Nodal activity in the node governs left-right asymmetry

Abstract

Nodal is expressed at the lateral edges of the mouse node, but its function in this "organizer" tissue remains unknown due to the early lethality of Nodal mutant embryos. Here we used a genetic strategy to selectively remove Nodal activity from the node. Embryos lacking Nodal in the node fail to initiate molecular asymmetry in the left lateral plate mesoderm and exhibit multiple left-right patterning defects. Nodal may also act as a short-range signal to establish a functional midline barrier. Our findings confirm that the mouse node is instrumental in initiating left-right axis specification and identify Nodal as the key morphogen regulating this process.

Figure 1

Characterization of the Nodal node enhancer. Ventral (C–J) and lateral (K) views of approximately 8.0-d embryos at the various somite (s) stages indicated. (A) Map of Nodal locus illustrating location of node enhancer element (red circle) and three LacZ reporter constructs used to make stable transgenic lines. Black box indicates position of exon 1 (K, Kpn I; B, Bgl II; N, Not I). (C) Nodal is expressed in the outermost ventral cells of the node at the 2-somite stage as assessed by the Nodal^lacZ reporter allele. Transgenic lines containing 2.7 kb (A,D) and 0.35 kb (A,E) of Nodal genomic sequence express β-gal reporter in the outermost ventral cells of the node. Removal of the 0.35 kb from the 2.7-kb transgene eliminates β-gal expression from the outermost cells of the node (A,F); some staining is seen in the central cells of the node. (B) Partial sequence of the minimal 0.35-kb fragment that drives Nodal expression in the node. Alignment of mouse and human sequences showing predicted Foxa2 binding site. Mismatches between the second and third bases and the Foxa2 consensus are compatible with Foxa2 binding (Overdier et al. 1994). (G) Whole-mount in situ hybridization showing Cre expression in the node of a transgenic line expressing Cre under the control of Tg 2.7 (the 2.7-kb node element). (H) Embryo carrying the Tg 2.7-Cre transgene and an allele of the Rosa-26 conditional reporter. Only a few cells show β-gal activity in the node (white arrow). At the 3-somite stage, Cre mRNA is expressed in the node (I), and Cre activity is detected throughout the outermost ventral cells of the node as visualized by β-gal activity (J). By the 6-somite stage, descendants of Nodal-expressing cells in the node are found exclusively in the notochordal plate (K‘). By 9.5 d (L) and 10.5 d (M), the node descendants are found in the notochord posterior to the hindlimb. No descendants are found in the gut or floor plate.

Figure 2

Targeted deletion of the Nodal node enhancer. (A) Map of Nodal genomic locus. (B) Targeting vector designed to replace 2.7 kb of Nodal sequence with a single loxP site (black triangle). (C) Targeted allele. (D) Recombined allele after Cre-mediated excision of the hygro selection cassette. (E) Southern hybridization of KpnI-digested DNA prepared from tail biopsies of heterozygous intercross offspring at weaning. DNA was hybridized with a 5′ external probe (A). (F) PCR analysis of yolk sac DNA prepared from 8.0-dpc embryos. Primers C2/D2 amplify a wild-type band of 220 bp, and primers A3/D2 amplify a 320-bp mutant band. K, KpnI; B, BglII.

Figure 3

Left-right patterning defects of the viscera of Nodal^Δ/− deletion mutants. Organs were dissected from control and deletion pups on the day of birth. Left (L) and right (R) are as indicated in A and B. Normally the stomach is situated on the left-hand side of the body (A), but in the deletion mutants the stomach position is randomized such that half the mutants have stomachs on the right (B). The spleen is apposed to the stomach wall (C). In mutants, the spleen is greatly reduced in size (D). In the normal lung, there is one lobe on the left and four on the right (E). In the mutants, there were three or four lobes on both sides, indicating a right pulmonary isomerism (F). At 9.5 d, expression of α-cardiac actin mRNA in the heart tube illustrates normal heart looping to the right (G). In Nodal^Δ/− mutants, looping was randomized, with 3/7 showing rightward and 4/7 showing leftward looping (H). In normal littermates, the apex of the heart points to the left (I), yet in mutants the heart apex is ambiguously positioned such that a proportion point to the left, some to the middle (J), and others to the left. Sections through mutant hearts reveal septation defects between the two atria (J). A, accessory lobe; CA, common atrium; Ca, caudal lobe; Cr, cranial lobe; L, left lobe; LA, left atrium; LV, left ventricle; M, medial lobe; OT, outflow tract; RA, right atrium; RV, right ventricle; Vn, ventricles.

Figure 4

Molecular asymmetry is not established in the lateral plate mesoderm of Nodal^Δ/− deletion mutants. Whole-mount in situ hybridization (A–C,F–L) and LacZ reporter staining (D,E) of 7.5-d (A–C) and 8.0-d (D–L) embryos. Lateral views with anterior to the left (A–C), caudal views (D–H), and rostral views (I–L) are shown. At the late head fold stage, Nodal is expressed in the node at the distal tip of the embryo (A); transverse sections through the node show that Nodal is expressed in the outermost ventral cells (A′;). In embryos homozygous for the Nodal^Δ/Δ allele, Nodal expression is downregulated in the node (B). In embryos trans-heterozygous for the deletion allele and a Nodal null allele (Nodal^Δ/−), Nodal expression is either entirely absent or negligible in the node (C). Control embryos between 2 and 8 somites express the Nodal^lacZ reporter in the node and the left LPM (D). In embryos carrying a copy of the deletion allele and the Nodal null reporter allele (Nodal^Δ/LacZ), Nodal^lacZ is only expressed in the node (E). Similarly Nodal mRNA is expressed in the node and left LPM in control embryos (F), but in Nodal^Δ/Δ homozygotes, low levels of Nodal are detected in the node yet normal levels of Nodal expression are observed in the left LPM (G). Reduced Nodal expression levels are also found in Nodal^Δ/− nodes, and Nodal expression is absent from the left LPM (H‘). Lefty2 is expressed in the left LPM and the left prospective floor plate of control embryos (I) but fails to be induced in either domain in the deletion mutants (J). Pitx2 is expressed in the head mesenchyme, body wall, and left LPM of control embryos (K,K′;). In the deletion mutants, Pitx2 is expressed in the head mesenchyme and body wall (L) but is absent from the left LPM (L‘). Normal expression of Pitx2 in the left-hand side mesenchyme of the developing heart (arrow in K") is absent in the deletion mutants (L"). llpm, left lateral plate mesoderm; n, node; nf, neural fold; nt, neural tube; pfp, prospective floor plate. Black arrows indicate Nodal expression domain.