Wdr68 Mediates Dorsal and Ventral Patterning Events for Craniofacial Development

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.

DCAF7/WDR68 is required for normal levels of DYRK1A and DYRK1B

“…Upon more careful analysis, we found that the level of WDR68 is significantly induced in DM (Fig 1A and 1A’, compare lane 3 to lane 1, p<0.05). Consistent with our previous report [23], WDR68 is not detected above background in Δwdr68-9 cells relative to NT1 control cells (Fig 1A and 1A’, compare lane 2 to lane 1 and lane 4 to lane 3, p<0.01). Next, we examined the level of DYRK1A in NT1 control cells and similarly found that the level of DYRK1A is significantly higher in DM than GM (Fig 1A and 1A”, compare lane 3 to lane 1, p<0.01).…”

“…C2C12 cells grow rapidly when cultured in growth medium (GM), but arrest cellular growth and differentiate into multinucleated myoblasts in low-serum/differentiation medium (DM) [52, 53]. WDR68 is important for this developmental transition [25], and we previously generated Δwdr68 mouse C2C12 sublines and non-targeted (NT1) control cells [23]. Upon more careful analysis, we found that the level of WDR68 is significantly induced in DM (Fig 1A and 1A’, compare lane 3 to lane 1, p<0.05).…”

“…Western blots were performed as previously described [23, 25]. Briefly, cell extracts were made from 10cm or 6-well plates of cells in GM or Differentiation Medium (DM) (DMEM; 2% Horse serum; 100ug/mL pen/strep).…”

“…The C2C12 NT1 and Δwdr68 cells were previously described [23]. Additional sublines were generated as previously described [23, 49]. Briefly, pLentiCRISPRv2-mdyrk1a1 was generated by annealing the oligonucleotides CRISPR-mdyrk1a-1f: and CRISPR-mdyrk1a-1r: .…”

“…Originally identified in plants for a role in anthocyanin biosynthesis [21], WDR68 is a 342 amino acid length protein composed of five WD40 repeats that modeling suggests forms a seven-blade ß-propeller structure [22]. In zebrafish, Wdr68 is important for embryonic development of the upper and lower jaws [3, 23–25]. WDR68 has also been identified as a DDB1 and CUL4-associated factor (DCAF), thus implicating it in the ubiquitin-mediated regulation of protein stability [26].…”