Nubp2 is required for cranial neural crest survival in the mouse

Abstract

The N-ethyl-N-nitrosourea (ENU) ←forward genetic screen is a useful tool for the unbiased discovery of novel mechanisms regulating developmental processes. We recovered the dorothy mutation in such a screen designed to recover recessive mutations affecting craniofacial development in the mouse. Dorothy embryos die prenatally and exhibit many striking phenotypes commonly associated with ciliopathies, including a severe midfacial clefting phenotype. We used exome sequencing to discover a missense mutation in nucleotide binding protein 2 (Nubp2) to be causative. This finding was confirmed by a complementation assay with the dorothy allele and an independent Nubp2 null allele (Nubp2^null). We demonstrated that Nubp2 is indispensable for embryogenesis. NUBP2 is implicated in both the cytosolic iron/sulfur cluster assembly pathway and negative regulation of ciliogenesis. Conditional ablation of Nubp2 in the neural crest lineage with Wnt1-cre recapitulates the dorothy craniofacial phenotype. Using this model, we found that the proportion of ciliated cells in the craniofacial mesenchyme was unchanged, and that markers of the SHH, FGF, and BMP signaling pathways are unaltered. Finally, we show evidence that the phenotype results from a marked increase in apoptosis within the craniofacial mesenchyme.

Keywords: Cilia; ENU mutagenesis; Neural crest; Nubp2.

Figure 1:. The Dorothy mutants have variable craniofacial phenotypes.

Whole-mount and histological analysis of E16.5 control (A-C) and dorothy (D-F) mutant embryos. Mutants consistently present with midfacial clefting, micromelia and oligodactyly. The nasal cartilage (black arrows) is underdeveloped and ectopically located. Accumulation of blood or other fluid under the epithelium are also often noted (red asterisk). (F) Coronal sections also reveal cleft palate (black asterisks). Tooth buds are present but tooth development appears delayed (yellow dotted outlines). (G-N) Dorothy mutants with more severe phenotypes are developmentally arrested at the start of organogenesis (G-M), and often show signs of pervasive hematoma (red arrows). (Scale bars in B,C,E,F = 1mm, G-N = 200μm).

Figure 2:. The dorothy mutation is an allele of Nubp2.

(A) Amino acid sequence of NUPB2 in multiple model organisms. Nonpolar side chains (valine and isoleucine) at the position of the affected residue (red box, highlighted in yellow) are heavily conserved and located in close proximity to known functional Iron-interaction sites (highlighted in purple). In the dorothy mutant (p.Val209Asp; NUBP2^V209D), this is replaced with an aspartate residue (red) harboring an electrically charged side chain. (B) Sanger sequencing confirms that Nubp2:c.626T>A is homozygous in embryos with the dorothy phenotype. (C,D) transfection of HEK293T cells with pAcGFP-C1-hNUBP2 (C) and pAcGFP-C1-hNUBP2^DOR (D) expression constructs revealed that the corresponding fusion proteins were both expressed and localized in roughly the same pattern, suggesting that the Nubp2^dor mutation does not preclude translation of NUBP2 protein.

Figure 3:. Conditional ablation of Nubp2 in the neural crest lineage causes midfacial defects and recapitulates the craniofacial phenotype of the dorothy mutant.

(A-H) Wnt1-cre; Nubp2^cKO embryos recovered at E15.5 have severe craniofacial abnormalities including midline clefting and mandibular hypoplasia. (C, G) H&E stained coronal sections demonstrate palatal shelves fail to elevate. (D, H) higher magnification reveals that the cartilage primordium of the nasal septum is severely truncated (black dotted outline), while cartilage primordium of the nasal capsule lateral to the septum (yellow dotted outline) appears to be completely absent in the mutant. (I-L) At E9.5 Wnt1-cre; Nubp2^cKO mutants are indistinguishable from wild-type (M-P) but at E10.5 the phenotype becomes apparent with hypoplastic nasal prominences, maxillary and mandibular prominences). (Scale bars in C, G = 500μm; D,H=50μm; I-P 100μm.)

Figure 4:. Craniofacial mesenchymal cells have normal primary cilia and centrioles.

(A-H) representitive fields showing immunostaining with fluorescent antibodies against γ-Tubulin and Arl13b in E9.5 craniofacial mesenchyme. No gross abnormalities are observed in mutant cilia (white arrows) or centrioles (white arrowheads). Mitotic cells can be observed with two centrioles (white M). (I) a minor, but statistically significant increase in the proportion of cilia per cell was measured in mutants. (J) There was no observed difference in the proportion of centrioles per cell in mutants. Scale bars: 5μm.

Figure 5:. Major developmental signaling pathways are unaffected in Wnt1-cre; Nubp2^cKO embryos.

(A-L) Whole mount in situ hybridizations of E9.5 embryos and (M) normalized TPM values derived from RNA-sequencing of dissected E9.5 heads show that the SHH (A-D), FGF (E-H) and BMP (I-L) signaling pathways are unaltered at E9.5 in Wnt1-cre; Nubp2^cKO embryos (Scale bars =100μm).

Figure 6:. Loss of Nubp2 leads to decreased survival of craniofacial neural crest.

(A-E’) immunofluorescent staining for Phosphorylated Histone 3 (PHH3) at E9.5 (A-C; p=0.453) and E10.5 (D-F; p=0.060) shows no significant change in the proportion of proliferating Wnt1-cre; Nubp2^cKO craniofacial mesenchymal cells at either stage. (G-L) in contrast, staining for CC3 at E9.5 (G-I; p=0.040) shows a slight increase in apoptotic mesenchyme and by E10.5 (J-L; p<0.0001) there is a striking and statistically significant increase. Scale bars: 100μm.