Fig. 7: Modulation of CDC42 signaling by a ubiquitin-based effector-to-inhibitor switch coordinates ectodermal patterning and neurulation in the vertebrate head.

Model depicting how CRL3-KLHL4-mediated restriction of anterior CDC42 signaling regulates ectodermal patterning. Canonically, CDC42 is activated by specific GEFs, including the GIT1-β-PIX complex, and inactivated by dedicated GAPs. The activated, GTP-bound form of CDC42 can bind and stimulate effector proteins, including PAK1, to mediate cytoskeletal and cell polarity-based downstream signaling. (i) During normal development, tissue-specific expression of CRL3-KLHL4 allows restriction of these canonical CDC42 signaling pathways in the anterior ectoderm of the gastrula. This inhibition relies on the CDC42-activating complex GIT1-β-PIX, which recruits CRL3-KLHL4 to its substrate PAK1. Ubiquitylation converts this canonical CDC42 effector kinases into a CDC42 inhibitor to restrict downstream signaling. This negative regulation ensures proper spatial ectodermal cell-fate specification, neural plate folding, and ultimately faithful development of the skin, the nervous system, and the craniofacial complex. (ii) Loss of CRL3-KLHL4 activity results in aberrant development. Hypomorphic mutations in CUL3 or KLHL4 impair CRL3-KLHL4 complex assembly and PAK1 ubiquitylation. This causes reduction of the negative feedback system and excessive CDC42 downstream signaling, which compromises ectodermal patterning, neural plate folding, and contributes to central nervous system and craniofacial phenotypes observed in patients with CUL3 and KLHL4 variants. Model was in part created using BioRender.com.