Initiation and propagation of posterior to anterior (PA) waves in zebrafish left-right development

Abstract

One of the most highly conserved steps in left-right patterning is asymmetric gene expression in lateral plate mesoderm (LPM). Here, we quantitatively describe the timing of the posterior to anterior (PA) wave-like propagation of zebrafish southpaw (Nodal) and pitx2 in LPM and lefty1 in the midline. By altering the timing of the PA wave, we provide evidence that the PA wave in the LPM instructs brain asymmetry. We find that initiation of pitx2 in LPM and lefty1 in midline depends on Southpaw, and that casanova (sox32) and two Nodal inhibitors, lefty1 and charon, have distinct roles upstream of PA wave initiation. Surprisingly, Casanova, endoderm and Kupffer's Vesicle are not required for normal timing of southpaw initiation and PA propagation. In contrast, lefty1 morphants display precocious asymmetric initiation of southpaw with an intrinsic left-hand orientation, whereas charon morphants have premature initiation without LR orientation, indicating distinct roles for these Nodal antagonists.

Fig. 1

Quantification of the Posterior to Anterior propagation of spaw, pitx2 and lft1. (A) At 15 SS, the anterior boundary of LPM spaw was at somite 7. Green arrowhead: anterior expression boundary of spaw. Red arrowhead: somite corresponding to the anterior expression boundary of spaw, pitx2 or lft1. (B) At 15 SS, the anterior boundary of LPM pitx2 expression was at somite 10. (C and D) lft1 expression in the midline was bi-phasic. (C) In the first phase, static lft1 expression was present in posterior midline before the LPM spaw expression at 10 SS. At about 15 SS, (D), the second propagation phase of lft1 expression started. Green arrow: spaw expression. Blue arrowhead: anterior expression boundary of lft1 expression. (A and B) anterior to the left. (C and D) anterior to the top. (E) Quantitative analysis of PA propagation of spaw (n=213), pitx2 (n=74) and lft1 (n=124). X axis is the development stages defined by total number of somite pairs. Y axis is the position of anterior expression boundary of each gene. A negative number means the anterior expression domain is anterior to the somitogenesis region. Almost 100% of embryos had detectable expression of spaw and pitx2 at stage 13 SS, 14 SS respectively. Error bars are ± standard error.

Fig. 2

The PA propagation of pitx2 and lft1 depends on spaw function. In WT (A) pitx2 is expressed in LPM (black arrow) and in Rohon-Beard cells (black arrowhead). In spaw morphants (B), pitx2 is not expressed in LPM but is expressed in Rohon-Beard cells (black arrowhead). Embryos were at 18 SS; anterior to left. Spaw expression is normal in WT (C) and spaw morphants (D) in the peri-KV bilateral domains (green arrow). In contrast, left-sided LPM spaw expression (green arrowhead) and lft1 midline expression (blue arrow) seen in WT (C) is absent in spaw morphants (D). Embryos were 16 SS; anterior to the top.

Fig. 3

Distinct bilateral phenotypes in cas, cha and lft1 morphants. (A) Standard analysis of LPM spaw expression categorized as left-side expression (Red), bilateral expression (green), absent expression (blue) and reversed expression (yellow). (B–D) Bilateral spaw expression in LPM is categorized as (B) the left side boundary more anterior than that of the right side (left side leading); (C) both sides with equal anterior boundaries (equivalent); (D) right side boundary was more anterior than that of the left side (right side leading). Green arrowhead: leading side spaw expression boundary. (E) Quantification of cas, cha and lft1 morphants with bilateral phenotype. (F–H) lft1 morphants retain LR bias at 9–10 SS. (F) spaw expression in the left LPM (n=28) (G) spaw expression in the right LPM (n=5) (H) spaw was not expressed in WT (n=86/88) at this stage. Green arrow: spaw expression.

Fig. 4

cas, cha and lft1 morphants have normal PA wave rates but distinct timing of initiation. (A–D) PA propagation profiles of wildtype and MO injected embryos. In each graph, the green line is wild type; red line morphants. Error bars were ± standard error. (A) Control (Ctrl) MO (n=104) vs WT (n=74). (B) cas MO (n=111) vs WT (n=102). (C) lft1 MO (n=193) vs WT (n=241). (D) cha MO (n=193) vs WT (n=256).

Fig. 5

Correlation between orientation of the leading edge of spaw in the LPM and the orientation of lft1 in the brain in lft1 morphants. (A) Majority of the morphants had bilaterally equivalent spaw expression in LPM (n=16) and bilateral lft1 expression in brain. (B) In the morphants with stronger spaw expression in the left LPM, lft1 expression was observed only in the left side of the brain (n=12). (C) In morphants with stronger spaw in the right LPM, lft1 expression was observed only in the right side of the brain (n=3). Red dot line: midline; blue arrow: lft1; green arrow: spaw.