Figure 4. Both Crabp2a and Cyp26a1 attenuate noise in krox20 expression and facilitate rhombomere boundary sharpening.

(A) Representative Z projections of r3 and r5 (dorsal views, anterior to the left) analyzed by hybridization chain reaction (HCR) for krox20 (r3, rhombomere 3; r5, rhombomere 5; A, anterior; P, posterior). We performed all HCR analyses on raw 3D data and later generated Z-projections and enhanced contrast to simplify presentation. Colors correspond to total krox20 RNA in each cell as measured by total fluorescence intensity bracketed for maximum and minimum for the 5 conditions and represented in a linear scale. (B) Mean-centered analysis of krox20 expression of a subset of cells for r3 and r5 from 3 randomly selected embryos for each condition. (C) Sharpness indices of the r3/r4 boundary (blue) and r4/r5 boundary (red) for embryos from each of the treatment conditions. Bars correspond to s.d. (D) Analysis of the variance in boundary sharpness from the quantification in (C). All perturbations yielded significant differences from wild-type controls, as noted in the Statistical Analysis. Therefore no asterisks were included to indicate columns representing statistical significance.

DOI: http://dx.doi.org/10.7554/eLife.14034.010

Figure 4—figure supplement 1. Single embryos show highly variable mean levels of krox20 expression and boundary sharpness.

(A) Embryos were injected with mRNA coding for the membrane-bound GFP-CAAX and morpholinos (MO) against Crabp2a or Cyp26a1 or mRNA coding for Crabp2a-Myc or Cyp26a1-Myc or water (WT). Ef1α was used as the standard and a homogeneous collection of mRNA from 100 wildtype embryos was used as the reference for the △△Ct method. Experiments were run in triplicates and repeated 4 (four) times. (B) A sharpness index was calculated using the ratio between the measured length of the theoretical sharp boundary and the actual measured length of the boundary of krox20 expression, consistent with previous models (Zhang et al., 2012). In this example, sharpness of the r3-r4 boundary is the ratio of a/b and the index for the r4-r5 boundary is the ratio c/d.

DOI: http://dx.doi.org/10.7554/eLife.14034.011