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Fig. 3 Defects in cloacal morphogenesis and pronephros patterning in nr6a1a−/− and nr6a1a−/− nr6a1b−/− mutants. a Brightfield images of the cloaca at 120hpf in embryos of nr6a1a+/-nr6a1b+/- incross. Classification of the phenotype as normal if the cloaca presents a typical inverted U-shaped with a visible pronephric duct in its terminal part (black arrow), and a visible opening of the gut (black arrowhead); mildly affected if the lumen of the gut reaches the cloaca (black arrowhead) whereas the terminal part of the pronephric duct is not visible (red arrow); severely affected if both ends are not visible (red arrow and red arrowhead). Some severely affected embryos present dilation of the gastrointestinal tract (yellow stars), suggesting imperforation. 26 embryos were selected for genotyping (normal (n = 7); mildly affected (n = 9); severely affected (n = 10)) Embryos showing normal morphology are wildtype or heterozygous for nr6a1a and show variable nr6a1b genotypes. Affected embryos are all nr6a1a-/- with severely affected embryos being homozygous for both nr6a1a and nr6a1b. b Double whole-mount in situ hybridization with the somite boundary marker xirp2a and the pronephros segment markers trpm7, clcnk and gata3 in embryos from nr6a1a+/- incross. The width of one somite is used as reference to measure the length of the expression domain. Expression in nr6a1a-/- mutants compared to wildtype/heterozygotes is increased with a difference of + 2.3 somites (p < 0.0001) for gata3 (n = 4 for nr6a1a-/- and n = 8 for nr6a1a+/-or +/+),+1.3 somites (p = 0.0022) for clcnk (n = 5 for nr6a1a-/- and n = 6 for nr6a1a+/- or +/+), and − 1.4 somites (p < 0.0001) for trpm7 (n = 8 for nr6a1a-/- and n = 12 for nr6a1a+/- or +/+). Created in BioRender. Humaine, G.