Determination of left/right asymmetric expression of nodal by a left side-specific enhancer with sequence similarity to a lefty-2 enhancer

Abstract

The nodal gene is expressed on the left side of developing mouse embryos and is implicated in left/right (L-R) axis formation. The transcriptional regulatory regions of nodal have now been investigated by transgenic analysis. A node-specific enhancer was detected in the upstream region (-9.5 to -8.7 kb) of the gene. Intron 1 was also shown to contain a left side-specific enhancer (ASE) that was able to direct transgene expression in the lateral plate mesoderm and prospective floor plate on the left side. A 3. 5-kb region of nodal that contained ASE responded to mutations in iv, inv, and lefty-1, all genes that act upstream of nodal. The same 3. 5- kb region also directed expression in the epiblast and visceral endoderm at earlier stages of development. Characterization of deletion constructs delineated ASE to a 340-bp region that was both essential and sufficient for asymmetric expression of nodal. Several sequence motifs were found to be conserved between the nodal ASE and the lefty-2 ASE, some of which appeared to be essential for nodal ASE activity. These results suggest that similar transcriptional mechanisms underlie the asymmetric expression of nodal and of lefty-2 as well as the earlier expression of nodal in the epiblast and endoderm.

Figure 1

Localization of a NDE in the upstream region of nodal. (A) The structure of the nodal gene, with the arrow indicating the direction of transcription, is shown above those of three lacZ constructs. (E) EcoRI; (No) NotI; (Ns) NsiI; (S) SalI. The blue box indicates the lacZ gene; the open black and closed blue circles indicate the nodal proximal promoter and the hsp68 promoter, respectively. The NDE is shown in pink. In the summary of the expression data in this and subsequent figures, #1 indicates the number of embryos positive for X-gal staining (numbers in parentheses refer to the number of embryos showing the typical staining pattern), #2 indicates the number of embryos that contained the lacZ transgene but were negative for X-gal staining, and #3 indicates the total number of injected embryos that were recovered at E8.2. (L-LPM) Left LPM. (B) X-gal staining of transgenic embryos harboring constructs 5′-1, 5′-2, or NDEhsp. Weak staining in a few cells of the left LPM (arrowhead) was apparent with 5′-1. The 5′-2 construct gave rise to expression in the node, whereas NDEhsp yielded expression in the node and in ectopic sites. The left (L)–right (R) axis is indicated.

Figure 2

Mapping of the NDE in the upstream region of nodal. (A) Structures and activities of various lacZ constructs. (B) Posterior views of X-gal staining in embryos transgenic for the indicated constructs. X-gal staining in the node was observed with SN5.0, Δ1–33, Δ1–70, Δ2–1, and Δ2–77, but not with NS1.5, Δ1–21, or Δ2–37.

Figure 3

Localization of a left side-specific enhancer (ASE) in intron 1 of nodal. (A) Structures and activities of six constructs containing various portions of nodal downstream plus the proximal promoter region. The approximate location of the ASE is indicated by the red oval. (B) Anterior and posterior views as well as transverse sections of X-gal staining in embryos transgenic for 3′-1 or 3′-1hsp. In both types of transgenic embryos, the somatopleure (so) and splanchnopleure (sp) on the left side are positive for staining. In the embryo harboring 3′-1hsp, left-sided staining was also apparent in the PFP.

Figure 4

Expression of 3′-1, 3′-1hsp, and 5′-2 in permanent transgenic lines. (A) Permanent transgenic lines harboring 3′-1, 3′-1hsp, or 5′-2 were generated, and the expression of the transgenes was examined at various developmental stages. Lateral views are shown for embryos at E5.5, E6.5, E6.75, and E7.5; posterior views are shown for E8.0 embryos. The arrow indicate the anterior visceral endoderm. A and P indicate the anteroposterior axis. (B) Transverse sections of E6.5 embryos transgenic for 3′-1hsp. In the embryo harboring 3′-1hsp, X-gal staining was apparent in the posterior epiblast and in the anterior visceral endoderm (arrow).

Figure 5

Mapping of ASE to a 340-bp region of intron 1. (A) Structures and activities of various deletion mutants of 3′-1hsp. The approximate location of ASE is shown by the red oval. (B) X-gal staining in representative embryos. Constructs 5′Δ2–30, 5′Δ1–20, 3′Δ2–1, and 3′Δ107 induced left-sided X-gal staining in LPM and PFP, whereas 5′Δ1–12 and 3′Δ2–7 failed to give rise to X-gal staining.

Figure 6

ASE is essential and sufficient for L-R asymmetric expression of nodal. (A) Structures and activities of the lacZ constructs 3′-1, 3′-1ΔASE, 5′-1ASE, and 501/301hsp. (B) X-gal staining patterns of representative transgenic embryos. Constructs 3′-1, 5′-1ASE, and 501/301hsp yielded left-sided staining in the LPM, whereas 3′-1ΔASE did not. Construct 501/301hsp also gave rise to left-sided staining in PFP.

Figure 7

Response of the 3′-1hsp construct to iv, inv, and lefty-1 mutations. Expression of the 3′-1hsp transgene was examined in iv/iv (A), inv/inv (B), and lefty-1^−/− (C) mutant embryos. Anterior and posterior views, as well as a transverse section, are shown for each transgenic embryo. In iv/iv embryos (A), the staining in the LPM and PFP was either left-sided, right-sided or bilateral. In inv/inv embryos (B), the staining in the LPM and PFP was right-sided (embryo at left) or bilateral (embryos at middle and right). In lefty-1^−/− embryos, the staining in the LPM and PFP was initially normal (embryo at left). At later stages (embryos at middle and right), however, the staining appeared on the right LPM. The staining in the PFP, increased and became bilateral (C). In lefty-1^−/− embryos harboring 3′-1 transgene (D), X-gal staining in the PFP, which was not detected in the wild-type embryos (Fig. 4A), became apparent. X-gal staining pattern in LPM was similar to that in C. lacZ expression in the PFP always preceded that in the right LPM.

Figure 8

Comparison of the structures of nodal ASE and lefty-2 ASE. (A) Structural organization of nodal ASE (340 bp) and lefty-2 ASE (350 bp). Conserved sequence motifs are indicated by ovals of various colors. (B) Nucleotide sequences of nodal ASE and lefty-2 ASE. The positions of nodal deletion mutants shown in Figs. 5 and 9 are indicated by arrows.

Figure 9

Dissection of the 340-bp ASE region. (A) Structures and activities of 501/301hsp-based deletion mutants. Each nodal fragment was linked to the hsp68 promoter and lacZ. (B) X-gal staining patterns of representative transgenic embryos.

Figure 10

Models for transcriptional regulation of nodal (top) and of lefty-2 (bottom). The asymmetric expression of both nodal and lefty-2 is determined by similar ASE elements. LSE, a putative weak left side-specific enhancer, is also shown. (See text for further details).