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Proc Natl Acad Sci U S A. Oct 2, 2007; 104(40): 15765–15770.
Published online Sep 20, 2007. doi:  10.1073/pnas.0704344104
PMCID: PMC2000419
Developmental Biology

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.

Keywords: cilia, left–right asymmetry, posterior notochord

The organizer of vertebrate embryos is essential for the establishment of the body plan during gastrulation and induces and patterns surrounding tissues. The inducing properties of the organizer are regulated by a network of genes that are expressed in the organizer and its derivatives (14). The Not genes form a group of homeobox genes that are expressed in the organizer and notochord and function in specifying axial mesoderm in zebrafish and Xenopus embryos (5). During gastrulation, the mouse Not homeobox gene Noto (formerly called Not) is expressed in the anterior primitive streak/organizer and in the posterior extreme of the forming notochord [posterior notochord (PNC)] (6, 7), which at embryonic day (E)7.5 is referred to as the node, a shallow, crescent-shaped depression on the endodermal (ventral) side at the distal tip of the embryo (8). Noto acts downstream of the organizer genes Foxa2 and T and a null allele-disrupted normal notochord formation in and posterior to the lumbar region (7).

The node/PNC that emerges during gastrulation (and equivalent structures in other vertebrate classes) is crucial for the establishment of left–right asymmetry. In mouse and rabbit embryos, each cell on the ventral side of the PNC, which has a plate like appearance until well after the first somites form, carries a single primary cilium on its surface (8, 9). These primary cilia are unique because they are motile, but their axonemes contain only the nine regular-spaced peripheral doublet microtubules and lack the central microtubule pair that is usually present in motile cilia (10, 11). Functional cilia in the PNC, and in the equivalent gastrocoel roof plate and Kupffer's vesicle of Xenopus and zebrafish embryos, respectively, are critical for generating molecular asymmetry (1215). In the PNC, cilia rotate clockwise and generate a leftward nodal flow (15) that generates an asymmetric signal either by establishing a gradient of a secreted morphogen (15, 16), or by physically stimulating sensory monocilia on cells in the periphery of the node (17). Consistent with the pivotal role of these monocilia in left–right determination, mutations that affect the formation (15, 1824), sensory function (25), or motility (26, 27) of monocilia in the node region disrupt the normal generation of left–right asymmetry.

Information on the regulation of expression of proteins essential for cilia formation and function in the node region is still scarce: Rfx3 and Foxj1, members of the RFX and forkhead family of transcription factors, respectively, are expressed in the node and in many tissues containing ciliated cells (28, 29). Loss of RFX3 leads to short but ultrastructural apparently normal cilia, left–right defects, and reduced expression of D2lic, a cytoplasmic dynein implicated in intraflagellar transport (30). Loss of FOXJ1 causes absence of cilia and loss of left–right dynein (Dnahc11) expression in lung epithelial cells, and, in addition, left–right defects (29, 31), although apparently normal cilia were observed in the node region (29). Thus, it appears that, in the node region, the components important for ciliogenesis and cilia function are differently regulated. Here, we show that loss of Noto function in NotoGFP mice disrupts the formation and function of the PNC and cilia, and consequently the establishment of normal left–right asymmetry. Our results establish Noto as a key regulator of ciliogenesis in the mouse PNC upstream of Foxj1 and Rfx3.

Results

Defective Left–Right Determination in Noto Mutants.

Embryos homozygous for the null NotoGFP allele showed defects in the caudal notochord and occasionally also at more anterior levels, and most mutants died shortly after birth (7). Urogenital or anorectal malformations that can be associated with defective notochord development (32, 33) were not observed in homozygous NotoGFP newborns (7). Detailed inspection of the visceral organs now revealed laterality defects in a large fraction of mutants (Fig. 1 A–F and data not shown). E16.5 NotoGFP/GFP embryos displayed heterotaxia (n = 2 of 32), left (n = 11 of 32; Fig. 1D) and right (n = 4 of 32; Fig. 1E) isomerism of the lung, complete situs inversus (n = 9 of 32; Fig. 1F) and situs solitus (n = 6 of 32). Consistent with laterality defects, a significant portion of mutant fetuses at E18.5 showed obvious macroscopic abnormalities in the outflow tracts [supporting information (SI) Fig. 5] providing a plausible explanation for the high postnatal mortality reported previously (7).

Fig. 1.
Laterality defects in NotoGFP 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 ...

Consistent with the abnormal situs of visceral organs, heart looping (Fig. 1 G and H) and embryonic turning (Fig. 1 I and J), and the expression patterns of asymmetrically expressed genes were randomized (Fig. 1 K–Z). Nodal, which is expressed in the left lateral plate mesoderm (lpm) in wild-type E8.25 (3–6 somites) embryos was expressed normally (2 of 10), bilaterally (4 of 10), or in the right lpm (1 of 10), or expression was severely reduced in the lpm (3 of 10) of homozygous NotoGFP embryos (Fig. 1 K–N and S–V and data not shown). Lefty2 expression was similarly affected, mutant embryos showing normal left-sided (2 of 8), bilateral (2 of 8), right-sided (3 of 8), or severely down-regulated (1 of 8) expression (Fig. 1 O–V and data not shown). Lefty1 expression in the midline was present in the majority of the analyzed embryos (6 of 8), although at variable levels, and, in some cases, the contiguous expression domain was interrupted (data not shown). Also, expression of Pitx2, which acts downstream of nodal (34), was randomized in E8.75 mutants (7–10 somites), with normal left-sided (1 of 7), bilateral (4 of 7), right sided (1 of 7), and no expression (1 of 7; Fig. 1 W–Z and data not shown). Given the importance of the organizer for patterning the anterior–posterior neuraxis (3538) and Noto expression in the organizer (6), we analyzed anterior–posterior patterning in Noto E8.75 null mutant embryos. The expression patterns of Fgf8, Gbx2, Krox20, and Otx2 were indistinguishable in wild-type and Noto mutants (data not shown), suggesting that anterior–posterior patterning occurs normally. Collectively, these results indicated that Noto is essential for establishing left–right asymmetry and suggested that Noto function is required in the node before or during establishment of asymmetric gene expression.

Disrupted Node Structure in Noto Mutants.

In wild-type embryos at E8.0, the peripheral “crown cells” (39) of the node region express nodal and dante, with stronger expression on the left or right side, respectively (Fig. 2A, E, I, and M; see also refs. 40 and 41). At this stage, Noto expression was detected in the central cells of the PNC [also called “pit cells” (39)] and appeared largely nonoverlapping with these expression domains (Fig. 2Q). This interpretation was supported by double in situ hybridizations that showed that Noto and dante are coexpressed only in a subset of peripheral cells (arrowheads in Fig. 2R). In the majority of mutant embryos, expression domains of nodal and dante were irregular and variable, or consisted of scattered groups of cells only in the PNC region (Fig. 2 B–D, F–H, J–L, and N–P and data not shown), and dante-expressing cells were intermingled with cells expressing gfp from the Noto locus (Fig. 2 S–U), suggesting that typical PNC (or pit) cells and crown cells are present, but the node is disorganized. Consistently, by scanning electron microscopy, a distinct node was hardly detected in 12 of 21 E7.5–E8.0 mutant embryos, whereas the remainder had small and irregular nodes with no sharp border between pit and crown cells (Fig. 3Ae, Af, Ai, and Aj), or with pit cells separated into noncoherent groups (red circles in Fig. 3Aj).

Fig. 2.
Node/PNC defects in NotoGFP 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 ...
Fig. 3.
Abnormal node/PNC and cilia structure in NotoGFP 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 ...

Abnormal Cilia Structure in Noto Mutants.

Mutant PNC cells had monocilia and microvilli, although the number of cilia-bearing cells appeared reduced (Fig. 3 Ac, Ag, and Ak), and cilia were significantly shorter (Fig. 3B). Some cilia emerged from indentations of the plasma membrane (Fig. 3Ah), and microvilli were bundled and variable in length (Fig. 3Al), suggesting that the structure of cilia and microvilli is abnormal. Transmission electron microscopy of mutant embryos (n = 11) showed that some microvilli were branched (Fig. 3Ci), and mutant cilia contained a variable number (3–8, rarely 9) of partly disorganized or incomplete, irregularly arranged peripheral microtubular doublets with or without a central microtubule (Fig. 3 Ch, Cj, and Ck). In addition, dynein arms were frequently missing (Insets in Fig. 3Ck). Consistent with abnormal cilia structure, the majority of cilia were immotile in seven analyzed mutant embryos (SI Movies 1–3). The structure of the basal bodies appeared normal (Fig. 3Cg). However, of 41 basal bodies found in ultrathin sections, only 10 were located in the cortex, whereas 14 were obviously located abnormally deep in the cytoplasm (Fig. 3Cf and data not shown), and the location of the remainder could not unambiguously be determined in the available sections. Taken together, abnormal morphology in the node region and aberrant cilia structure in mutants indicate a role(s) for Noto in the regulation of PNC formation, ciliogenesis, and the establishment of left–right asymmetry.

Altered Expression of Genes Implicated in Ciliogenesis or Function.

In line with the abnormal location of basal bodies in Noto mutant PNC cells and defective basal body anchoring in lung epithelium cells in Foxj1 mutants (29, 42) Foxj1 transcripts were barely detectable in mutant node regions (Fig. 4 A–C), indicating that Noto acts upstream of Foxj1. Expression of the axonemal dynein Dnahc11, which is regulated by Foxj1 in lung epithelial cells (31) and required for cilia motility, was down-regulated (Fig. 4 D–F) and Dnahc5, an axonemal dynein that is expressed in the node and required for the formation of outer dynein arms (43), and Dnchc2, a component of retrograde intraflagellar transport required for normal cilia morphology (21, 44), were not detected in mutant nodes (Fig. 4 G–L). In addition, expression of the cytoplasmic dynein Dncl2b in mutant nodes was essentially abolished (Fig. 4 M–O). Transcripts of Nphp3, encoding a protein that might be involved in microtubule organization and is mutated in Nephronophtisis3 patients and normally expressed in the node region (45), were significantly down-regulated in mutant embryos (Fig. 4 P–R). Finally, Rfx3, which is required for normal cilia growth (30), was severely down-regulated in the node regions of Noto mutant embryos (Fig. 4 S–U). Collectively, these results establish Noto as a transcription factor upstream of Foxj1 and Rfx3 that is essential for the expression of multiple proteins required for cilia formation and function.

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.

Discussion

We show that the homeobox gene Noto is essential for cilia formation in the PNC and for left–right patterning. Noto acts upstream of Foxj1 and Rfx3 in this process and is required for the expression of various components important for axonemal assembly and function. In addition, Noto is required for PNC morphogenesis but seems to be dispensable for anterior–posterior patterning functions of the organizer.

A Conserved Function of Noto in Formation of “Organs of Asymmetry”.

The majority of Noto mutants showed variable defects in the PNC that ranged from complete absence over variable size and shape and disorganized distribution of crown and pit cells to apparently normal morphology in rare cases. These findings indicate a major function of Noto in node and posterior notochord morphogenesis and suggest an essential role in regulating migration, positioning, or separation of crown and pit cells. Because Noto is expressed in the pit cells, but only some crown cells, Noto function might predominantly be required in pit cells. Abnormal migration and positioning of notochord precursors might also underlie defective notochord development in Noto mutants at later stages. This aspect of Noto function appears to be conserved among mammals, amphibia, and teleost fish. In teleosts, a transient spherical ciliated structure called Kupffer's vesicle functions in the establishment of left–right asymmetry equivalent to the amphibian gastrocoel roof plate and the mammalian PNC (12, 13, 46, 47). In zebrafish embryos mutant for the Noto homologue flh, Kupffer's vesicles were abnormally small and irregularly shaped (48). Kupffer's vesicle is formed from a distinct population of cells that migrate at the leading edge of the embryonic shield (49), the zebrafish equivalent of the organizer (50). Similarly, the precursors of the mouse node and notochord represent a distinct cell population that resides posterior to the midgastrulation organizer in midstreak-stage mouse embryos (51). Thus, it appears that, in mammals and teleost fish, Noto/flh regulates aspects of cell behaviour in a group of cells that is associated with the organizer and fated to form a functionally analogous specialized structure required for left–right patterning. Asymmetry defects have been described in flh mutants but were attributed to the complete absence of the notochord (52). Our data and abnormal Kupffer's vesicles observed by Melby et al. (48) in flh mutants both support the view that abnormal Kupffer's vesicle formation contributes to the left–right patterning defects in flh mutants.

Noto as Regulator Of Nodal Ciliogenesis Upstream of Foxj1 and Rfx3.

A striking feature of the Noto phenotype is the highly variable abnormal structure of cilia. These abnormalities likely arise from the reduced or missing expression of genes encoding proteins that are essential for cilia motility or axonemal assembly or transport. Because mutations that affect single components required for the formation (15, 1824), sensory function (25), or motility (26, 27) of nodal monocilia, respectively, disrupt the normal generation of left–right asymmetry, cilia in Noto mutants are most likely nonfunctional. Our results suggest that Noto is pivotal for the coordinate expression of essential components of the functional axonemal complex in the mouse PNC. However, the role of Not homeobox genes in regulating ciliogenesis in amphibian and fish embryos is currently not clear, whereas it appears irrelevant in avian embryos, where cells in the notochord region equivalent to teleost Kupffer's vesicle, the amphibian gastrocoel roof plate, and the mammalian PNC do not exhibit monocilia (53) despite expression of Gnot1 and Gnot2 (5456).

It is unlikely that Noto acts as a direct regulator of all deregulated axonemal genes. Down-regulation of Foxj1 and Dnahc11, which is regulated by Foxj1 in respiratory epithelial cells (31), suggests that Foxj1 acts as a mediator of Noto function in the node. This notion is further supported by the abnormal position of basal bodies in node cells, which closely resembles the phenotype in lung epithelial cells in Foxj1 mutants (42). The severe reduction of Foxj1 indicates that Foxj1 acts downstream of Noto, although residual Foxj1 expression suggests that other factor(s) also activate Foxj1. In contrast, Rfx3 expression was virtually abolished in the node regions of mutant embryos, which might indicate that Noto is the major activator of Rfx3 expression in the node region. In conclusion, our results suggest a general requirement of Noto for assembly and function of cilia in vertebrate organs of asymmetry.

Methods

Mice.

NotoGFP/GFP mice were genotyped as described (7).

Histology and Scanning Electron Microscopy.

After whole-mount in situ hybridization, embryos were embedded in HISTORESIN embedding medium (Leica, Wetzlar, Germany). Sections were cut at 2 μm, counterstained with eosin, and analyzed with a DM5000B microscope, a DFC300FX camera, and FireCam software (Leica). For scanning electron microscopy, dissected embryos were immersion-fixed in 3% glutaraldehyde–0.1M cacodylate buffer (pH 7.4) at 4°C overnight, washed in 0.1 M cacodylate buffer (four times for 10 min each), postfixed in 2% osmium tetroxide in 0.1 M cacodylate buffer at room temperature in the dark, dehydrated in a graded acetone series, critical point dried, mounted and sputter coated with gold, and viewed under a SEM 505 microscope (Philips, Eindhoven, The Netherlands).

In Situ Hybridization.

Mutant and wild-type embryos were processed in parallel under identical conditions by standard procedures. Double in situ hybridization was done by simultaneous hybridization of a Digoxigenin-labeled dante and fluorescein-labeled gfp probe. Bound probes were detected by using HRP-coupled anti-DIG antibodies and Cy3 tyramid signal amplification (TSA PlusFluorescence System; PerkinElmer, Waltham, MA), followed by BM-purple staining using AP-conjugated anti-fluorescein antibodies. Images were obtained with a DM5000B microscope, a DFC300FX camera, and FireCam software (Leica) or a M420 microscope (Leica) and HC300Z camera (Fuji, Tokyo, Japan) and FujixPhotograb300Z software (Fuji). Images were processed by using Photoshop CS (Adobe, San Jose, CA). False color images were generated by inserting an inverted bright-field image of the BM purple staining into the green channel of the Cy3 image and adjustments of the green and red channel, respectively.

Transmission Electron Microscopy.

Standard procedures were applied for fixation (1.5% glutaraldehye and 1.5% paraformaldehyde in phosphate buffer) of mutant and wild-type embryos. Thin sectioning of selected serial semithin sections (57) enabled faithful localization and transmission electron microscopy of cilia and their related structures.

Cilia Measurements.

One hundred nine and 93 cilia from four wild-type and mutant embryos, respectively, were measured in scanning electronmicrographs by using the ImageJ software (National Institutes of Health, Bethesda, MD, http://rsb.info.nih.gov/ij/). Statistical analysis was done by using an unpaired t test with the Prism software (GraphPad, San Diego, CA).

Measurement of Cilia Motility.

Motility of cilia was analyzed by videomicroscopy essentially as described (13).

Supplementary Material

Supporting Information:

Acknowledgments

We thank José António Belo (Instituto Gulbenkian de Ciência, Oeiras, Portugal), Martin Blum (University of Hohenheim, Stuttgart, Germany), Martina Brückner (Yale University School of Medicine, New Haven, CT), Bénédicte Durand (Université Claude Bernard, Villeurbanne, France), Brian P. Hackett (Washington University School of Medicine, St. Louis, MO), Andreas Kispert (Medizinische Hochschule, Hannover, Germany), and Janet Rossant (Hospital for Sick Children Research Institute, Toronto, ON, Canada) for probes; Stephanie Groos and Gerhard Preiss for help with the scanning electron microscopy; Thomas Grieskamp for help with outflow tract analysis; Kerstin Brekerbohm and Peter Schwartz for help with the transmission electron microscopy; and Anne Camus and Jérome Collignon for teaching fluorescent in situ hybridization to A.B. This work was supported by a German Research Council grant (to A.G.).

Abbreviations

E(n)
embryonic day n
PNC
posterior notochord.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0704344104/DC1.

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