Fig 1.
Ccdc40lnks/lnks embryos show delayed then randomized expression of Lefty2 and Lefty1.
Lefty2 (A-D) and Lefty1 (E-H) expression in wild type (A, B, E, F) and Ccdc40lnks/lnks (C, D, G, H) embryos at the 3- to 4- (A, C, E, G) and 5- to 7- (B, D, F, H) somite stages. Lefty1 and Lefty2 is expressed in the L-LPM in wild type embryos at both stages and Lefty1 is also expressed in the midline. In Ccdc40lnks/lnks embryos, expression of Lefty1 and Lefty2 is not detected in the LPM at the 3- to 4-somite stages and expression is randomized between left-sided and bilateral at 5- to 7-somite stages. All embryos with bilateral expression of Lefty1 and Lefty2 showed weaker expression in the LPM. Expression of Lefty1 is weak in the midline of mutants at the 3- to 4-somite stage and mutants with bilateral Lefty1 expression show reduced expression in the midline. (I.) Quantitation of the number and percentage of embryos with absent (grey), left-sided (blue) and bilateral (magenta) Lefty1 or Lefty2 expression in the LPM. Left (L) and Right sides are labeled in panel A.
Fig 2.
LPM expression of Nodal is delayed and then randomized in Ccdc40lnks/lnks embryos.
Nodal expression in wild type (A-E) and Ccdc40lnks/lnks (F-J) embryos at the indicated somite stages. Expression of Nodal in the LPM begins at the 2-somite stage in wild type embryos, increases in 3-somite stage embryos and becomes reduced in 4-somite stage followed by extinction in 5- to 7-somite stage embryos. In Ccdc40lnks/lnks mutant embryos, expression is not detected at the 2-somite stage. Bilateral and weak expression is observed in some embryos at the 3-somite stage. (K.) Quantitation of the number and percentage of embryos with absent (grey), weak left-sided (light blue), strong left-sided (dark blue) or weak bilateral (magenta) Nodal expression in the LPM. Left (L) and Right sides are labeled in panel A.
Fig 3.
Midline signaling in Ccdc40lnks/lnks embryos.
A, B. Expression of Lefty1 in the midline is reduced in Ccdc40lnks/lnks embryos with bilateral (B) compared to L-LPM (A) expression of Lefty1. C-F. Double in situ hybridizations showing normal expression levels of Lefty2 (LPM) with either Brachyury (C, D) or Shh (E, F) in the midline of wild type (C, E) or Ccdc40lnks/lnks mutant embryos with left isomerism (D, F). G-R. Fluorescent immunocytochemistry demonstrating similar dorsal-ventral distribution of the indicated antigens in the spinal neural tube of wild type (G, I, K, M, O, Q) and Ccdc40lnks/lnks mutants (H, J, L, N, P, R). Data are representative of 2–3 embryos per genotype. Left (L) and Right sides are labeled in panel A.
Fig 4.
Asymmetric perinodal expression of Cerl2 and Nodal is delayed and then randomized in Ccdc40lnks/lnks embryos.
Nodal (A-F) and Cerl2 (G-L) expression around the node of wild type (A-C, G-I) or Ccdc40lnks/lnks embryos (D-F, J-L) at the indicated somite stages. M, N. Quantitation of the number and percentage of embryos with right > left (green), left > right (blue) or unbiased (left = right, L = R, magenta) perinodal Nodal (Q) or Cerl2 (R) expression. Left (L) and Right sides are labeled in panel A.
Fig 5.
Modification of the Ccdc40 mutant laterality phenotype with reduced Nodal gene dosage.
A, B. Beta-galactosidase activity in E8.5 NodalLacZ/+ (A) or Ccdc40lnks/lnks;NodalLacZ/+ (B) mutant embryos. Wild type embryos (A) show staining in the left (L) but not right (R) LPM indicating normal situs. (B) Beta-galactosidase staining in the LPM of the majority of Ccdc40lnks/lnks;NodalLacZ/+ mutant embryos was undetectable even with intense perinodal staining. C, D. Lefty2 is expressed in the L-LPM of E8.5 Ccdc40+/+;NodalLacZ/+ embryos but the majority of Ccdc40lnks/lnks;NodalLacZ/+ mutant embryos (D) fail to express Lefty2 in the LPM. E, F. Quantitation of Nodal-LacZ (E) and Lefty2 (F) expression in LPM of wild type and Ccdc40lnks/lnks;NodalLacZ/+ mutant embryos. Normal left-sided expression (Blue) and absent expression (Grey) in the LPM. G-J. Lungs dissected from E11.5 Ccdc40lnks mutant embryos. G. Normal situs in a Ccdc40lnks/lnks mutant with a single lobe of the left lung and four lobes of the right. I. Left isomerism in a Ccdc40lnks/lnks mutant showing bilateral single lobed lungs. The majority of Ccdc40lnks/lnks;NodalLacZ/+ mutant lungs showed either normal situs (not shown) or right isomerism (J). A small percentage of Ccdc40lnks/lnks (not shown) and Ccdc40lnks/lnks;NodalLacZ/+ (H) mutants showed situs inversus. K. Quantitation of the number and percentage of lungs showing normal situs (NS, blue), left isomerism (LI, magenta), right isomerism (RI, grey) or situs inversus (SI, green) in E11.5–15.5 Ccdc40lnks/lnks and Ccdc40lnks/lnks;NodalLacZ/+ mutants.
Fig 6.
Model for Generation of Left Isomerism in Ccdc40 versus Shh pathway mutants.
In 1-somite stage wild type embryos, Cerl2 expression (green) becomes asymmetric with reduced expression on the left side of the node in response to fluid flow. With reduced expression of its antagonist on the left, Nodal activity and expression (dark blue) increases on the left side and decreases on the right. In 2-somite stage wild type embryos, Nodal induce Nodal expression in the L-LPM and by the 3-somite stage robust expression of Nodal along with Lefty1 and Lefty2 (light blue) is detected in the L-LPM and Lefty1 in the midline (light blue). In 4-somite stage embryos Nodal expression is reduced but Lefty1 and Lefty2 are strongly expressed in the L-LPM. Lefty1 expression in the midline and Cerl2 around the node inhibits Nodal signaling in the R-LPM. With lack of nodal flow in Ccdc40lnks and other cilia mutants with intact Shh signaling, Cerl2 and Nodal expression remains symmetric. Continued Cerl2 mediated antagonism of Nodal results in reduced Nodal signaling around the node and Nodal and Lefty1/2 expression in the LPM is delayed till the 3- and 4-somite stages, respectively. In a proportion of embryos where symmetry is not broken, Nodal signaling is reduced and bilateral. This is in contrast to IFT mutants with defects in Shh signaling. In these embryos, Nodal signaling is robustly induced bilaterally compounded by failure to activate the midline barrier.