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. 2023 Aug 17;26(9):107665. doi: 10.1016/j.isci.2023.107665

Figure 7.

Figure 7

Loss-of-function experiments for tgfbi in the context of otic development

(A) Xenopus 16-cell blastomeres that are fated to primarily contribute to neural crest (NC) and pre-placodal ectoderm (PPE) are labeled in green, and were microinjected with antisense morpholino oligonucleotides (MO) with or without mRNA encoding human TGFBI to which the MOs do not bind. Left (L) and right (R) sides of embryo. Uninjected half of the embryo was used as control for phenotype assessment. In the right panel, sequences of the two alleles of Xenopus laevis tgfbi on the long (.L) and short (.S) chromosomes and human TGFBI are aligned. Red and blue regions indicate sites for MO binding, blue arrow marks the translation start site (ATG). hTGFBI 5′ sequence cannot bind the MOs and therefore could be used to rescue the effects of the MOs.

(B and C) Targeted MS analysis shows Tgfbi protein is not detected in OVs dissected from the MO injected sides of stage 34 larvae. Abundance of the reference protein Gapdh does not change significantly following Tgfbi KD (paired Student’s t test, p > 0.05) (C) MO injected embryos cultured to stage 32 and processed by ISH for dlx5 and pax2. While OV gene expression was normal on the control side (black arrow), it was greatly reduced on the MO-injected side (red arrow) of same larva in a large percentage of the cases (n = 63 larvae for dlx5, n = 39 larvae for pax2). Embryos injected with Tgfbi MOs plus the MO-insensitive human TGFBI mRNA showed a significantly reduced percentage of reduced dlx5 expression (p < 0.05, Chi-Squared test), demonstrating rescue of the morphant phenotype. MO: morpholino injection; MO Rescue: MOs + hTGFBI mRNA injection. Bar indicates 200 μm.

(D) Larvae were vibratome sectioned to measure OV and luminal volumes. In the image shown, dlx5 expression was reduced in the OV on the MO injected side (red arrow) compared to the control side (black arrow). hb, hindbrain.

(E) Otic and luminal volumes of MO injected sides represented as percent change in otic volume compared to control (uninjected) sides of the same larvae (paired Wilcoxon signed rank test, ∗, p < 0.05, Data are represented as mean ± SEM).