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. 2020 Oct 13;9:e59469. doi: 10.7554/eLife.59469

Figure 3. urp2 expression is Pkd2l1-independent and is important for the Reissner fiber-dependent straightening of the embryonic posterior axis.

(A) Adult wild-type and pkd2l1icm02/icm02 siblings were incrossed to generate related clutches that were analyzed at 30 hpf. (B) Representative pictures of wild-type (left) and pkd2l1icm02/icm02 embryos (right) at 30 hpf. Note that mutant embryos develop a straight posterior axis. Scale bar: 0.5 mm. (C) Representative immunohistochemistry for the Reissner fiber imaged from the spinal cord of a wild-type (top, one representative embryo out of 20) and a pkd2l1icm02/icm02 embryo (bottom, one representative embryo out of 20). Note that the Reissner fiber forms properly in the mutant. Scale bars: 10 µm. (D) qRT-PCR analysis of mRNA levels of pkd2l1, urp2, and urp1 in wild-type (white) and pkd2l1icm02/icm02 embryos (grey). Data are represented as mean ± SEM. N = 6 independent replicates for wild-type and four for pkd2l1icm02/icm02. Each point represents a single experimental replicate. ns p>0.05, *p<0.05 (unpaired t-test). (E) Representative pictures of scospondinicm15/icm15 mutant embryos at 48 hpf after one cell stage injections of a control mRNA alone or of a mix containing a control mRNA and urp2 mRNA (middle and bottom). Note that upon control injections, scospondinicm15/icm15 mutants display at typical curled-down phenotype, while urp2 overexpression can lead to straightened (middle) or slightly curled-up posterior axis (bottom). Scale bar: 0.5 mm. (F) Quantification at 48 hpf of curled-down frequency in embryos obtained from scospondinicm15/+ incrosses upon control mRNA injections (n = 70 curled-down animals out of 268) or urp2 mRNA overexpression (26 curled-down embryos out of 242). Data were collected from four independent clutches and represented as mean ± SEM. **p<0.01 (paired t-test). (G) Injected embryos were genotyped at 48 hpf based on the loss of a restriction site in the scospondin mutant allele leading to a band resistant to digestion (-/-). While mutant animals are exclusively curled-down in control conditions, urp2 mRNA overexpression leads to the detection of mutant animals displaying a straight body axis (blue arrow). See also Figure 3—figure supplement 1.

Figure 3—source data 1. Data for Figure 3D.

Figure 3.

Figure 3—figure supplement 1. urp2 overexpression recues body axis curvature defects in scospondin mutants.

Figure 3—figure supplement 1.

Summary of straight, curled-up and curled-down phenotypes detected in wild-type (+/+), scospondinicm15/+ (+/-) and scospondinicm15/icm15 mutant embryos (-/-) at 48 hpf after one cell stage injection of control mRNA (A) or control +urp2 mRNA (B). Note that two mutant (-/-) embryos were detected as straight and two as curled-up upon urp2 overexpression. Data were collected over three independent clutches.