During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. We now show that feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state were upregulated, leading initially to progenitor overspecification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT were increased, while Smad1 deletion in Nkx2-5 mutants rescued SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies were also rescued by Smad1 deletion. Our findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation, and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD.

Substantial insight has recently been achieved into the mechanisms responsible for the generation of left-right (L-R) asymmetry in the vertebrate body plan. However, the mechanism that underlies the initial breaking of symmetry has remained unclear. In the mouse, a leftward fluid flow on the ventral side of the node caused by the vortical motion of cilia (referred to as nodal flow) is implicated in symmetry breaking, but direct evidence for the role of this flow has been lacking. Here we describe the development of a system in which mouse embryos are cultured under an artificial fluid flow and with which we have examined how flow affects L-R patterning. An artificial rightward flow that was sufficiently rapid to reverse the intrinsic leftward nodal flow resulted in reversal of situs in wild-type embryos. The artificial flow was also able to direct the situs of mutant mouse embryos with immotile cilia. These results provide the first direct evidence for the role of mechanical fluid flow in L-R patterning.

Retinoic acid (RA), a derivative of vitamin A, plays a pivotal role in vertebrate development. The level of RA may be determined by the balance between its synthesis and degradation. We have examined the role of CYP26, a P450 enzyme that may degrade RA, by generating mutant mice that lack CYP26. CYP26 −/− mice exhibited anomalies, including caudal agenesis, similar to those induced by administration of excess RA. The concentration of endogenous RA, as revealed by marker gene activity, was markedly increased in the tailbud of the mutant animals, in which CYP26 is normally expressed. Expression of T (Brachyury) and Wnt3a in the tailbud was down-regulated in CYP26 −/− mice, which may underlie the caudal truncation. The lack of CYP26 also resulted in homeotic transformation of vertebrae as well as in misspecification of the rostral hindbrain associated with anterior expansion of RA-positive domains. These results suggest that local degradation of RA by CYP26 is required for establishing an uneven distribution of RA along the anterio-posterior axis, which is essential for patterning the hindbrain, vertebrae, and tailbud.

Diverse histone modifications are catalysed and recognized by various specific proteins, establishing unique modification patterns that act as transcription signals. In particular, histone H3 trimethylation at lysine 36 (H3K36me3) is associated with actively transcribed regions and has been proposed to provide landmarks for continuing transcription; however, the control mechanisms and functions of H3K36me3 in higher eukaryotes are unknown. Here we show that the H3K36me3-specific histone methyltransferase (HMTase) Wolf-Hirschhorn syndrome candidate 1 (WHSC1, also known as NSD2 or MMSET) functions in transcriptional regulation together with developmental transcription factors whose defects overlap with the human disease Wolf-Hirschhorn syndrome (WHS). We found that mouse Whsc1, one of five putative Set2 homologues, governed H3K36me3 along euchromatin by associating with the cell-type-specific transcription factors Sall1, Sall4 and Nanog in embryonic stem cells, and Nkx2-5 in embryonic hearts, regulating the expression of their target genes. Whsc1-deficient mice showed growth retardation and various WHS-like midline defects, including congenital cardiovascular anomalies. The effects of Whsc1 haploinsufficiency were increased in Nkx2-5 heterozygous mutant hearts, indicating their functional link. We propose that WHSC1 functions together with developmental transcription factors to prevent the inappropriate transcription that can lead to various pathophysiologies.

Unidirectional fluid flow plays an essential role in the breaking of left-right (L-R) symmetry in mouse embryos, but it has remained unclear how the flow is sensed by the embryo. We report that the Ca2+ channel Pkd2 is required specifically in the peri-nodal crown cells for sensing the nodal flow. Examination of mutant forms of Pkd2 shows that the ciliary localization of Pkd2 is essential for correct L-R patterning. Whereas Kif3a mutant embryos, which lack all cilia, failed to respond to an artificial flow, restoration of primary cilia in crown cells rescued the response to the flow. Our results thus suggest that nodal flow is sensed in a manner dependent on Pkd2 by the cilia of crown cells located at the edge of the node.

The past decade or so has seen rapid progress in our understanding of how left-right (LR) asymmetry is generated in vertebrate embryos. However, many important questions about this process remain unanswered. Although a leftward flow of extra-embryonic fluid in the node cavity (nodal flow) is likely to be the symmetry-breaking event, at least in the mouse embryo, it is not yet known how this flow functions or how the asymmetric signal generated in the node is transferred to the lateral plate. The final step in left-right patterning -translation of the asymmetric signal into morphology -is also little understood. IntroductionThere are two key steps that contribute to the early establishment of left-right (LR) patterning in the mouse. The first step is the symmetry-breaking event that takes place in the node around embryonic day (E) 7.5 of mouse development (Fig. 1). In this step, an asymmetric signal(s) that is generated in the node is transferred preferentially towards the left side of the lateral plate mesoderm (LPM). (This mesoderm is located in the lateral region of the earlysomite-stage mouse embryo and later contributes to the mesenchyme of various visceral organs.) The transfer of this signal results in the second step: the asymmetric expression of the gene Nodal in the left LPM (see Fig. 1A,B). Cells in the left LPM that receive Nodal signaling contribute to various visceral organs, such as the lung and heart, that develop left side-specific morphologies.In this review, we discuss our current understanding of the mechanism of left-right (LR) patterning during development. In particular, we focus on genetic data from the mouse; we do not discuss finding from studies in other vertebrates, except where specifically mentioned. [For recent reviews of the similarities and differences in LR patterning between the mouse and other vertebrates, see Levin and Tabin (Levin, 2005;Tabin, 2005).] Leftward fluid flow breaks LR symmetryAlthough there is some controversy concerning the initiation of LR asymmetry in other vertebrates (see Tabin, 2005), at least in the mouse, the breaking of LR symmetry is most likely to be achieved by the unidirectional flow of extra-embryonic fluid in the node (the node is an embryonic structure that is located at the midline, at the anterior tip of the primitive streak in mouse embryos, see Fig. 1B and Fig. 2A). This fluid flow is referred to as nodal flow (Nonaka et al., 1998). This leftward laminar flow of extra-embryonic fluid in the node cavity occurs at a speed (visualized with fluorescent beads, see Fig. 2D) of ~15 to 20 m/second and is generated by the rotational movement of 9+0 monocilia (these are cilia that have nine doublets of microtubules but that lack a pair of central microtubles), which protrude from cells located on the ventral side of the node into the node cavity (Sulik et al., 1994) (Fig. 2C). These 200-300 cilia rotate in the same direction (clockwise, as viewed from the ventral side) at a speed of 600 rpm (Nonaka et al., 1998). Nodal flow takes place for only a short ...

Tight junctions (TJs) connect epithelial cells and form a semipermeable barrier that only allows selective passage of ions and solutes across epithelia. Here we show that mice lacking EpCAM, a putative cell adhesion protein frequently overexpressed in human cancers, manifest intestinal barrier defects and die shortly after birth as a result of intestinal erosion. EpCAM was found to be highly expressed in the developing intestinal epithelium of wild-type mice and to localize to cell-cell junctions including TJs. Claudin-7 colocalized with EpCAM at cell-cell junctions, and the two proteins were found to associate with each other. Claudins 2, 3, 7, and 15 were down-regulated in the intestine of EpCAM mutant mice, with claudin-7 being reduced to undetectable levels. TJs in the mutant intestinal epithelium were morphologically abnormal with the network of TJ strands scattered and dispersed. Finally, the barrier function of the intestinal epithelium was impaired in the mutant animals. These results suggest that EpCAM contributes to formation of intestinal barrier by recruiting claudins to cell-cell junctions.

Determination of left-right asymmetry in mouse embryos is achieved by a leftward fluid flow (nodal flow) in the node cavity that is generated by clockwise rotational movement of 200-300 cilia in the node. The precise action of nodal flow and how much flow input is required for the robust read-out of left-right determination remains unknown. Here we show that a local leftward flow generated by as few as two rotating cilia is sufficient to break left-right symmetry. Quantitative analysis of fluid flow and ciliary rotation in the node of mouse embryos shows that left-right asymmetry is already established within a few hours after the onset of rotation by a subset of nodal cilia. Examination of various ciliary mutant mice shows that two rotating cilia are sufficient to initiate left-right asymmetric gene expression. our results suggest the existence of a highly sensitive system in the node that is able to sense an extremely weak unidirectional flow, and may favour a model in which the flow is sensed as a mechanical force.