Absence of the primary cilia formation gene Talpid3 impairs muscle stem cell function.

Skeletal muscle stem cells (MuSC) are crucial for tissue homoeostasis and repair after injury. Following activation, they proliferate to generate differentiating myoblasts. A proportion of cells self-renew, re-enter the MuSC niche under the basal lamina outside the myofiber and become quiescent. Quiescent MuSC have a primary cilium, which is disassembled upon cell cycle entry. Ex vivo experiments suggest cilia are important for MuSC self-renewal, however, their requirement for muscle regeneration in vivo remains poorly understood. Talpid3 (TA3) is essential for primary cilia formation and Hedgehog (Hh) signalling. Here we use tamoxifen-inducible conditional deletion of TA3 in MuSC (iSC-KO) and show that regeneration is impaired in response to cytotoxic injury. Depletion of MuSC after regeneration suggests impaired self-renewal, also consistent with an exacerbated phenotype in TA3iSC-KO mice after repeat injury. Single cell transcriptomics of MuSC progeny isolated from myofibers identifies components of several signalling pathways, which are deregulated in absence of TA3, including Hh and Wnt. Pharmacological activation of Wnt restores muscle regeneration, while purmorphamine, an activator of the Smoothened (Smo) co-receptor in the Hh pathway, has no effect. Together, our data show that TA3 and primary cilia are important for MuSC self-renewal and pharmacological treatment can efficiently restore muscle regeneration.

A transcriptional and regulatory map of mouse somite maturation.

The mammalian body plan is shaped by rhythmic segmentation of mesoderm into somites, which are transient embryonic structures that form down each side of the neural tube. We have analyzed the genome-wide transcriptional and chromatin dynamics occurring within nascent somites, from early inception of somitogenesis to the latest stages of body plan establishment. We created matched gene expression and open chromatin maps for the three leading pairs of somites at six time points during mouse embryonic development. We show that the rate of somite differentiation accelerates as development progresses. We identified a conserved maturation program followed by all somites, but somites from more developed embryos concomitantly switch on differentiation programs from derivative cell lineages soon after segmentation. Integrated analysis of the somitic transcriptional and chromatin activities identified opposing regulatory modules controlling the onset of differentiation. Our results provide a powerful, high-resolution view of the molecular genetics underlying somitic development in mammals.

Investigating chromatin accessibility during development and differentiation by ATAC-sequencing to guide the identification of cis-regulatory elements.

Mapping accessible chromatin across time scales can give insights into its dynamic nature, for example during cellular differentiation and tissue or organism development. Analysis of such data can be utilised to identify functional cis-regulatory elements (CRE) and transcription factor binding sites and, when combined with transcriptomics, can reveal gene regulatory networks (GRNs) of expressed genes. Chromatin accessibility mapping is a powerful approach and can be performed using ATAC-sequencing (ATAC-seq), whereby Tn5 transposase inserts sequencing adaptors into genomic DNA to identify differentially accessible regions of chromatin in different cell populations. It requires low sample input and can be performed and analysed relatively quickly compared with other methods. The data generated from ATAC-seq, along with other genomic approaches, can help uncover chromatin packaging and potential cis-regulatory elements that may be responsible for gene expression. Here, we describe the ATAC-seq approach and give examples from mainly vertebrate embryonic development, where such datasets have identified the highly dynamic nature of chromatin, with differing landscapes between cellular precursors for different lineages.

Characterising open chromatin in chick embryos identifies cis-regulatory elements important for paraxial mesoderm formation and axis extension.

Somites arising from paraxial mesoderm are a hallmark of the segmented vertebrate body plan. They form sequentially during axis extension and generate musculoskeletal cell lineages. How paraxial mesoderm becomes regionalised along the axis and how this correlates with dynamic changes of chromatin accessibility and the transcriptome remains unknown. Here, we report a spatiotemporal series of ATAC-seq and RNA-seq along the chick embryonic axis. Footprint analysis shows differential coverage of binding sites for several key transcription factors, including CDX2, LEF1 and members of HOX clusters. Associating accessible chromatin with nearby expressed genes identifies cis-regulatory elements (CRE) for TCF15 and MEOX1. We determine their spatiotemporal activity and evolutionary conservation in Xenopus and human. Epigenome silencing of endogenous CREs disrupts TCF15 and MEOX1 gene expression and recapitulates phenotypic abnormalities of anterior-posterior axis extension. Our integrated approach allows dissection of paraxial mesoderm regulatory circuits in vivo and has implications for investigating gene regulatory networks.

4D Live Imaging and Analysis of Chick Embryo Somites.

Avian (chick) embryos are an established and accessible model organism making them ideal for studying developmental processes. Chick embryos can be harvested from the egg and cultured allowing real-time observations and imaging. Here, we describe ex vivo culture and preparation of somite tissue followed by time-lapse multi-photon microscopy, image capture and processing. We applied this approach to perform live imaging of somites, the paired segments in vertebrate embryos that form in a regular sequence on either side of the neural tube, posteriorly from presomitic mesoderm (psm). Somites give rise to cell lineages of the musculoskeletal system in the trunk such as skeletal muscle, cartilage and tendon, as well as endothelial cells. Until recently it was not possible to observe the cellular dynamics underlying morphological transitions in live tissue, including in somites which undergo epithelial-to-mesenchymal transitions (EMT) during their differentiation. In addition to the experimental setup, we describe the analytical tools used for image processing.

The Pax6 master control gene initiates spontaneous retinal development via a self-organising Turing network.

Understanding how complex organ systems are assembled from simple embryonic tissues is a major challenge. Across the animal kingdom a great diversity of visual organs are initiated by a 'master control gene' called Pax6, which is both necessary and sufficient for eye development. Yet precisely how Pax6 achieves this deeply homologous function is poorly understood. Using the chick as a model organism, we show that vertebrate Pax6 interacts with a pair of morphogen-coding genes, Tgfb2 and Fst, to form a putative Turing network, which we have computationally modelled. Computer simulations suggest that this gene network is sufficient to spontaneously polarise the developing retina, establishing the first organisational axis of the eye and prefiguring its further development. Our findings reveal how retinal self-organisation may be initiated independently of the highly ordered tissue interactions that help to assemble the eye in vivo These results help to explain how stem cell aggregates spontaneously self-organise into functional eye-cups in vitro We anticipate these findings will help to underpin retinal organoid technology, which holds much promise as a platform for disease modelling, drug development and regenerative therapies.

4D imaging reveals stage dependent random and directed cell motion during somite morphogenesis.

Somites are paired embryonic segments that form in a regular sequence from unsegmented mesoderm during vertebrate development. Although transient structures they are of fundamental importance as they generate cell lineages of the musculoskeletal system in the trunk such as cartilage, tendon, bone, endothelial cells and skeletal muscle. Surprisingly, very little is known about cellular dynamics underlying the morphological transitions during somite differentiation. Here, we address this by examining cellular rearrangements and morphogenesis in differentiating somites using live multi-photon imaging of transgenic chick embryos, where all cells express a membrane-bound GFP. We specifically focussed on the dynamic cellular changes in two principle regions within the somite, the medial and lateral domains, to investigate extensive morphological transformations. Furthermore, by using quantitative analysis and cell tracking, we capture for the first time a directed movement of dermomyotomal progenitor cells towards the rostro-medial domain of the dermomyotome, where skeletal muscle formation initiates.

miR-133-mediated regulation of the Hedgehog pathway orchestrates embryo myogenesis.

Skeletal myogenesis serves as a paradigm to investigate the molecular mechanisms underlying exquisitely regulated cell fate decisions in developing embryos. The evolutionarily conserved miR-133 family of microRNAs is expressed in the myogenic lineage, but how it acts remains incompletely understood. Here, we performed genome-wide differential transcriptomics of miR-133 knockdown (KD) embryonic somites, the source of vertebrate skeletal muscle. These analyses, performed in chick embryos, revealed extensive downregulation of Sonic hedgehog (Shh) pathway components: patched receptors, Hedgehog interacting protein and the transcriptional activator Gli1. By contrast, Gli3, a transcriptional repressor, was de-repressed and confirmed as a direct miR-133 target. Phenotypically, miR-133 KD impaired myotome formation and growth by disrupting proliferation, extracellular matrix deposition and epithelialization. Together, these observations suggest that miR-133-mediated Gli3 silencing is crucial for embryonic myogenesis. Consistent with this idea, we found that activation of Shh signalling by either purmorphamine, or KD of Gli3 by antisense morpholino, rescued the miR-133 KD phenotype. Thus, we identify a novel Shh/myogenic regulatory factor/miR-133/Gli3 axis that connects epithelial morphogenesis with myogenic fate specification.

microRNAs in skeletal muscle development.

A fundamental process during both embryo development and stem cell differentiation is the control of cell lineage determination. In developing skeletal muscle, many of the diffusible signaling molecules, transcription factors and more recently non-coding RNAs that contribute to this process have been identified. This has facilitated advances in our understanding of the molecular mechanisms underlying the control of cell fate choice. Here we will review the role of non-coding RNAs, in particular microRNAs (miRNAs), in embryonic muscle development and differentiation, and in satellite cells of adult muscle, which are essential for muscle growth and regeneration. Some of these short post-transcriptional regulators of gene expression are restricted to skeletal muscle, but their expression can also be more widespread. In addition, we discuss a few examples of long non-coding RNAs, which are numerous but much less well understood.

Expression of myogenic regulatory factors in chicken embryos during somite and limb development.

The expression of the myogenic regulatory factors (MRFs), Myf5, MyoD, myogenin (Mgn) and MRF4 have been analysed during the development of chicken embryo somites and limbs. In somites, Myf5 is expressed first in somites and paraxial mesoderm at HH stage 9 followed by MyoD at HH stage 12, and Mgn and MRF4 at HH stage 14. In older somites, Myf5 and MyoD are also expressed in the ventrally extending myotome prior to Mgn and MRF4 expression. In limb muscles a similar temporal sequence is observed with Myf5 expression detected first in forelimbs at HH stage 22, MyoD at HH stage 23, Mgn at HH stage 24 and MRF4 at HH stage 30. This report describes the precise time of onset of expression of each MRF in somites and limbs during chicken embryo development, and provides a detailed comparative timeline of MRF expression in different embryonic muscle groups.

Klhl31 attenuates ß-catenin dependent Wnt signaling and regulates embryo myogenesis.

Klhl31 is a member of the Kelch-like family in vertebrates, which are characterized by an amino-terminal broad complex tram-track, bric-a-brac/poxvirus and zinc finger (BTB/POZ) domain, carboxy-terminal Kelch repeats and a central linker region (Back domain). In developing somites Klhl31 is highly expressed in the myotome downstream of myogenic regulators (MRF), and it remains expressed in differentiated skeletal muscle. In vivo gain- and loss-of-function approaches in chick embryos reveal a role of Klhl31 in skeletal myogenesis. Targeted mis-expression of Klhl31 led to a reduced size of dermomyotome and myotome as indicated by detection of relevant myogenic markers, Pax3, Myf5, myogenin and myosin heavy chain (MF20). The knock-down of Klhl31 in developing somites, using antisense morpholinos (MO), led to an expansion of Pax3, Myf5, MyoD and myogenin expression domains and an increase in the number of mitotic cells in the dermomyotome and myotome. The mechanism underlying this phenotype was examined using complementary approaches, which show that Klhl31 interferes with ß-catenin dependent Wnt signaling. Klhl31 reduced the Wnt-mediated activation of a luciferase reporter in cultured cells. Furthermore, Klhl31 attenuated secondary axis formation in Xenopus embryos in response to Wnt1 or ß-catenin. Klhl31 mis-expression in the developing neural tube affected its dorso-ventral patterning and led to reduced dermomyotome and myotome size. Co-transfection of a Wnt3a expression vector with Klhl31 in somites or in the neural tube rescued the phenotype and restored the size of dermomyotome and myotome. Thus, Klhl31 is a novel modulator of canonical Wnt signaling, important for vertebrate myogenesis. We propose that Klhl31 acts in the myotome to support cell cycle withdrawal and differentiation.

Interactions between FGF18 and retinoic acid regulate differentiation of chick embryo limb myoblasts.

During limb development Pax3 positive myoblasts delaminate from the hypaxial dermomyotome of limb level somites and migrate into the limb bud where they form the dorsal and ventral muscle masses. Only then do they begin to differentiate and express markers of myogenic commitment and determination such as Myf5 and MyoD. However the signals regulating this process remain poorly characterised. We show that FGF18, which is expressed in the distal mesenchyme of the limb bud, induces premature expression of both Myf5 and MyoD and that blocking FGF signalling also inhibits endogenous MyoD expression. This expression is mediated by ERK MAP kinase but not PI3K signalling. We also show that retinoic acid (RA) can inhibit the myogenic activity of FGF18 and that blocking RA signalling allows premature induction of MyoD by FGF18 at HH19. We propose a model where interactions between FGF18 in the distal limb and retinoic acid in the proximal limb regulate the timing of myogenic gene expression during limb bud development.

myomiR-dependent switching of BAF60 variant incorporation into Brg1 chromatin remodeling complexes during embryo myogenesis.

Myogenesis involves the stable commitment of progenitor cells followed by the execution of myogenic differentiation, processes that are coordinated by myogenic regulatory factors, microRNAs and BAF chromatin remodeling complexes. BAF60a, BAF60b and BAF60c are structural subunits of the BAF complex that bind to the core ATPase Brg1 to provide functional specificity. BAF60c is essential for myogenesis; however, the mechanisms regulating the subunit composition of BAF/Brg1 complexes, in particular the incorporation of different BAF60 variants, are not understood. Here we reveal their dynamic expression during embryo myogenesis and uncover the concerted negative regulation of BAF60a and BAF60b by the muscle-specific microRNAs (myomiRs) miR-133 and miR-1/206 during somite differentiation. MicroRNA inhibition in chick embryos leads to increased BAF60a or BAF60b levels, a concomitant switch in BAF/Brg1 subunit composition and delayed myogenesis. The phenotypes are mimicked by sustained BAF60a or BAF60b expression and are rescued by morpholino knockdown of BAF60a or BAF60b. This suggests that myomiRs contribute to select BAF60c for incorporation into the Brg1 complex by specifically targeting the alternative variants BAF60a and BAF60b during embryo myogenesis, and reveals that interactions between tissue-specific non-coding RNAs and chromatin remodeling factors confer robustness to mesodermal lineage determination.

Smad1 transcription factor integrates BMP2 and Wnt3a signals in migrating cardiac progenitor cells.

In vertebrate embryos, cardiac progenitor cells (CPCs) undergo long-range migration after emerging from the primitive streak during gastrulation. Together with other mesoderm progenitors, they migrate laterally and then toward the ventral midline, where they form the heart. Signals controlling the migration of different progenitor cell populations during gastrulation are poorly understood. Several pathways are involved in the epithelial-to-mesenchymal transition and ingression of mesoderm cells through the primitive streak, including fibroblast growth factors and wingless-type family members (Wnt). Here we focus on early CPC migration and use live video microscopy in chicken embryos to demonstrate a role for bone morphogenetic protein (BMP)/SMA and MAD related (Smad) signaling. We identify an interaction of BMP and Wnt/glycogen synthase kinase 3 beta (GSK3ß) pathways via the differential phosphorylation of Smad1. Increased BMP2 activity altered migration trajectories of prospective cardiac cells and resulted in their lateral displacement and ectopic differentiation, as they failed to reach the ventral midline. Constitutively active BMP receptors or constitutively active Smad1 mimicked this phenotype, suggesting a cell autonomous response. Expression of GSK3ß, which promotes the turnover of active Smad1, rescued the BMP-induced migration phenotype. Conversely, expression of GSK3ß-resistant Smad1 resulted in aberrant CPC migration trajectories. De-repression of GSK3ß by dominant negative Wnt3a restored normal migration patterns in the presence of high BMP activity. The data indicate the convergence of BMP and Wnt pathways on Smad1 during the early migration of prospective cardiac cells. Overall, we reveal molecular mechanisms that contribute to the emerging paradigm of signaling pathway integration in embryo development.

Many routes to the same destination: lessons from skeletal muscle development.

The development and differentiation of vertebrate skeletal muscle provide an important paradigm to understand the inductive signals and molecular events controlling differentiation of specific cell types. Recent findings show that a core transcriptional network, initiated by the myogenic regulatory factors (MRFs; MYF5, MYOD, myogenin and MRF4), is activated by separate populations of cells in embryos in response to various signalling pathways. This review will highlight how cells from multiple distinct starting points can converge on a common set of regulators to generate skeletal muscle.