irx3b activity is requisite for distal early (DE) segment identity, and the absence of retinoic acid (RA) and irx3b activity generates nephrons with a distal late (DL) -only tubule identity. A: Whole-mount in situ hybridization analysis for nephron segment markers (purple) and mhc (red) at the 28 somite stage in wild-type embryos, irx3b morpholino (MO) -injected embryos, lib homozygous mutants, lib homozygous mutants injected with irx3b MO, diaminobenzaldehyde (DEAB) -treated embryos, and DEAB-treated embryos injected with irx3b MO. Embryos are shown in lateral views with anterior to the left. Black brackets and arrows indicated expression domains, and numbers correspond to somite position. irx3b knockdown in wild-types and lib mutants was associated with DE segment abrogation, expansion of the proximal convoluted tubule (PCT) and proximal straight tubule (PST) segments as well as the corpuscle of Stannius (CS) population. In DEAB-treated embryos, which normally only form DE and DL tubule segments, irx3b knockdown was associated with expansion of the DL while the PD is unchanged. B: In DEAB-treated embryos, irx3b expression marked the DE domain that becomes defined by expression of slc12a1. DEAB-treated embryos injected with irx3b MO showed expression of irx3b in the same region of the pronephros, suggesting that DE regionalization had been established, but that in the absence of irx3b DE-specific solute transporter expression does not ensue. C: Summary of nephron segmentation with respect to embryo somite number. Asterisks in the proximal DL of DEAB-treated wild-types injected with irx3b MO indicate partial regionalization that is blocked/prevented in these animals.
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