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Abstract
The vertebrate Six (Sine oculis homeobox) family of homeodomain transcription factors plays critical roles in the development of several organs. Six1 plays a central role in cranial placode development, including the precursor tissues of the inner ear, as well as other cranial sensory organs and the kidney. In humans, mutations in SIX1 underlie some cases of Branchio-oto-renal (BOR) syndrome, which is characterized by moderate-to-severe hearing loss. We utilized CRISPR/Cas9 technology to establish a six1 mutant line in Xenopus tropicalis that is available to the research community. We demonstrate that at larval stages, the six1-null animals show severe disruptions in gene expression of putative Six1 target genes in the otic vesicle, cranial ganglia, branchial arch, and neural tube. At tadpole stages, six1-null animals display dysmorphic Meckel''s, ceratohyal, and otic capsule cartilage morphology. This mutant line will be of value for the study of the development of several organs as well as congenital syndromes that involve these tissues.
HD 084409 Eunice Kennedy Shriver National Institute of Child Health and Human Development, DE 026434 NIDCR NIH HHS, OD 010997 NIH Office of the Director, OD 030008 NIH Office of the Director, R01 HD084409 NICHD NIH HHS , R24 OD030008 NIH HHS , P40 OD010997 NIH HHS , R01 DE026434 NIDCR NIH HHS
zFigure 1. Generation of six1 mutant X. tropicalis line. (a) Schematic outline of genomic six1 locus and Six1 protein. Yellow arrowhead demarcates the relative location of sgRNA target sequence and mutations in this region are expected to disrupt the protein–protein interaction domain (SD). (b) Alignment of wild-type and Xtr.six1em2Horb mutant nucleotide sequence with the −28 bp deletion. Underlined region defines where the mutant and wild-type sequence are identical after the 28 bp deletion. Amino acid sequence is included to show frameshift with resulting stop codon 36 amino acids downstream.
Figure 2. In situ hybridization of six1+/− and six1 −/− X. tropicalis larvae. (a) Left image: pax2 expression in a six1+/− larva. Staining is strong in the otic vesicle (ov), retina (ret), and neural tube (nt). Right image: pax2 expression in a six1−/− larva is indistinguishable from the heterozygous sibling. (b) Left image: eya2 expression in a six1+/− larva. Staining is strong in ov, olfactory placode (olf), VII and IX/X cranial ganglia, nephric mesoderm (ne) and hypaxial muscle precursors (hyp). Middle image: eya2 expression in a six1−/− larva with a partial effect. ov (red arrow) and VIIg staining appears normal, ne and hyp staining appears reduced, and IX/Xg and olf staining is not detectable. Right image: eya2 expression in a six1−/− larva with a severe effect. Only ov staining (red arrow) is detected and ov is smaller than normal. (c) Left image: tbx1 expression in a six1+/− larva. Staining is strong in ventral ov and branchial arches (ba). Middle image: tbx1 expression in a six1−/− larva with a partial effect. ov (red arrow) and ba staining is reduced but not missing. Right image: tbx1 expression in a six1−/− larva with a complete loss of staining in the ov (red arrow) and ba. (d) Left image: dlx5 expression in a six1+/− larva. Staining is strong in ov, ba, and olf. Right image: Staining in ov (red arrow), ba and olf is not detected. (e) Left image: irx1 expression in a six1+/− larva. Staining is strong in ov and nt. Right image: irx1 expression in a six1−/− larva. Staining in ov (red arrow) and nt is not detected. (f) Left image: sobp expression in a six1+/− larva. Staining is strong in ov, VIIg (cg), olf, and nt. Right image: sobp expression in a six1−/− larva. Staining in ov (red arrow), cg, olf, and nt is not detected. (g) Left image: rnf150 expression in a six1+/− larva. Staining is strong in ov, ba, ret, and nt. Right image: rnf150 expression in a six1−/− larva. Staining in ov (red arrow), ret, and nt is not detected, and greatly reduced in ba. (h) Left image: pick1 expression in a six1+/− larva. Staining is strong in ov, ba, ret, and nt. Right image: pick1 expression in a six1−/− larva. Staining in ov (red arrow), ba, ret, and nt is not detected. All views are lateral with anterior to the right and dorsal to the top.
Figure 3.Changes in gene expression after loss of one (six1+/−) or two (six1−/−) alleles of six1. (a) Sections through the otic vesicle (ov) of six1 -heterozygous (left) and six1-null larva processed for eya2 expression. Note the smaller domain of eya2 expression in the null ov. (b) Sections through the facial ganglion placode (VIIg) of six1-heterozygous (left) and six1-null larva processed for eya2 expression. Note the discontinuous nature of eya2 expression in the null VIIg. (c) Sections through the branchial arches (ba) of six1-heterozygous (left) and six1-null larva processed for tbx1 expression. Note the lack of tbx1 expression in the null ba. (d) Sections through the ov of six1-heterozygous (left) and six1-null larva processed for tbx1 expression. Note the lack of tbx1 expression in the null ov. (e) Sections through the ov of six1-heterozygous (left) and six1-null larva processed for irx1 expression. Note the lack of irx1 expression in the null neural tube (nt) and ov. (f) Sections through the ov of six1-heterozygous (left) and six1-null larva processed for sobp expression. Note the lack of sobp expression in the null ventral nt, VIIg, and ov. (g) Sections through the ov of six1-heterozygous (left) and six1-null larva processed for rnf150 expression. Note the lack of rnf150 expression in the null nt and ov. (h) Sections through the ov of six1-heterozygous (left) and six1-null larva processed for pick1 expression. Note the lack of pick1 expression in the null nt and ov. All sections are in the transverse plane oriented with dorsal to the top.
Figure 4.Loss of six1 causes craniofacial dysmorphologies. (a and b) Whole-mount Alcian blue staining of Xenopus tropicalis tadpoles shows that while six1-heterozygotes (six1+/−) present no detectable defects in the branchial arch-derived craniofacial cartilages, six1-nulls (six1−/−) have hypoplastic and deformed Meckel's (m), quadrate (q), ceratohyal (ch), and branchial arch (b) cartilages and hypoplastic otic capsule (oc). (c and d) Transverse section of tadpoles in (a) and (b) at the level of the otic capsule (oc) showing hypoplasia of this structure in six1-null tadpoles (d). hb, hindbrain. Bar. 100 μm. (e and f) Whole-mount Alcian blue staining of E14.5 mouse embryos showing absent otic capsule (oc*) and deformed Meckel's cartilage (m) in Six1-null embryos.
