Andrea Münsterberg

Ectopic Pax-3 activates MyoD and Myf-5 expression in embryonic mesoderm and neural tissue.

To understand how the skeletal muscle lineage is induced during vertebrate embryogenesis, we have sought to identify the regulatory molecules that mediate induction of the myogenic regulatory factors MyoD and Myf-5. In this work, we demonstrate that either signals from the overlying ectoderm or Wnt and Sonic hedgehog signals can induce somitic expression of the paired box transcription factors, Pax-3 and Pax-7, concomitant with expression of Myf-5 and prior to that of MyoD. Moreover, infection of embryonic tissues in vitro with a retrovirus encoding Pax-3 is sufficient to induce expression of MyoD, Myf-5, and myogenin in both paraxial and lateral plate mesoderm in the absence of inducing tissues as well as in the neural tube. Together, these findings imply that Pax-3 may mediate activation of MyoD and Myf-5 in response to muscle-inducing signals from either the axial tissues or overlying ectoderm and identify Pax-3 as a key regulator of somitic myogenesis.

Cell Movement Patterns during Gastrulation in the Chick Are Controlled by Positive and Negative Chemotaxis Mediated by FGF4 and FGF8

During gastrulation in amniotes, epiblast cells ingress through the primitive streak and migrate away to form endodermal, mesodermal, and extraembryonic structures. Here we analyze the detailed movement trajectories of cells emerging at different anterior-posterior positions from the primitive streak, using in vivo imaging of the movement of GFP-tagged streak cells. Cells emerging at different anterior-posterior positions from the streak show characteristic cell migration patterns, in response to guidance signals from neighboring tissues. Streak cells are attracted by sources of FGF4 and repelled by sources of FGF8. The observed movement patterns of anterior streak cells can be explained by an FGF8-mediated chemorepulsion of cells away from the streak followed by chemoattraction toward an FGF4 signal produced by the forming notochord.

SPECIFIC REQUIREMENTS OF MRFS FOR THE EXPRESSION OF MUSCLE SPECIFIC MICRORNAS, MIR-1, MIR-206 AND MIR-133

The expression of three microRNAs, miR-1, miR-206 and miR-133 is restricted to skeletal myoblasts and cardiac tissue during embryo development and muscle cell differentiation, which suggests a regulation by muscle regulatory factors (MRFs). Here we show that inhibition of C2C12 muscle cell differentiation by FGFs, which interferes with the activity of MRFs, suppressed the expression of miR-1, miR-206 and miR-133. To further investigate the role of myogenic regulators (MRFs), Myf5, MyoD, Myogenin and MRF4 in the regulation of muscle specific microRNAs we performed gain and loss-of-function experiments in vivo, in chicken and mouse embryos. We found that directed expression of MRFs in the neural tube of chicken embryos induced ectopic expression of miR-1 and miR-206. Conversely, the lack of Myf5 but not of MyoD resulted in a loss of miR-1 and miR-206 expression. Taken together our results demonstrate differential requirements of distinct MRFs for the induction of microRNA gene expression during skeletal myogenesis.

Negative Feedback Regulation of FGF Signaling Levels by Pyst1/MKP3 in Chick Embryos

BACKGROUND The importance of endogenous antagonists in intracellular signal transduction pathways is becoming increasingly recognized. There is evidence in cultured mammalian cells that Pyst1/MKP3, a dual specificity protein phosphatase, specifically binds to and inactivates ERK1/2 mitogen-activated protein kinases (MAPKs). High-level Pyst1/Mkp3 expression has recently been found at many sites of known FGF signaling in mouse embryos, but the significance of this association and its function are not known. RESULTS We have cloned chicken Pyst1/Mkp3 and show that high-level expression in neural plate correlates with active MAPK. We show that FGF signaling regulates Pyst1 expression in developing neural plate and limb bud by ablating and/or transplanting tissue sources of FGFs and by applying FGF protein or a specific FGFR inhibitor (SU5402). We further show by applying a specific MAP kinase kinase inhibitor (PD184352) that Pyst1 expression is regulated via the MAPK cascade. Overexpression of Pyst1 in chick embryos reduces levels of activated MAPK in neural plate and alters its morphology and retards limb bud outgrowth. CONCLUSIONS Pyst1 is an inducible antagonist of FGF signaling in embryos and acts in a negative feedback loop to regulate the activity of MAPK. Our results demonstrate both the importance of MAPK signaling in neural induction and limb bud outgrowth and the critical role played by dual specificity MAP kinase phosphatases in regulating developmental outcomes in vertebrates.

Wiring diagrams: regulatory circuits and the control of skeletal myogenesis

During the past year, targeted mutagenesis in mice has begun to clarify the roles of individual members of the MyoD family of myogenic regulators in vertebrate development. In this review, we discuss these studies both in the context of tissue interactions necessary to induce skeletal muscle precursor cells during embryogenesis and the molecular circuitry that regulates the terminal differentiation of these cells.

Fin development in a cartilaginous fish and the origin of vertebrate limbs.

Recent fossil finds and experimental analysis of chick and mouse embryos highlighted the lateral fin fold theory, which suggests that two pairs of limbs in tetrapods evolved by subdivision of an elongated single fin. Here we examine fin development in embryos of the primitive cartilaginous fish, Scyliorhinus canicula (dogfish) using scanning electron microscopy and investigate expression of genes known to be involved in limb positioning, identity and patterning in higher vertebrates. Although we did not detect lateral fin folds in dogfish embryos, Engrailed-1 expression suggests that the body is compartmentalized dorso-ventrally. Furthermore, specification of limb identity occurs through the Tbx4 and Tbx5 genes, as in higher vertebrates. In contrast, unlike higher vertebrates, we did not detect Shh transcripts in dogfish fin-buds, although dHand (a gene involved in establishing Shh) is expressed. In S. canicula, the main fin axis seems to lie parallel to the body axis. ‘Freeing’ fins from the body axis and establishing a separate ‘limb’ axis has been proposed to be a crucial step in evolution of tetrapod limbs. We suggest that Shh plays a critical role in this process.

Feedback interactions between MKP3 and ERK MAP kinase control scleraxis expression and the specification of rib progenitors in the developing chick somite.

Cells in the early vertebrate somite receive cues from surrounding tissues, which are important for their specification. A number of signalling pathways involved in somite patterning have been described extensively. By contrast, the interactions between cells from different regions within the somite are less well characterised. Here, we demonstrate that myotomally derived FGFs act through the MAPK signal transduction cascade and in particular, ERK1/2 to activate scleraxis expression in a population of mesenchymal progenitor cells in the dorsal sclerotome. We show that the levels of active, phosphorylated ERK protein in the developing somite are crucial for the expression of scleraxis and Mkp3. MKP3 is a dual specificity phosphatase and a specific antagonist of ERK MAP kinases and we demonstrate that in somites Mkp3 transcription depends on the presence of active ERK. Therefore, MKP3 and ERK MAP kinase constitute a negative feedback loop activated by FGF in sclerotomal progenitor cells. We propose that tight control of ERK signalling strength by MKP3 is important for the appropriate regulation of downstream cellular responses including the activation of scleraxis. We show that increased or decreased levels of phosphorylated ERK result in the loss of scleraxis transcripts and the loss of distal rib development, highlighting the importance of the MKP3-ERK-MAP kinase mediated feedback loop for cell specification and differentiation.

Pax1 and Pax9 activate Bapx1 to induce chondrogenic differentiation in the sclerotome

We have previously shown that the paired-box transcription factors Pax1 and Pax9 synergistically act in the proper formation of the vertebral column. Nevertheless, downstream events of the Pax1/Pax9 action and their target genes remain to be elucidated. We show, by analyzing Pax1;Pax9 double mutant mice, that expression of Bapx1 in the sclerotome requires the presence of Pax1 and Pax9, in a gene dose-dependent manner. By using a retroviral system to overexpress Pax1 in chick presomitic mesoderm explants, we show that Pax1 can substitute for Shh in inducing Bapx1 expression and in initiating chondrogenic differentiation. Furthermore, we demonstrate that Pax1 and Pax9 can transactivate regulatory sequences in the Bapx1 promoter and that they physically interact with the Bapx1 promoter region. These results strongly suggest that Bapx1 is a direct target of Pax1 and Pax9. Together, we conclude that Pax1 and Pax9 are required and sufficient for the chondrogenic differentiation of sclerotomal cells.

Vertebrate limb development--the early stages in chick and mouse.

More news this year about FGFs and their roles in vertebrate limb initiation; Wnt signalling is shown for the first time to be another component of the signalling cascade involved in early limb formation. Ectodermal compartments that control apical ridge formation were previously described in chick embryos and are now shown to exist in mouse embryos; Engrailed1 is expressed in the ventral ectodermal compartment but experiments in both chick and mouse show that it is not responsible for compartment specification.

MicroRNA regulation of the paired-box transcription factor Pax3 confers robustness to developmental timing of myogenesis

Commitment of progenitors in the dermomyotome to myoblast fate is the first step in establishing the body musculature. Pax3 is a crucial transcription factor, important for skeletal muscle development and expressed in myogenic progenitors in the dermomyotome of developing somites and in migratory muscle progenitors that populate the limb buds. Down-regulation of Pax3 is essential to ignite the myogenic program, including up-regulation of myogenic regulators, Myf-5 and MyoD. MicroRNAs (miRNAs) confer robustness to developmental timing by posttranscriptional repression of genetic programs that are related to previous developmental stages or to alternative cell fates. Here we demonstrate that the muscle-specific miRNAs miR-1 and miR-206 directly target Pax3. Antagomir-mediated inhibition of miR-1/miR-206 led to delayed myogenic differentiation in developing somites, as shown by transient loss of myogenin expression. This correlated with increased Pax3 and was phenocopied using Pax3-specific target protectors. Loss of myogenin after antagomir injection was rescued by Pax3 knockdown using a splice morpholino, suggesting that miR-1/miR-206 control somite myogenesis primarily through interactions with Pax3. Our studies reveal an important role for miR-1/miR-206 in providing precision to the timing of somite myogenesis. We propose that posttranscriptional control of Pax3 downstream of miR-1/miR-206 is required to stabilize myoblast commitment and subsequent differentiation. Given that mutually exclusive expression of miRNAs and their targets is a prevailing theme in development, our findings suggest that miRNA may provide a general mechanism for the unequivocal commitment underlying stem cell differentiation.