Retinoic acid activates myogenesis in vivo through Fgf8 signalling

Retinoic acid (RA) has been shown to regulate muscle differentiation in vitro. Here, we have investigated the role of RA signalling during embryonic myogenesis in zebrafish. We have altered RA signalling from gastrulation stages onwards by either inhibiting endogenous RA synthesis using an inhibitor of retinaldehyde dehydrogenases (DEAB) or by addition of exogenous RA. DEAB reduces expression of the myogenic markers myoD and myogenin in somites, whereas RA induces increased expression of these genes and strongly induces premature myoD expression in the presomitic mesoderm (psm). The expression dynamics of myf5 in presomitic and somitic mesoderm suggest that RA promotes muscle differentiation, a role supported by the fact that RA activates expression of fast myosin, while DEAB represses it. We identify Fgf8 as a major relay factor in RA-mediated activation of myogenesis. We show that fgf8 expression in somites and anterior psm is regulated by RA, and find that in the absence of Fgf8 signalling in the acerebellar mutant RA fails to promote myoD expression. We propose that, in the developing embryo, localised synthesis of RA by Raldh2 in the anterior psm and in somites activates fgf8 expression which in turn induces the expression of myogenic genes and fast muscle differentiation.     

WNT signalling in the immune system: WNT is spreading its wings

WNT proteins are secreted morphogens that are required for basic developmental processes, such as cell-fate specification, progenitor-cell proliferation and the control of asymmetric cell division, in many different species and organs. In blood and immune cells, WNT signalling controls the proliferation of progenitor cells and might also affect the cell-fate decisions of stem cells. Recent studies indicate that WNT proteins also regulate effector T-cell development, regulatory T-cell activation and dendritic-cell maturation. WNT signalling seems to function as a universal mechanism in leukocytes to establish a pool of undifferentiated cells for further selection, effector-cell maturation and terminal differentiation. WNT signalling is therefore subject to strict molecular control, and dysregulated WNT signalling is implicated in the development of haematological malignancies.     

Oscillations of the snail genes in the presomitic mesoderm coordinate segmental patterning and morphogenesis in vertebrate somitogenesis

The segmented body plan of vertebrate embryos arises through segmentation of the paraxial mesoderm to form somites. The tight temporal and spatial control underlying this process of somitogenesis is regulated by the segmentation clock and the FGF signaling wavefront. Here, we report the cyclic mRNA expression of Snail 1 and Snail 2 in the mouse and chick presomitic mesoderm (PSM), respectively. Whereas Snail genes' oscillations are independent of NOTCH signaling, we show that they require WNT and FGF signaling. Overexpressing Snail 2 in the chick embryo prevents cyclic Lfng and Meso 1 expression in the PSM and disrupts somite formation. Moreover, cells mis-expressing Snail 2 fail to express Paraxis, remain mesenchymal, and are thereby inhibited from undergoing the epithelialization event that culminates in the formation of the epithelial somite. Thus, Snail genes define a class of cyclic genes that coordinate segmentation and PSM morphogenesis.     

FGF signaling acts upstream of the NOTCH and WNT signaling pathways to control segmentation clock oscillations in mouse somitogenesis

Fibroblast growth factor (FGF) signaling plays a crucial role in vertebrate segmentation. The FGF pathway establishes a posterior-to-anterior signaling gradient in the presomitic mesoderm (PSM), which controls cell maturation and is involved in the positioning of segmental boundaries. In addition, FGF signaling was shown to be rhythmically activated in the PSM in response to the segmentation clock. Here, we show that conditional deletion of the FGF receptor gene Fgfr1 abolishes FGF signaling in the mouse PSM, resulting in an arrest of the dynamic cyclic gene expression and ultimately leading to an arrest of segmentation. Pharmacological treatments disrupting FGF signaling in the PSM result in an immediate arrest of periodic WNT activation, whereas NOTCH-dependent oscillations stop only during the next oscillatory cycle. Together, these experiments provide genetic evidence for the role of FGF signaling in segmentation, and identify a signaling hierarchy controlling clock oscillations downstream of FGF signaling in the mouse.     

BMP inhibition stimulates WNT-dependent generation of chondrogenic mesoderm from embryonic stem cells

WNT and bone morphogenetic protein (BMP) signaling are known to stimulate hemogenesis from pluripotent embryonic stem (ES) cells. However, osteochondrogenic mesoderm was generated effectively when BMP signaling is kept to a low level, while WNT signaling was strongly activated. When mesoderm specification from ES cells was exogenous factor dependent, WNT3a addition supported the generation of cardiomyogenic cells expressing lateral plate/extraembryonic mesoderm genes, and this process involved endogenous BMP activities. Exogenous BMP4 showed a similar effect that depended on endogenous WNT activities. However, neither factor induced robust chondrogenic activity. In support, ES cell differentiation in the presence of either WNT3a or BMP4 was associated with elevated levels of both Bmp and Wnt mRNAs, which appeared to provide sufficient levels of active BMPs and WNTs to promote the nonchondrogenic mesoderm specification. The osteochondrogenic mesoderm expressed PDGFRα, which also expressed genes that mark somite and rostral presomitic mesoderm. A strong WNT signaling was required for generating the mesodermal progeny, while approximately 50- to 100-fold lower concentration of WNT3a was sufficient for specifying axial mes(end)oderm. Thus, depending on the dose and cofactor (BMP), WNT signaling stimulates the generation of different biological activities and specification of different types of mesodermal progeny from ES cells.     

Vertebrate segmentation: is cycling the rule?

Vertebrate segmentation initiates with the subdivision of the paraxial mesoderm into a regular array of somites. Recent evidence suggests that the segmentation clock - a biochemical oscillator acting in the unsegmented paraxial mesoderm cells in most vertebrates - controls cyclic Notch signalling, resulting in periodic formation of somite boundaries.     

A beta-catenin gradient links the clock and wavefront systems in mouse embryo segmentation

Rhythmic production of vertebral precursors, the somites, causes bilateral columns of embryonic segments to form. This process involves a molecular oscillator--the segmentation clock--whose signal is translated into a spatial, periodic pattern by a complex signalling gradient system within the presomitic mesoderm (PSM). In mouse embryos, Wnt signalling has been implicated in both the clock and gradient mechanisms, but how the Wnt pathway can perform these two functions simultaneously remains unclear. Here, we use a yellow fluorescent protein (YFP)-based, real-time imaging system in mouse embryos to demonstrate that clock oscillations are independent of beta-catenin protein levels. In contrast, we show that the Wnt-signalling gradient is established through a nuclear beta-catenin protein gradient in the posterior PSM. This gradient of nuclear beta-catenin defines the size of the oscillatory field and controls key aspects of PSM maturation and segment formation, emphasizing the central role of Wnt signalling in this process.     

Retinoic-acid signalling in node ectoderm and posterior neural plate directs left-right patterning of somitic mesoderm

Somitogenesis requires bilateral rhythmic segmentation of paraxial mesoderm along the antero-posterior axis. The location of somite segmentation depends on opposing signalling gradients of retinoic acid (generated by retinaldehyde dehydrogenase-2; Raldh2) anteriorly and fibroblast growth factor (FGF; generated by Fgf8) posteriorly. Retinoic-acid-deficient embryos exhibit somite left-right asymmetry, but it remains unclear how retinoic acid mediates left-right patterning. Here, we demonstrate that retinoic-acid signalling is uniform across the left-right axis and occurs in node ectoderm but not node mesoderm. In Raldh2(-/-) mouse embryos, ectodermal Fgf8 expression encroaches anteriorly into node ectoderm and neural plate, but its expression in presomitic mesoderm is initially unchanged. The late stages of somitogenesis were rescued in Raldh2(-/-) mouse embryos when the maternal diet was supplemented with retinoic acid until only the 6-somite stage, demonstrating that retinoic acid is only needed during node stages. A retinoic-acid-reporter transgene marking the action of maternal retinoic acid in rescued Raldh2(-/-) embryos revealed that the targets of retinoic-acid signalling during somitogenesis are the node ectoderm and the posterior neural plate, not the presomitic mesoderm. Our findings suggest that antagonism of Fgf8 expression by retinoic acid occurs in the ectoderm and that failure of this mechanism generates excessive FGF8 signalling to adjacent mesoderm, resulting initially in smaller somites and then left-right asymmetry.     

How the community effect orchestrates muscle differentiation

The "community effect" is necessary for tissue differentiation. In the Xenopus muscle paradigm, e-FGF has been identified as a candidate community factor. Standley et al.1 now show that the community effect, mediated through FGF signalling, continues to be important at later stages of development in the posterior part of the embryo. In this region, the paraxial mesoderm is still undergoing segmentation into somites, which are the site of early skeletal muscle formation. Indeed, somitogenesis, together with the read-out of the Hox code, which confers anteroposterior positional identity, is regulated by FGF signalling. This raises the question of the co-ordination between these events and the community effect which orchestrates myogenesis.