PgmNr Z6274: Wdr68 modulates TGFβ interference with BMP signaling for lower jaw patterning.

Authors:
A. J. Martinez; T. Whitman; G. Alvarado; R. Shang; M. Yousefelahiye; Y. Yu; A. Pham; B. Wang; E. Alvarado; R. M. Nissen


Institutes
California State University Los Angeles, Los Angeles, CA.


Abstract:

Birth defects are among the leading causes of infant mortality and contribute substantially to illness and long-term disability. Craniofacial anomalies, excluding cleft lip and palate, occur in 1 out of every 1600 births in the United States. 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, defects in Bone Morphogenetic Protein (BMP) signaling are associated with cleft lip/palate, 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, wdr68 is required for both the ventral Meckel’s (M) cartilage and the dorsal Palatoquadrate (PQ). Previously, we identified the wdr68 gene as essential for edn1 expression in the zebrafish. Here we show that wdr68 activity is required between the 17-somites and prim-5 stages through experiments using an inducible Tg(hsp70l:GFP-Wdr68) zebrafish line. This identified developmental window overlaps with the known onset of edn1 expression at the 18-somites stage. Using an RNA rescue assay and in situ hybridization (ISH), we also found that ectopic edn1 mRNA can rescue the expression of the downstream target dlx6a in wdr68 mutants. We similarly examined the expression patterns of jag1b, hey1, and grem2 and found ventrally-expanded expression of these otherwise normally dorsally-restricted genes. Strikingly, we found that the BMP agonist ISL could partially rescue lower jaw formation and edn1 expression in wdr68 mutants. To elucidate the mechanism of action, we generated wdr68 deletions by CRISPR/Cas9 gene targeting in the BMP-responsive mouse C2C12 cell line. However, we found no defects in pSmad1/5 accumulation or BMP reporter induction in wdr68 mutant cells relative to non-target (NT1) controls. The Transforming Growth Factor Beta (TGFβ) signaling pathways are also important for several patterning events during early embryonic development, including proper craniofacial development. Notably, TGFβ can interfere with BMP signaling via Smad3 displacement of Smad4 from pSmad1/5 complexes. We examined this potential mechanism and found that TGFβ interference with BMP signaling was greater in wdr68 mutant cells relative to NT1 controls. To determine whether the interference mechanism might also act in vivo, we treated wdr68 mutant zebrafish embryos with the TGFβ signaling inhibitor SB431542 and found a partial rescue of craniofacial development. Together these data suggest an indirect role for Wdr68 in the BMP-Edn1-Jag1b signaling hierarchy through modulating TGFβ interference with BMP signaling.



ZFIN Genetics Index
1. dcaf7
2. tgfb1
3. bmp4
4. edn1
5. jag1b
6. hey1
7. grem2