Chung HA et al. (2014), Sp8 regulates inner ear development.

XB-ART-48815

Proc Natl Acad Sci U S A 2014 Apr 29;11117:6329-34. doi: 10.1073/pnas.1319301111.

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Sp8 regulates inner ear development.

Chung HA , Medina-Ruiz S , Harland RM .

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A forward genetic screen of N-ethyl-N-nitrosourea mutagenized Xenopus tropicalis has identified an inner ear mutant named eclipse (ecl). Mutants developed enlarged otic vesicles and various defects of otoconia development; they also showed abnormal circular and inverted swimming patterns. Positional cloning identified specificity protein 8 (sp8), which was previously found to regulate limb and brain development. Two different loss-of-function approaches using transcription activator-like effector nucleases and morpholino oligonucleotides confirmed that the ecl mutant phenotype is caused by down-regulation of sp8. Depletion of sp8 resulted in otic dysmorphogenesis, such as uncompartmentalized and enlarged otic vesicles, epithelial dilation with abnormal sensory end organs. When overexpressed, sp8 was sufficient to induce ectopic otic vesicles possessing sensory hair cells, neurofilament innervation in a thickened sensory epithelium, and otoconia, all of which are found in the endogenous otic vesicle. We propose that sp8 is an important factor for initiation and elaboration of inner ear development.

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Species referenced: Xenopus tropicalis
Genes referenced: actl6a ctnna1 dkk1 egr2 epha4 fgf8 foxj1 foxj1.2 gbx2.1 gbx2.2 isl1 maf mafb msx1 neurod1 oc90 otx2 pax2 pax8 pc.1 six1 sox2 sp8 tbx1 tbx2 tuba4b tubb2b
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	Fig. 1. Sp8 mRNA is reduced in ecl mutants. Gross morphology at stage 40 in WT (A) and ecl mutants (B and C). The inner ears in WT (A′) and mutants (B′ and C′) at stage 45. (Scale bar, 500 μm.) (D) Mapping interval of ecl mutants on the scaffold/chromosome 6. Parentheses indicate numbers of recombinants from 4,000 genotyped ecl tadpoles. Whole-mount in situ hybridization in WT (E and G) and ecl mutants (F and H). (Upper) Stage 26; (Lower) stage 33/34. (Inset) sp8 expression pattern in the OV of WT embryos. (I) qPCR analysis in WT and ecl embryos at stages 33/34 and 40. Error bars indicate SEM.
	Fig. 2. Depletion of Sp8 by MO and TALEN phenocopy ecl mutants. (A) Sp8 gene model and sequences of MO and TALEN target. Translation start site is marked in red. Sequences of MO, 5′ UTR, and TALEN binding sites are denoted in italic uppercase, lowercase, and underline, respectively. Domains of Sp, buttonhead box, and zinc finger are colored orange, blue, and green, respectively. (B–E) Phenotypes of sp8 TALEN-injected embryos. (F and G) Phenotypes of sp8 MO-, mRNA-, and TALEN-injected embryos. Number of injected embryos is shown in parentheses. (H) sp8 TALEN target amplicon and Cel1 target. (I) Cel I assay for non-homologous end joining events in- duced by sp8 TALEN. Red arrows indicate mismatch cleavages due to mutation at the target. L is the DNA size marker. (J) Regional injection of sp8 TALEN leads to loss of limbs. UC, uninjected control.

	Fig. 4. Molecular markers of the inner ear in sp8 MO- or TALEN-injected embryos. (A–J) Expression patterns of krox20, epha4, mafb, gbx2, and foxj1.2 in uninjected and sp8 TALEN- or MO-injected embryos. Arrows, uninjected side. Arrowheads, injected side. Anterior is up. (K–T) Expression domains of pax2, msx1, tbx1, sox2, and oc90 in WT and mutant embryos at stage 33/34.
	sp8 (Sp8 transcription factor) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 26, head region, lateral view, anterior left, dorsal up. Inset, enlarged view of otic vesicle staining.

	epha4 (EPH receptor A4) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior up.
	mafb (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior up.
	gbx2 (gastrulation brain homeobox 2, gene 1) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior up.
	foxj1.2 (forkhead box J1, gene 2) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior up.





	Ctnna1 Ab2 expression in the vestibulocochlear ganglion (CVG) and saccular macula (SM) of the otic vesicle of NF stage 46-47 Xenopus tropicalis embryo.

	Fig. S1. Otic vesicle sizes of wild type, ecl 0, and ecl 2 were measured at stage 40. To compare otic vesicle sizes of mutant embryos and wild-type siblings, embryos were anesthetized with 0.005% benzocaine and OV images were taken under the dissection microscope using Image-Pro 5.1.
	Fig. S2. ecl mutation is linked to “old” linkage group 2 of Wells et al. (3). Pooled diploid gynogenotes were genotyped using published SSLP markers and analyzed on 6–12% polyacrylamide gels. This figure shows the cosegregation of the linkage group (LG) 2 centromere-linked marker with the eclipse mutant embryos. Previous linkage group 2 is linkage group 6 in the new X. tropicalis linkage map (3). Whereas eclipse (ec) female possesses polymorphisms from both parents of Nigerian (N) and Ivory Coast (IC) for other linkage groups, half-tetrad (diploid) pools of mutant embryos show only the Nigerian-derived allele.
	Fig. S3. Both sp8 ATGMO and sp8 SBMO phenocopy ecl mutation. When injected bilaterally at the two-cell stage, both a translation blocking MO (ATGMO) and a splice-blocking MO (SBMO) phenocopied ecl mutants. Sp8 SBMO is designed to bind the sp8 exon 1/intron 1 junction. Sp8 MO represents these two MOs. The gross external morphology of these MO-injected embryos was normal but showed otic dysmorphogenesis with various penetrance (A′–D′). Enlarged otic vesicles were devoid of otoconia, whereas relatively normal sized otic vesicles had reduced, scattered, or fused otoconia. These embryos also showed circular and ventral swimming behaviors compared with their uninjected control or standard control MO (Con MO)-injected embryos that exhibit linear and dorsal-up swimming patterns. Phenotypes are summarized in E.
	Fig. S5. Number of Islet-1 positive cells in the sensory epithelium of wild-type and ecl embryos. Islet-1 positive cells were counted in cryosections of wild-type (n = 5) and ecl embryos (n = 5)
	Fig. S6. Gene expression patterns in sp8 depleted or overexpressing embryos. Sp8 MO/TALENs or mRNA were injected unilaterally at the two-cell stage with lacZ lineage tracer. To analyze expression patterns of marker genes, the embryos were fixed at the desired stages, and subjected to in situ hybridization. White arrows indicate endogenous expression of marker genes. Black arrows indicate ectopic or up-regulated expression of marker genes. (A and B) six1 expression, (C and D) msx1 expression, (E and F) neurod expression, and (G and H) sox2 expression. UC, uninjected control.
	Fig. S7. Number of pH3 positive cells and TUNEL positive cells in wild-type and ecl embryos. Wild-type and mutant embryos were collected at stage 30 and fixed in 4 morpholine propane sulfonic acid, EGTA, magnesium sulfate, and formaldehyde and subject to phospho histone 3 antibody staining and TUNEL assays as described (6).
	Fig. S8. Genetic interaction of sp8 and pax2, pax8, and fgf8 was evaluated by oc90 expression in single-injected or coinjected embryos. No strong interactions or synergies were observed. The number of analyzed embryos is in parentheses. Serial dilution of each factor determined the lowest dose of mRNA, which induced ectopic otic tissues. Microinjection of pax2, pax8, or fgf8 alone resulted in the development of OVs that were reduced or enlarged in size, but did not induce ectopic otic tissues. A higher concentration of pax2 has a potential to produce ectopic OV, which partially agrees with previous studies (1). When 12.5 pg of pax2 was coinjected with sp8 mRNA (12.5 pg), there was an approximately fourfold increase in the number of embryos that show ectopic oc90 expression domain, compared with a single injection of 12.5 pg of either sp8 or pax2 (which usually led to otic vesicle reduction). At higher pax2/sp8 combined con- centrations, however, the domain of oc90 expression more frequently developed enlarged otic tissues. Unlike pax2/sp8 coinjection, 7% of pax8/sp8-coinjected embryos showed ectopic otic tissues and this is similar to sp8 (6%) single injections. Pax8/sp8 coinjection resulted in reduction of oc90 expression in 33% of embryos, which is higher than sp8 (18%) and pax8 (12%) single injections. Fgf8/sp8 coinjection most frequently reduced oc90 expression, more frequently than single injections of sp8 and fgf8, 18% and 15%, respectively.
	Fig. S9. Ectopic otic vesicle formation by activation of Wnt signaling is reduced when sp8 expression is down-regulated. Ectopic otic vesicle formation was evaluated by oc90 expression. Embryos incubated with 6-bromoindirubin-3′-oxime (BIO) from stage 13/14 to stage 28 were subject to in situ hybridization analysis. Whereas control embryos showed normal oc90 expression (A), BIO-treated embryos showed increased or ectopic oc90 expression (B–D). To address whether Wnt-induced ectopic otic vesicle formation requires sp8, embryos receiving sp8 ATGMO or sp8 TALEN injection at the two-cell stage were incubated with (E) or without (F) BIO from stage 13/14 to stage 28. Interestingly, BIO-induced ectopic otic vesicles were not observed in sp8 MO or TALEN injected embryos (E), indicating sp8 is required for the Wnt-mediated ectopic otic vesicle formation. Black arrows indicate ectopic oc90 expression. In addition, we observed that oc90 expression was retained on the dkk1-injected side (H) in comparision to the uninjected side (G), suggesting that the absence of sp8 expression in dkk1- injected embryos is not due to the loss of otic vesicle tissue.
	oc90 (otoconin 90) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, head region, anterior left, dorsal up.

References [+] :

Bell, Sp8 is crucial for limb outgrowth and neuropore closure. 2003, Pubmed