Left-right patterning of the mouse lateral plate requires nodal produced in the node

Initial determination of left-right (L-R) polarity in mammalian embryos takes place in the node. However, it is not known how asymmetric signals are generated in the node and transferred to the lateral plate mesoderm (LPM). Mice homozygous for a hypomorphic Nodal allele (Nodal(neo)) were generated and found to exhibit L-R defects, including right isomerism. Although the mutant embryos express Nodal at gastrulation stages, the subsequent expression of this gene in the node and left LPM is lost. A transgene that conferred Nodal expression specifically in the node rescued the L-R defects of the Nodal(neo/neo) embryos. Conversely, ectopic expression of the Nodal inhibitor Lefty2 in the node of Nodal(neo/+) embryos resulted in a phenotype similar to that of the Nodal(neo/neo) mutant. These results indicate that Nodal produced in the node is required for expression of Nodal and other left side-specific genes in the LPM.     

BMP signaling positively regulates Nodal expression during left right specification in the chick embryo

Exogenous application of BMP to the lateral plate mesoderm (LPM) of chick embryos at the early somite stage had a positive effect on Nodal expression. BMP applications into the right LPM were followed by a rapid activation of Nodal, while applications into the left LPM resulted in expansion of the normal domain of Nodal expression. Conversely, blocking of BMP signaling by Noggin in the left LPM interfered with the activation of Nodal expression. These results support a positive role for endogenous BMP on Nodal expression in the LPM. We also report that BMP positively regulates the expression of Caronte, Snail and Cfc in both the left and right LPM. BMP-treated embryos had molecular impairment of the midline with downregulation of Lefty1, Brachyury and Shh but we also show that the midline defect was not sufficient to induce ectopic Nodal expression. We discuss our findings in the context of the known molecular control of the specification of left-right asymmetry.     

Left-right asymmetry in BMP4 signalling pathway during chick gastrulation

Determination of the left-right (L-R) axis was shown recently to implicate several genes, among which TGFbeta-related molecules such as Activin betaB, lefty1 and 2 and Nodal. We show here that Bmp4 and its signal transduction pathway partners BMPR IA and Smad1 are transiently expressed on the right side of Hensen's node, when L-R polarity is being established. Moreover, Smad1 is expressed asymmetrically in the nascent notochord. These observations suggest a role for a BMP4-dependent autocrine or paracrine mechanism during early L-R determination.     

Nodal expression in the uterus of the mouse is regulated by the embryo and correlates with implantation

Nodal, a transforming growth factor beta (TGFB) superfamily member, plays a critical role during early embryonic development. Recently, components of the Nodal signaling pathway were characterized in the human uterus and implicated in the tissue remodeling events during menstruation. Furthermore, the Nodal inhibitor, Lefty, was identified in the mouse endometrium during pregnancy, and its overexpression led to implantation failure. Nonetheless, the precise function and mechanism of Nodal signaling during pregnancy remains unclear. In order to elucidate the potential roles Nodal plays in these processes, we have generated a detailed profile of maternal Nodal expression in the mouse uterus throughout pregnancy. NODAL, although undetectable during the nonpregnant estrus cycle, was localized throughout the glandular epithelium of the endometrium during the peri-implantation period. Interestingly, Nodal expression generated a banding pattern along the proximal-distal axis of the uterine horn on Day 4.5 that directly correlated with blastocyst implantation. Embryo transfer experiments indicate the embryo regulates Nodal expression in the uterus and directs its expression at the time of implantation, restricting NODAL to the sites between implantation crypts. During the later stages of pregnancy, Nodal exhibits a dynamic expression profile that suggests a role in regulating the endometrial response to decidualization and associated trophoblast invasion.     

Rab23 regulates Nodal signaling in vertebrate left–right patterning independently of the Hedgehog pathway

Asymmetric fluid flow in the node and Nodal signaling in the left lateral plate mesoderm (LPM) drive left–right patterning of the mammalian body plan. However, the mechanisms linking fluid flow to asymmetric gene expression in the LPM remain unclear. Here we show that the small GTPase Rab23, known for its role in Hedgehog signaling, plays a separate role in Nodal signaling and left–right patterning in the mouse embryo. Rab23 is not required for initial symmetry breaking in the node, but it is required for expression of Nodal and Nodal target genes in the LPM. Microinjection of Nodal protein and transfection of Nodal cDNA in the embryo indicate that Rab23 is required for the production of functional Nodal signals, rather than the response to them. Using gain- and loss-of function approaches, we show that Rab23 plays a similar role in zebrafish, where it is required in the teleost equivalent of the mouse node, Kupffer?s vesicle. Collectively, these data suggest that Rab23 is an essential component of the mechanism that transmits asymmetric patterning information from the node to the LPM.     

Chick CFC controls Lefty1 expression in the embryonic midline and nodal expression in the lateral plate

Members of the EGF-CFC family of proteins have recently been implicated as essential cofactors for Nodal signaling. Here we report the isolation of chick CFC and describe its expression pattern, which appears to be similar to Cfc1 in mouse. During early gastrulation, chick CFC was asymmetrically expressed on the left side of Hensen's node as well as in the emerging notochord, prechordal plate, and lateral plate mesoderm. Subsequently, its expression became confined to the heart fields, notochord, and posterior mesoderm. Implantation experiments suggest that chick CFC expression in the lateral plate mesoderm is dependent on BMP signaling, while in the midline its expression depends on an Activin-like signal. The asymmetric expression domain within Hensen's node was not affected by application of FGF8, Noggin, or Shh antibody. Implantation of cells expressing human or mouse CFC2, or chick CFC on the right side of Hensen's node randomized heart looping without affecting expression of genes involved in left-right axis formation, including SnR, Nodal, Car, or Pitx2. Application of antisense oligodeoxynucleotides to the midline of Hamburger-Hamilton stage 4-5 embryos also randomized heart looping, but in contrast to the overexpression experiments, antisense oligodeoxynucleotide treatment resulted in bilateral expression of Nodal, Car, Pitx2, and NKX3.2, whereas Lefty1 expression in the midline was transiently lost. Application of the antisense oligodeoxynucleotides to the lateral plate mesoderm abolished Nodal expression. Thus, chick CFC seems to have a dual function in left-right axis formation by maintaining Nodal expression in the lateral plate mesoderm and controlling expression of Lefty1 expression in the midline territory.     

Anteriorward shifting of asymmetric Xnr1 expression and contralateral communication in left-right specification in Xenopus

Transient asymmetric Nodal signaling in the left lateral plate mesoderm (L LPM) during tailbud/early somitogenesis stages is associated in all vertebrates examined with the development of stereotypical left-right (L-R) organ asymmetry. In Xenopus, asymmetric expression of Nodal-related 1 (Xnr1) begins in the posterior L LPM shortly after the initiation of bilateral perinotochordal expression in the posterior tailbud. The L LPM expression domain rapidly shifts forward to cover much of the flank of the embryo before being progressively downregulated, also in a posterior-to-anterior direction. The mechanisms underlying the initiation and propagation of Nodal/Xnr1 expression in the L LPM, and its transient nature, are not well understood. Removing the posterior tailbud domain prevents Xnr1 expression in the L LPM, consistent with the idea that normal embryos respond to a posteriorly derived asymmetrically acting positive inductive signal. The forward propagation of asymmetric Xnr1 expression occurs LPM-autonomously via planar tissue communication. The shifting is prevented by Nodal signaling inhibitors, implicating an underlying requirement for Xnr1-to-Xnr1 induction. It is also unclear how asymmetric Nodal signals are modulated during L-R patterning. Small LPM grafts overexpressing Xnr1 placed into the R LPM of tailbud embryos induced the expression of the normally L-sided genes Xnr1, Xlefty, and XPitx2, and inverted body situs, demonstrating the late-stage plasticity of the LPM. Orthogonal Xnr1 signaling from the LPM strongly induced Xlefty expression in the midline, consistent with recent findings in the mouse and demonstrating for the first time in another species conservation in the mechanism that induces and maintains the midline barrier. Our findings suggest that there is long-range contralateral communication between L and R LPM, involving Xlefty in the midline, over a substantial period of tailbud embryogenesis, and therefore lend further insight into how, and for how long, the midline maintains a L versus R status in the LPM.     

Sulfated glycosaminoglycans are necessary for Nodal signal transmission from the node to the left lateral plate in the mouse embryo

Situs-specific organogenesis in the mouse results from leftward fluid flow in the node cavity and subsequent left-sided expression of Nodal in the lateral plate mesoderm (LPM). Nodal expression at the node is essential for the subsequent asymmetric Nodal expression in the left LPM, but the precise role of Nodal produced at the node has remained unknown. We have now investigated how the Nodal signal is transferred from the node to the LPM. Externally supplied Nodal protein failed to signal to the LPM, suggesting that the Nodal signal is transferred to the LPM via an internal route rather than an external one. Transgenic rescue experiments showed that the Nodal co-receptor Cryptic (Cfc1) is required only in the LPM, not at the node, for asymmetric Nodal expression in the LPM, indicating that the Nodal signal is not relayed indirectly between the node and LPM. Nodal interacts in vitro with sulfated glycosaminoglycans (GAGs), which are specifically localized to the basement membrane-like structure between the node and LPM in the mouse embryo. Inhibition of sulfated GAG biosynthesis prevents Nodal expression in the LPM. These data suggest that Nodal produced at the node might travel directly to the LPM via interaction with sulfated GAGs.     

The role of an endogenous PKA inhibitor, PKIalpha, in organizing left-right axis formation

Protein kinase inhibitor (PKI) is an endogenous inhibitor of cAMP-dependent protein kinase A (PKA). We have found that the alpha-isoform of PKI (PKIalpha) is asymmetrically expressed along the left-right (L-R) axis in chick embryos. At stage 6, PKIalpha is expressed on the right side of the node, and this asymmetric expression continues until stage 7+. After stage 8, PKIalpha expression returns symmetric. Treatment of embryos with antisense PKIalpha oligonucleotides increased the incidence of reversed heart looping. Antisense oligonucleotides also induced ectopic expression of the left-specific genes Nodal and Pitx2, and suppressed the expression of the right-specific gene SnR in the right lateral plate mesoderm. Similarly, treatment with PKA activators forskolin and Sp-cAMPs resulted in both reversed heart looping and bilateral expression of NODAL: Ectopic activin induced PKIalpha on the left side of the node, while ectopic Shh and anti-Shh antibody had no effect on PKIalpha expression. Taken together, these data suggest that PKIalpha induced by an activin-like molecule, through the inhibition of PKA activity, suppresses the Nodal-Pitx2 pathway on the right side of the body.     

Distinct requirements for extra-embryonic and embryonic bone morphogenetic protein 4 in the formation of the node and primitive streak and coordination of left-right asymmetry in the mouse

In the mouse and chick embryo, the node plays a central role in generating left-right (LR) positional information. Using several different strategies, we provide evidence in the mouse that bone morphogenetic protein 4 (Bmp4) is required independently in two different sites for node morphogenesis and for LR patterning. Bmp4 expression in the trophoblast-derived extra-embryonic ectoderm is essential for the normal formation of the node and primitive streak. However, tetraploid chimera analysis demonstrates that Bmp4 made in epiblast-derived tissues is required for robust LR patterning, even when normal node morphology is restored. In the absence of embryonic Bmp4, the expression of left-side determinants such as Nodal and Lefty2 is absent in the left lateral plate mesoderm (LPM). Noggin-mediated inhibition of Bmp activity in cultured wild-type embryos results in suppression of Nodal expression in the LPM. Thus, unlike previous models proposed in the chick embryo in which Bmp4 suppresses left-sided gene expression, our results suggest that Bmp acts as a positive facilitator of the left-sided molecular cascade and is required for Nodal induction and maintenance in the left LPM.     

BMP2 is a positive regulator of Nodal signaling during left-right axis formation in the chicken embryo

A model of left-right axis formation in the chick involves inhibition of bone morphogenetic proteins by the antagonist Car as a mechanism of upregulating Nodal in the left lateral plate mesoderm. By contrast, expression of CFC, a competence factor, which is absolutely required for Nodal signaling in the lateral plate mesoderm is dependent on a functional BMP signaling pathway. We have therefore investigated the relationship between BMP and Nodal in further detail. We implanted BMP2 and Noggin-expressing cells into the left lateral plate and paraxial mesoderm and observed a strong upregulation of Nodal and its target genes Pitx2 and Nkx3.2. In addition Cfc, the Nodal type II receptor ActrIIa and Snr were found to depend on BMP signaling for their expression. Comparison of the expression domains of Nodal, Bmp2, Car and Cfc revealed co-expression of Nodal, Cfc and Bmp2, while Car and Nodal only partially overlapped. Ectopic application of BMP2, Nodal, and Car as well as combinations of this signaling molecules to the right lateral plate mesoderm revealed that BMP2 and Car need to synergize in order to specify left identity. We propose a novel model of left-right axis formation, which involves BMP as a positive regulator of Nodal signaling in the chick embryo.     

文昌鱼左右体轴建立机制的研究进展

两侧对称动物左右体轴建立机制研究是发育生物学领域重要的基础科学问题之一。文昌鱼(amphioxus)由于其特殊的进化地位以及与脊椎动物相似的胚胎发育模式和身体构筑方式,是研究动物左右体轴建立机制的理想模式物种。近年来随着文昌鱼室内全人工繁育技术、高效显微注射技术和基因敲除技术的建立,国内外学者在左右体轴建立机制研究上取得了丰硕的成果。本文从文昌鱼胚胎左右不对称发育特点出发,总结了近期文昌鱼左右体轴建立方面取得的研究进展,并提出了文昌鱼左右体轴调控网络图:纤毛运动导致Hh蛋白在文昌鱼中不对称分布(L相似文献   

Chordin is required for neural but not axial development in sea urchin embryos

The oral-aboral (OA) axis in the sea urchin is specified by the TGFβ family members Nodal and BMP2/4. Nodal promotes oral specification, whereas BMP2/4, despite being expressed in the oral territory, is required for aboral specification. This study explores the role of Chordin (Chd) during sea urchin embryogenesis. Chd is a secreted BMP inhibitor that plays an important role in axial and neural specification and patterning in Drosophila and vertebrate embryos. In Lytechinus variegatus embryos, Chd and BMP2/4 are functionally antagonistic. Both are expressed in overlapping domains in the oral territory prior to and during gastrulation. Perturbation shows that, surprisingly, Chd is not involved in OA axis specification. Instead, Chd is required both for normal patterning of the ciliary band at the OA boundary and for development of synaptotagmin B-positive (synB) neurons in a manner that is reciprocal with BMP2/4. Chd expression and synB-positive neural development are both downstream from p38 MAPK and Nodal, but not Goosecoid. These data are summarized in a model for synB neural development.     

Expression cloning of Xenopus Os4, an evolutionarily conserved gene, which induces mesoderm and dorsal axis.

Multiple factors, including members of the FGF, TGF beta, and Wnt family of proteins, are important mediators in the regulation of dorsal-ventral pattern formation during vertebrate development. By using an expression cloning approach to identify novel factors that could regulate dorsal-ventral patterning in the Xenopus embryo, we isolated the Xenopus homologue of the human Os4 gene by virtue of its ability to induce a secondary dorsal axis. While Os4 homologues have been identified in a variety of species, and human Os4 is overexpressed in human tumors, the biological function of Os4 is unknown. To explore the mechanism by which Xenopus Os4 (XOs4) induces a secondary dorsal axis, we used Xenopus explant and whole-embryo assays. The secondary axis induced by XOs4 is distinct from that induced by activation of Wnt or FGF pathways but similar to that induced by inhibition of BMP signaling or activation of an Activin pathway. However, XOs4 did not inhibit BMP signaling in dissociated animal cap explants, indicating that XOs4 does not inhibit BMP signaling. Similar to activation of an Activin-like pathway, expression of XOs4 induces molecular markers for mesoderm in animal cap explants, although expression of gastrula-stage mesodermal markers was very weak and substantially delayed. Yet, XOs4 does not require activity of the Activin signal-transduction pathway for mesoderm induction as dominant-negative components of the Activin/Nodal/Vg1 pathway did not prevent XOs4-mediated induction of mesodermal derivatives. Finally, like Activin/Nodal/Vg1 pathways, XOs4 requires FGF signaling for expression of mesoderm markers. Results presented in this study demonstrate that XOs4 can induce mesoderm and dorsalize ventral mesoderm resulting in ectopic dorsal axis formation, suggesting a role for this large evolutionarily conserved gene family in early development.     

Defective Nodal and Cerl2 expression in the Arl13b(hnn) mutant node underlie its heterotaxia

Specification of the left-right axis during embryonic development is critical for the morphogenesis of asymmetric organs such as the heart, lungs, and stomach. The first known left-right asymmetry to occur in the mouse embryo is a leftward fluid flow in the node that is created by rotating cilia on the node surface. This flow is followed by asymmetric expression of Nodal and its inhibitor Cerl2 in the node. Defects in cilia and/or fluid flow in the node lead to defective Nodal and Cerl2 expression and therefore incorrect visceral organ situs. Here we show the cilia protein Arl13b is required for left right axis specification as its absence results in heterotaxia. We find the defect originates in the node where Cerl2 is not downregulated and asymmetric expression of Nodal is not maintained resulting in symmetric expression of both genes. Subsequently, Nodal expression is delayed in the lateral plate mesoderm (LPM). Symmetric Nodal and Cerl2 in the node could result from defects in either the generation and/ or the detection of Nodal flow, which would account for the subsequent defects in the LPM and organ positioning.