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Wdr68 Mediates Dorsal and Ventral Patterning Events for Craniofacial Development

View Article: PubMed Central - PubMed

ABSTRACT

Birth defects are among the leading causes of infant mortality and contribute substantially to illness and long-term disability. Defects in Bone Morphogenetic Protein (BMP) signaling are associated with cleft lip/palate. Many craniofacial syndromes are caused by defects in signaling pathways that pattern the cranial neural crest cells (CNCCs) along the dorsal-ventral axis. For example, auriculocondylar syndrome is caused by impaired Endothelin-1 (Edn1) signaling, and Alagille syndrome is caused by defects in Jagged-Notch signaling. The BMP, Edn1, and Jag1b pathways intersect because BMP signaling is required for ventral edn1 expression that, in turn, restricts jag1b to dorsal CNCC territory. In zebrafish, the scaffolding protein Wdr68 is required for edn1 expression and subsequent formation of the ventral Meckel’s cartilage as well as the dorsal Palatoquadrate. Here we report that wdr68 activity is required between the 17-somites and prim-5 stages, that edn1 functions downstream of wdr68, and that wdr68 activity restricts jag1b, hey1, and grem2 expression from ventral CNCC territory. Expression of dlx1a and dlx2a was also severely reduced in anterior dorsal and ventral 1st arch CNCC territory in wdr68 mutants. We also found that the BMP agonist isoliquiritigenin (ISL) can partially rescue lower jaw formation and edn1 expression in wdr68 mutants. However, we found no significant defects in BMP reporter induction or pSmad1/5 accumulation in wdr68 mutant cells or zebrafish. The Transforming Growth Factor Beta (TGF-β) signaling pathway is also known to be important for craniofacial development and can interfere with BMP signaling. Here we further report that TGF-β interference with BMP signaling was greater in wdr68 mutant cells relative to control cells. To determine whether interference might also act in vivo, we treated wdr68 mutant zebrafish embryos with the TGF-β signaling inhibitor SB431542 and found partial rescue of edn1 expression and craniofacial development. While ISL treatment failed, SB431542 partially rescued dlx2a expression in wdr68 mutants. Together these findings reveal an indirect role for Wdr68 in the BMP-Edn1-Jag1b signaling hierarchy and dorso-anterior expression of dlx1a/2a.

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Related in: MedlinePlus

TGF-β interference with BMP signaling is enhanced in cells lacking Wdr68 expression.A) Isolation of Wdr68/Dcaf7 knock-out C2C12 cell sublines and expression levels in growth medium (GM) versus differentiation medium (Diff). Panel A1) Lanes 1 and 4, Wdr68 protein was detected in the control NT1 cells. Lanes 2 and 5, Δwdr68-5 lacks wildtype Wdr68 protein expression. Lanes 3 and 6, Δwdr68-9 lacks wildtype Wdr68 protein expression. Panel A2) β-tubulin expression was used as a loading control and did not differ substantially between lanes. Panel A3) pYap1 levels did not differ substantially between lanes. Panel A4) Total Yap1 levels did not differ substantially between lanes. B) pSmad1/5 induction was not substantially altered in Δwdr68-5 or Δwdr68-9 sublines. Panel B1) pSmad1/5 levels in control (NT1) or Δwdr68-5 (5) cells after 1 hour of exposure to 0, 1, 10, or 100ng/mL BMP4 in DM. Panel B2) β-tubulin expression was used as a loading control and did not differ substantially between lanes. Panel B3) pSmad1/5 levels in control (NT1) or Δwdr68-9 (5) cells after 1 hour of exposure to 0, 1, 10, or 100ng/mL BMP4 in DM. Panel B4) β-tubulin expression was used as a loading control and did not differ substantially between lanes. C) Transient transfection of NT1, Δwdr68-5, and Δwdr68-9 sublines with BRE-Luc and SV40-Renilla plasmids and induced with 0, 1, 10, or 100ng/mL BMP4 in GM. No significant differences were found between control and deletion sublines. Representative experiment shown from at least 3 independent trials. D) Transient transfection of NT1, Δwdr68-5, and Δwdr68-9 sublines with BRE-Luc and SV40-Renilla plasmids, induced with 10ng/mL BMP4, and then challenged with 0, 0.1, 1.0, or 10ng/mL TGF-®1. At 10ng/mL TGF-®1 interference with BRE-Luc activity was significantly greater in the Δwdr68-5 and Δwdr68-9 sublines relative to NT1 controls (* = p < 0.002). Representative experiment shown from at least 3 independent trials. E-H) Immunofluorescence detection of pSmad1/5 in prim-12 stage zebrafish embryos raised at 32°C. E) wildtype sibling embryo. F) wdr68hi3812/hi3812 mutant embryo. G) DMSO-treated wildtype sibling. H) ISL-treated wildtype sibling.
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pone.0166984.g006: TGF-β interference with BMP signaling is enhanced in cells lacking Wdr68 expression.A) Isolation of Wdr68/Dcaf7 knock-out C2C12 cell sublines and expression levels in growth medium (GM) versus differentiation medium (Diff). Panel A1) Lanes 1 and 4, Wdr68 protein was detected in the control NT1 cells. Lanes 2 and 5, Δwdr68-5 lacks wildtype Wdr68 protein expression. Lanes 3 and 6, Δwdr68-9 lacks wildtype Wdr68 protein expression. Panel A2) β-tubulin expression was used as a loading control and did not differ substantially between lanes. Panel A3) pYap1 levels did not differ substantially between lanes. Panel A4) Total Yap1 levels did not differ substantially between lanes. B) pSmad1/5 induction was not substantially altered in Δwdr68-5 or Δwdr68-9 sublines. Panel B1) pSmad1/5 levels in control (NT1) or Δwdr68-5 (5) cells after 1 hour of exposure to 0, 1, 10, or 100ng/mL BMP4 in DM. Panel B2) β-tubulin expression was used as a loading control and did not differ substantially between lanes. Panel B3) pSmad1/5 levels in control (NT1) or Δwdr68-9 (5) cells after 1 hour of exposure to 0, 1, 10, or 100ng/mL BMP4 in DM. Panel B4) β-tubulin expression was used as a loading control and did not differ substantially between lanes. C) Transient transfection of NT1, Δwdr68-5, and Δwdr68-9 sublines with BRE-Luc and SV40-Renilla plasmids and induced with 0, 1, 10, or 100ng/mL BMP4 in GM. No significant differences were found between control and deletion sublines. Representative experiment shown from at least 3 independent trials. D) Transient transfection of NT1, Δwdr68-5, and Δwdr68-9 sublines with BRE-Luc and SV40-Renilla plasmids, induced with 10ng/mL BMP4, and then challenged with 0, 0.1, 1.0, or 10ng/mL TGF-®1. At 10ng/mL TGF-®1 interference with BRE-Luc activity was significantly greater in the Δwdr68-5 and Δwdr68-9 sublines relative to NT1 controls (* = p < 0.002). Representative experiment shown from at least 3 independent trials. E-H) Immunofluorescence detection of pSmad1/5 in prim-12 stage zebrafish embryos raised at 32°C. E) wildtype sibling embryo. F) wdr68hi3812/hi3812 mutant embryo. G) DMSO-treated wildtype sibling. H) ISL-treated wildtype sibling.

Mentions: To further examine whether Wdr68 might directly impact BMP signaling, we used CRISPR/Cas9 gene targeting technology to generate loss-of-function deletions in the Wdr68/Dcaf7 locus in mouse C2C12 cells. Western blot analysis and DNA sequencing confirmed the generation of two independently isolated mutant sublines, Δwdr68-5 and Δwdr68-9 (Fig 6A and S4B Fig) as well as a non-target control (NT1) subline. The transcriptional co-activator Yap is reported to be important for BMP signaling in mammalian cells [58, 59]. In flies, the ortholog of Wdr68, Riquiqui (Riq), is also reported to positively regulate the Yap ortholog yorkie through its interaction with the kinase Minibrain (Mnb) that negatively regulates the Hippo signaling pathway kinase Warts (Wts) [60]. Therefore, we examined both total Yap and pYap levels in the control, Δwdr68-5, and Δwdr68-9 sublines but found no significant differences between them (Fig 6A). To further characterize the sublines, we examined the levels of pSmad1/5 after a one-hour treatment of the cells with 0, 1, 10, or 100ng/mL BMP4 and found no significant reproducible differences between the sublines (Fig 6B). To determine whether Wdr68/Dcaf7 might generally facilitate BMP signaling in a functional assay, we transfected the control and deletion sublines with the BRE-Luc reporter plasmid [55] along with a SV40-Renilla plasmid, and generated a dose-response curve to BMP4 ligand (Fig 6C). All relative light unit responses were normalized to the vehicle-treated control subline (Fig 6C, leftmost column). Overall, no significant decreases in fold-induction were found in the Δwdr68-5 and Δwdr68-9 sublines relative to the control subline (Fig 6C grey bars versus black bars). Immunofluorescence analysis also revealed little consistent difference in pSmad1/5 levels between wildtype and wdr68hi3812/hi3812 mutant zebrafish embryos (Fig 6E and 6F). As expected, ISL treatment did consistently increase pSmad1/5 signal (compare Fig 6H to 6G). Thus, Wdr68/Dcaf7 does not appear to directly modulate BMP signaling pathway activity.


Wdr68 Mediates Dorsal and Ventral Patterning Events for Craniofacial Development
TGF-β interference with BMP signaling is enhanced in cells lacking Wdr68 expression.A) Isolation of Wdr68/Dcaf7 knock-out C2C12 cell sublines and expression levels in growth medium (GM) versus differentiation medium (Diff). Panel A1) Lanes 1 and 4, Wdr68 protein was detected in the control NT1 cells. Lanes 2 and 5, Δwdr68-5 lacks wildtype Wdr68 protein expression. Lanes 3 and 6, Δwdr68-9 lacks wildtype Wdr68 protein expression. Panel A2) β-tubulin expression was used as a loading control and did not differ substantially between lanes. Panel A3) pYap1 levels did not differ substantially between lanes. Panel A4) Total Yap1 levels did not differ substantially between lanes. B) pSmad1/5 induction was not substantially altered in Δwdr68-5 or Δwdr68-9 sublines. Panel B1) pSmad1/5 levels in control (NT1) or Δwdr68-5 (5) cells after 1 hour of exposure to 0, 1, 10, or 100ng/mL BMP4 in DM. Panel B2) β-tubulin expression was used as a loading control and did not differ substantially between lanes. Panel B3) pSmad1/5 levels in control (NT1) or Δwdr68-9 (5) cells after 1 hour of exposure to 0, 1, 10, or 100ng/mL BMP4 in DM. Panel B4) β-tubulin expression was used as a loading control and did not differ substantially between lanes. C) Transient transfection of NT1, Δwdr68-5, and Δwdr68-9 sublines with BRE-Luc and SV40-Renilla plasmids and induced with 0, 1, 10, or 100ng/mL BMP4 in GM. No significant differences were found between control and deletion sublines. Representative experiment shown from at least 3 independent trials. D) Transient transfection of NT1, Δwdr68-5, and Δwdr68-9 sublines with BRE-Luc and SV40-Renilla plasmids, induced with 10ng/mL BMP4, and then challenged with 0, 0.1, 1.0, or 10ng/mL TGF-®1. At 10ng/mL TGF-®1 interference with BRE-Luc activity was significantly greater in the Δwdr68-5 and Δwdr68-9 sublines relative to NT1 controls (* = p < 0.002). Representative experiment shown from at least 3 independent trials. E-H) Immunofluorescence detection of pSmad1/5 in prim-12 stage zebrafish embryos raised at 32°C. E) wildtype sibling embryo. F) wdr68hi3812/hi3812 mutant embryo. G) DMSO-treated wildtype sibling. H) ISL-treated wildtype sibling.
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Related In: Results  -  Collection

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Show All Figures
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pone.0166984.g006: TGF-β interference with BMP signaling is enhanced in cells lacking Wdr68 expression.A) Isolation of Wdr68/Dcaf7 knock-out C2C12 cell sublines and expression levels in growth medium (GM) versus differentiation medium (Diff). Panel A1) Lanes 1 and 4, Wdr68 protein was detected in the control NT1 cells. Lanes 2 and 5, Δwdr68-5 lacks wildtype Wdr68 protein expression. Lanes 3 and 6, Δwdr68-9 lacks wildtype Wdr68 protein expression. Panel A2) β-tubulin expression was used as a loading control and did not differ substantially between lanes. Panel A3) pYap1 levels did not differ substantially between lanes. Panel A4) Total Yap1 levels did not differ substantially between lanes. B) pSmad1/5 induction was not substantially altered in Δwdr68-5 or Δwdr68-9 sublines. Panel B1) pSmad1/5 levels in control (NT1) or Δwdr68-5 (5) cells after 1 hour of exposure to 0, 1, 10, or 100ng/mL BMP4 in DM. Panel B2) β-tubulin expression was used as a loading control and did not differ substantially between lanes. Panel B3) pSmad1/5 levels in control (NT1) or Δwdr68-9 (5) cells after 1 hour of exposure to 0, 1, 10, or 100ng/mL BMP4 in DM. Panel B4) β-tubulin expression was used as a loading control and did not differ substantially between lanes. C) Transient transfection of NT1, Δwdr68-5, and Δwdr68-9 sublines with BRE-Luc and SV40-Renilla plasmids and induced with 0, 1, 10, or 100ng/mL BMP4 in GM. No significant differences were found between control and deletion sublines. Representative experiment shown from at least 3 independent trials. D) Transient transfection of NT1, Δwdr68-5, and Δwdr68-9 sublines with BRE-Luc and SV40-Renilla plasmids, induced with 10ng/mL BMP4, and then challenged with 0, 0.1, 1.0, or 10ng/mL TGF-®1. At 10ng/mL TGF-®1 interference with BRE-Luc activity was significantly greater in the Δwdr68-5 and Δwdr68-9 sublines relative to NT1 controls (* = p < 0.002). Representative experiment shown from at least 3 independent trials. E-H) Immunofluorescence detection of pSmad1/5 in prim-12 stage zebrafish embryos raised at 32°C. E) wildtype sibling embryo. F) wdr68hi3812/hi3812 mutant embryo. G) DMSO-treated wildtype sibling. H) ISL-treated wildtype sibling.
Mentions: To further examine whether Wdr68 might directly impact BMP signaling, we used CRISPR/Cas9 gene targeting technology to generate loss-of-function deletions in the Wdr68/Dcaf7 locus in mouse C2C12 cells. Western blot analysis and DNA sequencing confirmed the generation of two independently isolated mutant sublines, Δwdr68-5 and Δwdr68-9 (Fig 6A and S4B Fig) as well as a non-target control (NT1) subline. The transcriptional co-activator Yap is reported to be important for BMP signaling in mammalian cells [58, 59]. In flies, the ortholog of Wdr68, Riquiqui (Riq), is also reported to positively regulate the Yap ortholog yorkie through its interaction with the kinase Minibrain (Mnb) that negatively regulates the Hippo signaling pathway kinase Warts (Wts) [60]. Therefore, we examined both total Yap and pYap levels in the control, Δwdr68-5, and Δwdr68-9 sublines but found no significant differences between them (Fig 6A). To further characterize the sublines, we examined the levels of pSmad1/5 after a one-hour treatment of the cells with 0, 1, 10, or 100ng/mL BMP4 and found no significant reproducible differences between the sublines (Fig 6B). To determine whether Wdr68/Dcaf7 might generally facilitate BMP signaling in a functional assay, we transfected the control and deletion sublines with the BRE-Luc reporter plasmid [55] along with a SV40-Renilla plasmid, and generated a dose-response curve to BMP4 ligand (Fig 6C). All relative light unit responses were normalized to the vehicle-treated control subline (Fig 6C, leftmost column). Overall, no significant decreases in fold-induction were found in the Δwdr68-5 and Δwdr68-9 sublines relative to the control subline (Fig 6C grey bars versus black bars). Immunofluorescence analysis also revealed little consistent difference in pSmad1/5 levels between wildtype and wdr68hi3812/hi3812 mutant zebrafish embryos (Fig 6E and 6F). As expected, ISL treatment did consistently increase pSmad1/5 signal (compare Fig 6H to 6G). Thus, Wdr68/Dcaf7 does not appear to directly modulate BMP signaling pathway activity.

View Article: PubMed Central - PubMed

ABSTRACT

Birth defects are among the leading causes of infant mortality and contribute substantially to illness and long-term disability. Defects in Bone Morphogenetic Protein (BMP) signaling are associated with cleft lip/palate. Many craniofacial syndromes are caused by defects in signaling pathways that pattern the cranial neural crest cells (CNCCs) along the dorsal-ventral axis. For example, auriculocondylar syndrome is caused by impaired Endothelin-1 (Edn1) signaling, and Alagille syndrome is caused by defects in Jagged-Notch signaling. The BMP, Edn1, and Jag1b pathways intersect because BMP signaling is required for ventral edn1 expression that, in turn, restricts jag1b to dorsal CNCC territory. In zebrafish, the scaffolding protein Wdr68 is required for edn1 expression and subsequent formation of the ventral Meckel&rsquo;s cartilage as well as the dorsal Palatoquadrate. Here we report that wdr68 activity is required between the 17-somites and prim-5 stages, that edn1 functions downstream of wdr68, and that wdr68 activity restricts jag1b, hey1, and grem2 expression from ventral CNCC territory. Expression of dlx1a and dlx2a was also severely reduced in anterior dorsal and ventral 1st arch CNCC territory in wdr68 mutants. We also found that the BMP agonist isoliquiritigenin (ISL) can partially rescue lower jaw formation and edn1 expression in wdr68 mutants. However, we found no significant defects in BMP reporter induction or pSmad1/5 accumulation in wdr68 mutant cells or zebrafish. The Transforming Growth Factor Beta (TGF-&beta;) signaling pathway is also known to be important for craniofacial development and can interfere with BMP signaling. Here we further report that TGF-&beta; interference with BMP signaling was greater in wdr68 mutant cells relative to control cells. To determine whether interference might also act in vivo, we treated wdr68 mutant zebrafish embryos with the TGF-&beta; signaling inhibitor SB431542 and found partial rescue of edn1 expression and craniofacial development. While ISL treatment failed, SB431542 partially rescued dlx2a expression in wdr68 mutants. Together these findings reveal an indirect role for Wdr68 in the BMP-Edn1-Jag1b signaling hierarchy and dorso-anterior expression of dlx1a/2a.

No MeSH data available.


Related in: MedlinePlus