific organ formation ( Carl et al . , 2007; Matsui and Bessho , 2012 ) . Our results suggest that forced expression of Hif-3α2 impairs LR axis development at the step of KV organogenesis/function . This conclusion is supported by the findings that Hif-3α2 overexpression altered KV morphology and spaw expression , while it did not affect DFC appearance or DFC clustering . We provided several lines of evidence suggesting that Hif-3α2 impairs KV development by inhibiting the canonical Wnt/β-catenin signaling pathway . These include: i ) Hif-3α2 overexpression decreases Wnt target gene expression while it had no effect on Fgf- , Hedgehog- , and Notch target genes; ii ) Co-expression of Hif-3α2 inhibits Wnt3 and β-catenin-induced gene expression and dorsalization; iii ) genetic deletion of Hif-3α2 ( without affecting Hif-3α1 expression and function ) increases Topflash reporter activity and Wnt target gene expression; iv ) MO-based knockdown had similar effects in elevating Wnt signaling . These data strongly suggest that Hif-3α2 is a negative regulator of canonical Wnt signaling . Wnt signaling plays multiple roles in vertebrate axis specification ( see Hikasa and Sokol , 2013 ) . While the maternal Wnt/β-catenin signaling contributes to the dorsoventral axis establishment in early embryogenesis , the zygotic Wnt/β-catenin signaling is involved in anteroposterior axis specification . It has been suggested that these two different roles are mediated by two distinct sets of target genes ( Hikasa and Sokol , 2013 ) . As discussed earlier , canonical Wnt/β-catenin signaling also plays an important role in KV development and the establishment of the LR axis in zebrafish . Our results showed that overexpression of Hif-3α2 acts at the step of KV development . This may be related to the fact that it takes several hours for the injected mRNA to be translated into protein , and for resulting biological activities to manifest . Since Hif-3α2 mRNA is maternally deposited , the endogenous Hif-3α2 may act earlier . Indeed , we observed a significant increase in maternal Wnt signaling and elevated mortality in early stages in the mutant embryos . Although deletion of Hif-3α2 by either genetic mutations or gene knockdown results in elevated Wnt/β-catenin signaling , the two approaches gave rise to very different phenotypes . The F3 hif-3αΔ42 mutant embryos had significantly elevated mortality in early stages . This increase in embryo mortality was also observed in F3 hif-3αΔ20 mutant embryos . In contrast , no mortality increase was detected in the morphants . In both genetic alleles , the vast majority of the surviving embryos looked morphologically normal . The morphants , however , displayed phenotypes characteristic of excessive Wnt signaling . There are several possible explanations for the different phenotype . It is conceivable that the lack of a body patterning phenotype in the surviving mutant embryos may be due to activation of compensatory mechanisms . Such compensation mechanisms have been demonstrated recently in zebrafish and accounted for the different phenotypes found in the egfl7-/- genetic mutants and Egfl7 morphants ( Rossi et al . , 2015 ) . In support of this view , the hif-3αΔ42 mutant embryos displayed a transient and dynamic activation of Wnt signaling , while a sustained elevation in Wnt signaling was observed in the morphants . A concomitant reduction in BMP signaling was observed in the hif-3αΔ42 embryos but not in the morphants . Furthermore , the hif-3αΔ42 mutant embryos were more sensitive to the Wnt activating agents Lithium and BIO . Another possible explanation is the differential effects of genetic mutations and MO knockdown on the maternal Wnt signaling . The maternal Wnt signaling is significantly elevated in the F3 hif-3αΔ42 mutant embryos , as indicated by the increased boz expression , while no such increase was observed in the morphants . It is also possible that Hif-3α2 may have other activities in vivo . In fact , our results show that Hif-3α2 can regulate gene expression via its TAD domain . We cannot exclude the possibility Hif-3α2 may affect other pathways directly or indirectly . A biochemical and functional interaction between an HIF-3α isoform and Wnt signaling has not been reported before . However , HIF-1α has been shown to both inhibit and activate Wnt signaling in mammals . Kaidi et al . ( Kaidi et al . , 2007 ) reported that hypoxia inhibits the β-catenin/TCF4 complex formation and activity , resulting in a G1 arrest in cultured colon cancer cells . These authors proposed that HIF-1α acts by competing with TCF4 for β-catenin binding . Lim et al . ( Lim et al . , 2008 ) reported that HIF-1α inhibits Wnt signaling by binding to human arrest defective 1 ( hARD1 ) , which is responsible for β-catenin acetylation . Another mechanism was reported by Chen et al . ( 2013 ) . They showed that HIF-1α indirectly inhibits the Wnt signaling pathway by inducing the expression of a Wnt antagonist Sclerostin in MC3T3 osteoblastic cells . In a very recent paper , Majmundar et al . demonstrated that tissue-specific knockout of HIF-1α in mice has little effect on embryonic and fetal myogenesis but increases adult muscle regeneration ( Majmundar et al . , 2015 ) . HIF-1α acts by repressing canonical Wnt signaling in adult skeletal muscle . HIF-1α has also been shown to activate Wnt signaling in mammals . Using β-Gal Wnt reporter and Hif-1a conditional knockout mouse lines , Mazumdar et al . ( 2010 ) Mazumdar et al . ( 2010 ) showed that HIF-1α increases Wnt/β-catenin signaling activity in murine embryonic mesencephalon and adult hippocampal neuronal stem cells and precursor cells in vitro and in vivo . These authors further showed that HIF-1α activates canonical Wnt signaling by increasing the expression levels of β-catenin and its downstream effectors LEF-1 and TCF-1 . Subsequently , Jeong and Pack reported that HIF-1α increases β-catenin expression level and action in SH-SY5Y human neuroblastoma cells ( Jeong and Park , 2013 ) . Medley et al . reported that hypoxia activates Wnt signaling in undifferentiated iPS cells ( Medley et al . , 2013 ) . As in the case of HIF-1α , HIF-2α has been shown to inhibit or/and enhance Wnt/β-catenin signaling ( Choi et al . , 2010; Santoyo-Ramos et al . , 2014 ) . Another mechanism by which HIF-1α and -2α can active the Wnt signaling pathway was reported by Yuen et al . recently ( Yuen et al . , 2014 ) . They reported that HIF-1α and -2α directly bind to the promoters of the Wnt7a and 7b genes and induce their expression in murine oligodendrocytes . Our study is different from the above studies in that Hif-3α2 is an oxygen-insensitive and constitutively expressed Hif-3α isoform and therefore functions under both normoxia and hypoxia . Importantly , our biochemical analysis results revealed that Hif-3α2 directly binds to β-catenin and promotes the degradation of the nuclear β-catenin complex . This action is specific , as shown by the fact that Hif-3α2 overexpression had no effect on the level of Tcf3 , a β-catenin suppressor . This mechanism is distinct from those reported for HIF-1α and -2α . In addition to the GSK3β/β-TrCP-mediated degradation mentioned earlier , β-catenin is also subject to negative regulation by pVHL . The β-catenin degradation induced by pVHL is dependent on the presence of Jade-1 , an E3 ubiquitin ligase ( Chitalia et al . , 2008 ) . These two pathways are unlikely to be involved in the Hif-3α2-induced degradation for several reasons . First , both β-TrCP and Jade-1 bind to the N-terminal region of β-catenin in a phosphorylation-dependent manner and predominantly degrade β-catenin in the cytosol . Our results showed that Hif-3α2 inhibits the activity of β-cateninΔN , a constitutively active β-catenin mutant lacking the N-terminal sequence . Second , two GSK-3β inhibitors , BIO and lithium , did not inhibit Hif-3α2-induced β-catenin degradation . Finally , Hif-3α2 not only reduces the levels of β-catenin but also decreases the levels of Tcf-4 and Lef1 . GST-Hif-3α2 did not directly interact with Tcf-4 and Lef1 . Therefore , Hif-3α2 probably binds to β-catenin , which in turn interacts with Tcf-4 and Lef1 . It is unclear whether other factors may be involved in the physical association between Hif-3α2 and β-catenin and whether post-translational modification of Hif-3α2 and/or β-catenin is involved its their binding . β-catenin can shuttle in and out of the nucleus ( Krieghoff et al . , 2006 ) . The results of our cell fractionation experiment showed that Hif-3α2 expression resulted in a marked decrease in the nuclear β-catenin levels without notable changes in the cytosolic β-catenin levels . In general , the fate of active β-catenin in the nucleus is not well understood . A recent study reported that the RING finger E3 ubiquitin ligase c-Cbl preferentially targets the active nuclear β-catenin for degradation by binding to the ARM domains of β-catenin ( Chitalia et al . , 2013 ) . Wnt activation promotes c-Cbl phosphorylation at Y371 , and the phosphorylated c-Cbl dimerizes , translocates into the nucleus and promotes nuclear active β-catenin degradation during the Wnt-on phase ( Shivanna et al . , 2015 ) . TRIM33 , another E3 ubiquitin ligase , has been reported to interact with and ubiquitylate nuclear β-catenin , and promote nuclear β-catenin degradation in a GSK-3β and β-TrCP-independent manner ( Xue et al . , 2015 ) . It is unknown where c-Cbl and/or TRIM33 mediated the degradation of the β-catenin/Tfc4/Left1 complex . Future studies will be needed to determine the molecular mechanism ( s ) responsible for the Hif-3α2-induced β-catenin complex destabilization . Another intriguing and potentially important finding made in this study is that Hif-3α2 has HRE-dependent transcriptional activity . The current dogma is that HIFs act as α/β heterodimers ( Semenza , 2012 ) . The bHLH domain in HIFα is known to be involved in DNA binding , and the PAS-A and PAS-B domains are proposed to be involved in dimerization with HIF-β and target gene specificity ( Erbel et al . , 2003; Simon and Keith , 2008 ) . Although Hif-3α2 has a TAD , it lacks the bHLH and PAS domains . While our findings suggest that Hif-3α2 has transcriptional activity under normoxia , it is not clear whether this action requires HIF-β . Makino et al . ( 2001 ) reported that mouse IPAS could not form dimers with HIF-β . Rather , it formed a complex with HIF-1α but this complex did bind to an HRE . Using co-immunoprecipitation assays , Maynard et al . ( 2005 ) showed that HIF-3α4 interacted with HIF-1α when transfected into HEK293 cells . HIF-3α4 also formed a complex with HIF-β . The HIF-3α4/HIF-β complex did not appear to be capable of binding to an HRE , but it inhibited the binding of the HIF-1α/β complex to the HRE in a dose-dependent fashion . A similar relationship between HIF-3α4 and HIF-2α was reported ( Maynard et al . , 2007 ) . The structure of mouse IPAS and human HIF-3α4 is very different from zebrafish Hif-3α2 . Mouse IPAS and human HIF-3α4 contain bHLH , PAS-A and -B , and PAC domains but lack the ODD/T-NAD and LIZP domains . In contract , zebrafish Hif-3α2 contains N-TAD and LZIP only . The LZIP domain is only found in HIF-3α among the 3 HIFα isoforms ( Duan , 2015 ) . LZIP is the dimerization domain of the ATF-6/CREB subfamily of bZIPs ( a class of eukaryotic transcription factors ) ( Vinson et al . , 1989 ) and it facilitates dimerization and in some cases higher oligomerization of proteins ( Lu et al . , 1997 ) . LZIP containing regulatory proteins include c-Fos and c-Jun and the LZIP domains in Fos and Jun are necessary for the formation of the AP1 heterodimer ( Ransone et al . , 1989 ) . The LZIP domains in Fos and Jun have also been shown to bind to cAMP-responsive elements as a homodimer and can activate transcription from CRE-containing reporter genes ( Lu et al . , 1997; Lu et al . , 1998 ) . Our available data showed that the ΔLZIP mutant had stronger transcriptional activity . Future studies are needed to elucidate whether Hif-3α2 exerts its HRE-dependent activity independently of Hif β and to determine the functional relationships between Hif-3α2 , Hif-3α1 , and other Hifα proteins . In summary , Hif-3α2 represents a novel and O2-insensitive HIF/Hif-3α variants . Hif-3α2 has two distinct functions: activating HRE-dependent gene expression and binding to β-catenin and destabilizing the nuclear β-catenin complex . The corresponding region of human HIF-3α possesses both activities , suggesting that both actions are evolutionarily conserved . In agreement with this notion , Wnt3a knockout mice exhibit randomized internal organ positioning across the midline ( Nakaya et al . , 2005 ) . The Hif-3α-/- mice had enlarged embryonic right atrium and right ventricle and this phenotype became more prominent in neonatal mice and continues until the adult stage ( Yamashita et al . , 2008 ) . In contrast , no enlargement was observed in the left ventricle in these mutant mice ( Yamashita et al . , 2008 ) . Our mutation analysis results suggested that different structural element ( s ) are responsible for these two distinct biological actions . Interestingly , the full-length Hif-3α1 , while exhibiting strong HRE-dependent transcriptional activity , does not inhibit β-catenin activity . It is possible that there are structural element ( s ) in the full-length Hif-3α proteins that mask this important activity . In support of this view , we found that while the full-length human HIF-3α9 has no β-catenin inhibitory activity , deletion of its N-terminal sequence can unmask this ability ( Figure 10—figure supplement 2 ) . An earlier study suggested that there are structural element ( s ) in the full-length human HIF-3α inhibiting its ubiquitylation ( Maynard et al . , 2003 ) . Sequence analysis of human and fish HIF/Hifαs indicate that the residues critical for promoting β-catenin degradation are conserved in human and fish HIF/Hifαs . It is possible that all HIFαs may have the ability to interact with β-catenin . Future studies will determine whether this activity is conserved in the full-length HIFαs and reveal how they are regulated . This would explain the inconsistent and even opposite results regarding the interactions between HIF-1/2α and Wnt/β-catenin signaling .
The data shown are means ± SE . Differences among groups were analyzed by one-way or two-way ANOVA or by Student’s t test using GraphPad Prism . Significance was accepted at P < 0 . 05 or lower .
Hypoxia-inducible factors ( HIFs ) , while best known for their roles in the hypoxic response , have oxygen-independent roles in early development with poorly defined mechanisms . Here , we report a novel Hif-3α variant , Hif-3α2 , in zebrafish . Hif-3α2 lacks the bHLH , PAS , PAC , and ODD domains , and is expressed in embryonic and adult tissues independently of oxygen availability . Hif-3α2 is a nuclear protein with significant hypoxia response element ( HRE ) -dependent transcriptional activity . Hif-3α2 overexpression not only decreases embryonic growth and developmental timing but also causes left-right asymmetry defects . Genetic deletion of Hif-3α2 by CRISPR/Cas9 genome editing increases , while Hif-3α2 overexpression decreases , Wnt/β-catenin signaling . This action is independent of its HRE-dependent transcriptional activity . Mechanistically , Hif-3α2 binds to β-catenin and destabilizes the nuclear β-catenin complex . This mechanism is distinct from GSK3β-mediated β-catenin degradation and is conserved in humans . These findings provide new insights into the oxygen-independent actions of HIFs and uncover a novel mechanism regulating Wnt/β-catenin signaling .
Proteins known as hypoxia-inducible factors ( HIFs ) are important in animals when the amount of oxygen in the air or water drops . These proteins switch on genes that help cells and tissues adapt to the shortage in oxygen , for example , by stimulating the production of red blood cells . Each HIF is made up of two subunits called α and β that only bind to each other when the oxygen levels drop . This two-subunit complex , or 'dimer' , then activates a set of genes by binding to a stretch of DNA known as the hypoxia response element . HIFs also play important roles in many different stages of animal development . There are many different HIF proteins that are each present at different developmental stages; this has made them difficult to study . Zhang et al . have found a new form of HIF-3α in zebrafish – called Hif-3α2 . The experiments show that this α subunit is not regulated by oxygen , but may still be able to activate genes that have the hypoxia response element . When Hif-3α2 was injected into zebrafish embryos , the body pattern that is normally set up in embryogenesis was disrupted . Further experiments revealed that Hif-3α2 regulates embryo development by destabilizing a protein called β-catenin . This inhibits a cell communication system called Wnt/β-catenin signaling . Zhang et al . also show that the two distinct activities of Hif-3α2 – binding to the hypoxia response element and destabilizing β-catenin – involve two different regions of the protein . Together , Zhang et al . ’s findings show that zebrafish Hif-3α2 combines some conventional features of HIF proteins with a unique developmental role . It is likely that human Hif-3α may also work in a similar way , so future studies will focus on understanding the molecular mechanisms responsible for these distinct roles .