The mouse homeobox gene Noto regulates node morphogenesis, notochordal ciliogenesis, and left right patterning

Abstract

The mouse homeobox gene Noto represents the homologue of zebrafish floating head (flh) and is expressed in the organizer node and in the nascent notochord. Previous analyses suggested that Noto is required exclusively for the formation of the caudal part of the notochord. Here, we show that Noto is also essential for node morphogenesis, controlling ciliogenesis in the posterior notochord, and the establishment of laterality, whereas organizer functions in anterior-posterior patterning are apparently not compromised. In mutant embryos, left-right asymmetry of internal organs and expression of laterality markers was randomized. Mutant posterior notochord regions were variable in size and shape, cilia were shortened with highly irregular axonemal microtubuli, and basal bodies were, in part, located abnormally deep in the cytoplasm. The transcription factor Foxj1, which regulates the dynein gene Dnahc11 and is required for the correct anchoring of basal bodies in lung epithelial cells, was down-regulated in mutant nodes. Likewise, the transcription factor Rfx3, which regulates cilia growth, was not expressed in Noto mutants, and various other genes important for cilia function or assembly such as Dnahc5 and Nphp3 were down-regulated. Our results establish Noto as an essential regulator of node morphogenesis and ciliogenesis in the posterior notochord, and suggest Noto acts upstream of Foxj1 and Rfx3.

Fig. 1.

Laterality defects in Noto^GFP mutants (A–F) Situs of visceral organs in wild-type (A and C) and mutant (B and D–F) newborns (A and B) and E16.5 embryos (C–F). Heart looping (G and H) and embryonic turning (I and J) in wild-type (G and I) and mutant (H and J) embryos are shown. Age of the embryos is indicated to the left. (K–Z) Whole-mount in situ hybridization of wild-type and three different representative mutant embryos with probes (indicated to the left) for asymmetrically expressed genes. R ↔ L indicates the orientation of the embryos. Genotypes are indicated at the top.

Fig. 2.

Node/PNC defects in Noto^GFP mutants. (A–H) Pictures of the node after whole-mount in situ hybridization with probes for nodal (A–D) and dante (E–H). (I–Q) Eosin-counterstained sections of plastic-embedded embryos after whole-mount in situ hybridization. (R–U) False-color double (fluorescent–colorimetric) whole-mount in situ hybridization with dante (red) and gfp (green; false color). The arrowheads in R point to cells with overlapping dante and gfp expression. L ↔ R indicates the orientation of the embryos. Genotypes are indicated at the top.

Fig. 3.

Abnormal node/PNC and cilia structure in Noto^GFP mutants. (A) Scanning electron micrographs of E7.5 wild-type (Aa–Ad) and mutant (Ae–Al) embryos. Dotted circles in Aa, Ae, and Ai) indicate the node region. Red circles in Aj point to noncoherent groups of pit cells. Arrowhead in (Ah) points to a cilium emerging from an indentation of the plasma membrane. Arrowheads in Al point to bundled and elongated microvilli and to a shortened cilium, respectively. (B) Reduction of cilia length in mutant embryos (P < 0.0001) compared with wild-type. Error bars indicate standard deviation. (C) Transmission electron micrographs of E7.5 wild-type (Cc–Ce) and mutant (Cf–Ck) embryos. In Ca and Cb, the plane of sectioning is shown. Cc and Cf show saggital sections through a cilium and the corresponding basal body. The arrowhead in Cf points to an abnormally located basal body. The microvilli shown in Ci are elongated and branched (arrowhead). The structure of basal bodies in transverse sections Cd and Cg appeared normal. Transverse sections of mutant cilia (Ch, Cj, and Ck) show variably arranged microtubuli with or without a central microtubular structure instead of the normal wild-type (Ce) arrangement of 9 + 0 tubuli. Arrowheads in Ce point to inner and outer dynein arms that are frequently missing in mutant cilia Ck. The cilium in Ch emerges from an abnormally located basal body. (Scale bars in A represent 1 μm in Ad, Ah, and Al; 2 μm in Ac, Ag, and Ak; 10 μm in Ab, Af, and Aj; and 50 μm in Aa, Ae, and Ai; scale bars in C represent 300 nm in Cc, Cf, and Ci and 100 nm in Cd, Ce, Cg, Ch, Cj, and Ck).

Fig. 4.

Expression of potential Noto target genes. Whole-mount in situ hybridization of E8.0 wild-type and mutant embryos. Genotypes are indicated at the top, used probes are indicated on the left. Pictures are taken from the node region.