Fig. 1: CRL3-KLHL4 controls ectodermal patterning and neurulation in the vertebrate head.

a Model of a substrate-engaged CRL3 complex with an example crystal structure of a CUL3-BTB interface (pdb: 4APF) highlighting the positions of developmental disease-causing missense variants in CUL3. b Schematic overview of the IP/MS (i) and the exome sequencing screen (ii) for novel CRL3-BTB complexes with functions during ectodermal differentiation, identifying CRL3-KLHL4 and CRL3-KLHL36 as hits. c Immunoblot analysis of FLAG-IP fractions from HEK293T cells reveals that the KLHL4 p.I287V patient variant reduces CUL3 binding. Ectopically expressed KLHL4 is present in two isoforms (short and long), likely originating from differential usage of start codons (Supplementary Fig. 2n). n = 4 biological replicates, error bars = s.d., CUL3: ****P < 0.0001, HA: P = 0.3640, unpaired t test. d Schematic overview of the neural conversion differentiation paradigm. CNS = central nervous system. e CRL3-KLHL4 is required for neural conversion. Control or KLHL4-depleted CRISPRi iPSCs expressing sgRNA-resistant and doxycycline-inducible wild-type (WT) or patient variant (I287V) KLHL4^FLAG were treated with doxycycline (dox) and neural conversion for 6 days as indicated. Immunoblotting shows decreased expression of CNS and neural crest markers upon KLHL4 depletion that is rescued by WT but not p.I287V KLHL4. ACTIN = loading control. Immunoblots = 2 biological replicates. f Fluorescein-labeled translation-blocking morpholinos were electroporated into the ectoderm at gastrula stage (HH4) by treating one side with a KLHL4 Morpholino (K4) and the other side with a control morpholino (Co). The embryos were incubated until the neural folds stage (HH7-8). The experimental side was compared to the control side, and control embryos were treated with Co on both sides. g Loss of KLHL4 reduces anterior expression Dlx5, Msx1, and Myc-N as shown by HCR fluorescent in situ hybridization. Loss of KLHL4 is rescued by co-expression of WT KLHL4, but not with the patient mutation KLHL4 I287V. The arrows point to the neural folds on the experimental (red) and control (white) side. h Quantification of fluorescence intensities (Dlx5 and Msx1: n = 4–9 embryos as indicated by the number in the respective bar, Dlx5: P = 0.000237; Msx1: P = 0.0004, one-way ANOVA). Scale bar = 250 μm.