A local subset of mesenchymal cells expressing the transcription factor Osr1 orchestrates lymph node initiation.

During development, lymph node (LN) initiation is coordinated by lymphoid tissue organizer (LTo) cells that attract lymphoid tissue inducer (LTi) cells at strategic positions within the embryo. The identity and function of LTo cells during the initial attraction of LTi cells remain poorly understood. Using lineage tracing, we demonstrated that a subset of Osr1-expressing cells was mesenchymal LTo progenitors. By investigating the heterogeneity of Osr1+ cells, we uncovered distinct mesenchymal LTo signatures at diverse anatomical locations, identifying a common progenitor of mesenchymal LTos and LN-associated adipose tissue. Osr1 was essential for LN initiation, driving the commitment of mesenchymal LTo cells independent of neural retinoic acid, and for LN-associated lymphatic vasculature assembly. The combined action of chemokines CXCL13 and CCL21 was required for LN initiation. Our results redefine the role and identity of mesenchymal organizer cells and unify current views by proposing a model of cooperative cell function in LN initiation.

Extraocular muscle stem cells exhibit distinct cellular properties associated with non-muscle molecular signatures.

Skeletal muscle stem cells (MuSCs) are recognised as functionally heterogeneous. Cranial MuSCs are reported to have greater proliferative and regenerative capacity when compared with those in the limb. A comprehensive understanding of the mechanisms underlying this functional heterogeneity is lacking. Here, we have used clonal analysis, live imaging and single cell transcriptomic analysis to identify crucial features that distinguish extraocular muscle (EOM) from limb muscle stem cell populations. A MyogeninntdTom reporter showed that the increased proliferation capacity of EOM MuSCs correlates with deferred differentiation and lower expression of the myogenic commitment gene Myod. Unexpectedly, EOM MuSCs activated in vitro expressed a large array of extracellular matrix components typical of mesenchymal non-muscle cells. Computational analysis underscored a distinct co-regulatory module, which is absent in limb MuSCs, as driver of these features. The EOM transcription factor network, with Foxc1 as key player, appears to be hardwired to EOM identity as it persists during growth, disease and in vitro after several passages. Our findings shed light on how high-performing MuSCs regulate myogenic commitment by remodelling their local environment and adopting properties not generally associated with myogenic cells.

Interplay between Pitx2 and Pax7 temporally governs specification of extraocular muscle stem cells.

Gene regulatory networks that act upstream of skeletal muscle fate determinants are distinct in different anatomical locations. Despite recent efforts, a clear understanding of the cascade of events underlying the emergence and maintenance of the stem cell pool in specific muscle groups remains unresolved and debated. Here, we invalidated Pitx2 with multiple Cre-driver mice prenatally, postnatally, and during lineage progression. We showed that this gene becomes progressively dispensable for specification and maintenance of the muscle stem (MuSC) cell pool in extraocular muscles (EOMs) despite being, together with Myf5, a major upstream regulator during early development. Moreover, constitutive inactivation of Pax7 postnatally led to a greater loss of MuSCs in the EOMs compared to the limb. Thus, we propose a relay between Pitx2, Myf5 and Pax7 for EOM stem cell maintenance. We demonstrate also that MuSCs in the EOMs adopt a quiescent state earlier that those in limb muscles and do not spontaneously proliferate in the adult, yet EOMs have a significantly higher content of Pax7+ MuSCs per area pre- and post-natally. Finally, while limb MuSCs proliferate in the mdx mouse model for Duchenne muscular dystrophy, significantly less MuSCs were present in the EOMs of the mdx mouse model compared to controls, and they were not proliferative. Overall, our study provides a comprehensive in vivo characterisation of MuSC heterogeneity along the body axis and brings further insights into the unusual sparing of EOMs during muscular dystrophy.

Overlapping functions of SIX homeoproteins during embryonic myogenesis.

Four SIX homeoproteins display a combinatorial expression throughout embryonic developmental myogenesis and they modulate the expression of the myogenic regulatory factors. Here, we provide a deep characterization of their role in distinct mouse developmental territories. We showed, at the hypaxial level, that the Six1:Six4 double knockout (dKO) somitic precursor cells adopt a smooth muscle fate and lose their myogenic identity. At the epaxial level, we demonstrated by the analysis of Six quadruple KO (qKO) embryos, that SIX are required for fetal myogenesis, and for the maintenance of PAX7+ progenitor cells, which differentiated prematurely and are lost by the end of fetal development in qKO embryos. Finally, we showed that Six1 and Six2 are required to establish craniofacial myogenesis by controlling the expression of Myf5. We have thus described an unknown role for SIX proteins in the control of myogenesis at different embryonic levels and refined their involvement in the genetic cascades operating at the head level and in the genesis of myogenic stem cells.

Local retinoic acid signaling directs emergence of the extraocular muscle functional unit.

Coordinated development of muscles, tendons, and their attachment sites ensures emergence of functional musculoskeletal units that are adapted to diverse anatomical demands among different species. How these different tissues are patterned and functionally assembled during embryogenesis is poorly understood. Here, we investigated the morphogenesis of extraocular muscles (EOMs), an evolutionary conserved cranial muscle group that is crucial for the coordinated movement of the eyeballs and for visual acuity. By means of lineage analysis, we redefined the cellular origins of periocular connective tissues interacting with the EOMs, which do not arise exclusively from neural crest mesenchyme as previously thought. Using 3D imaging approaches, we established an integrative blueprint for the EOM functional unit. By doing so, we identified a developmental time window in which individual EOMs emerge from a unique muscle anlage and establish insertions in the sclera, which sets these muscles apart from classical muscle-to-bone type of insertions. Further, we demonstrate that the eyeballs are a source of diffusible all-trans retinoic acid (ATRA) that allow their targeting by the EOMs in a temporal and dose-dependent manner. Using genetically modified mice and inhibitor treatments, we find that endogenous local variations in the concentration of retinoids contribute to the establishment of tendon condensations and attachment sites that precede the initiation of muscle patterning. Collectively, our results highlight how global and site-specific programs are deployed for the assembly of muscle functional units with precise definition of muscle shapes and topographical wiring of their tendon attachments.

Transcriptome and epigenome diversity and plasticity of muscle stem cells following transplantation.

Adult skeletal muscles are maintained during homeostasis and regenerated upon injury by muscle stem cells (MuSCs). A heterogeneity in self-renewal, differentiation and regeneration properties has been reported for MuSCs based on their anatomical location. Although MuSCs derived from extraocular muscles (EOM) have a higher regenerative capacity than those derived from limb muscles, the molecular determinants that govern these differences remain undefined. Here we show that EOM and limb MuSCs have distinct DNA methylation signatures associated with enhancers of location-specific genes, and that the EOM transcriptome is reprogrammed following transplantation into a limb muscle environment. Notably, EOM MuSCs expressed host-site specific positional Hox codes after engraftment and self-renewal within the host muscle. However, about 10% of EOM-specific genes showed engraftment-resistant expression, pointing to cell-intrinsic molecular determinants of the higher engraftment potential of EOM MuSCs. Our results underscore the molecular diversity of distinct MuSC populations and molecularly define their plasticity in response to microenvironmental cues. These findings provide insights into strategies designed to improve the functional capacity of MuSCs in the context of regenerative medicine.

Muscle-selective RUNX3 dependence of sensorimotor circuit development.

The control of all our motor outputs requires constant monitoring by proprioceptive sensory neurons (PSNs) that convey continuous muscle sensory inputs to the spinal motor network. Yet the molecular programs that control the establishment of this sensorimotor circuit remain largely unknown. The transcription factor RUNX3 is essential for the early steps of PSNs differentiation, making it difficult to study its role during later aspects of PSNs specification. Here, we conditionally inactivate Runx3 in PSNs after peripheral innervation and identify that RUNX3 is necessary for maintenance of cell identity of only a subgroup of PSNs, without discernable cell death. RUNX3 also controls the sensorimotor connection between PSNs and motor neurons at limb level, with muscle-by-muscle variable sensitivities to the loss of Runx3 that correlate with levels of RUNX3 in PSNs. Finally, we find that muscles and neurotrophin 3 signaling are necessary for maintenance of RUNX3 expression in PSNs. Hence, a transcriptional regulator that is crucial for specifying a generic PSN type identity after neurogenesis is later regulated by target muscle-derived signals to contribute to the specialized aspects of the sensorimotor connection selectivity.

A knock-in mouse line conditionally expressing the tumor suppressor WTX/AMER1.

WTX/AMER1 is an important developmental regulator, mutations in which have been identified in a proportion of patients suffering from the renal neoplasm Wilms' tumor and in the bone malformation syndrome Osteopathia Striata with Cranial Sclerosis (OSCS). Its cellular functions appear complex and the protein can be found at the membrane, within the cytoplasm and the nucleus. To understand its developmental and cellular function an allelic series for Wtx in the mouse is crucial. Whereas mice carrying a conditional knock out allele for Wtx have been previously reported, a gain-of-function mouse model that would allow studying the molecular, cellular and developmental role of Wtx is still missing. Here we describe the generation of a novel mouse strain that permits the conditional activation of WTX expression. Wtx fused to GFP was introduced downstream a stop cassette flanked by loxP sites into the Rosa26 locus by gene targeting. Ectopic WTX expression is reported after crosses with several Cre transgenic mice in different embryonic tissues. Further, functionality of the fusion protein was demonstrated in the context of a Wtx null allele.

Embryonic founders of adult muscle stem cells are primed by the determination gene Mrf4.

Skeletal muscle satellite cells play a critical role during muscle growth, homoeostasis and regeneration. Selective induction of the muscle determination genes Myf5, Myod and Mrf4 during prenatal development can potentially impact on the reported functional heterogeneity of adult satellite cells. Accordingly, expression of Myf5 was reported to diminish the self-renewal potential of the majority of satellite cells. In contrast, virtually all adult satellite cells showed antecedence of Myod activity. Here we examine the priming of myogenic cells by Mrf4 throughout development. Using a Cre-lox based genetic strategy and novel highly sensitive Pax7 reporter alleles compared to the ubiquitous Rosa26-based reporters, we show that all adult satellite cells, independently of their anatomical location or embryonic origin, have been primed for Mrf4 expression. Given that Mrf4Cre and Mrf4nlacZ are active exclusively in progenitors during embryogenesis, whereas later expression is restricted to differentiated myogenic cells, our findings suggest that adult satellite cells emerge from embryonic founder cells in which the Mrf4 locus was activated. Therefore, this level of myogenic priming by induction of Mrf4, does not compromise the potential of the founder cells to assume an upstream muscle stem cell state. We propose that embryonic myogenic cells and the majority of adult muscle stem cells form a lineage continuum.

Functional specialisation and coordination of myonuclei.

Myofibres serve as the functional unit for locomotion, with the sarcomere as fundamental subunit. Running the entire length of this structure are hundreds of myonuclei, located at the periphery of the myofibre, juxtaposed to the plasma membrane. Myonuclear specialisation and clustering at the centre and ends of the fibre are known to be essential for muscle contraction, yet the molecular basis of this regionalisation has remained unclear. While the 'myonuclear domain hypothesis' helped explain how myonuclei can independently govern large cytoplasmic territories, novel technologies have provided granularity on the diverse transcriptional programs running simultaneously within the syncytia and added a new perspective on how myonuclei communicate. Building upon this, we explore the critical cellular and molecular sources of transcriptional and functional heterogeneity within myofibres, discussing the impact of intrinsic and extrinsic factors on myonuclear programs. This knowledge provides new insights for understanding muscle development, repair, and disease, but also opens avenues for the development of novel and precise therapeutic approaches.

Identification of bipotent progenitors that give rise to myogenic and connective tissues in mouse.

How distinct cell fates are manifested by direct lineage ancestry from bipotent progenitors, or by specification of individual cell types is a key question for understanding the emergence of tissues. The interplay between skeletal muscle progenitors and associated connective tissue cells provides a model for examining how muscle functional units are established. Most craniofacial structures originate from the vertebrate-specific neural crest cells except in the dorsal portion of the head, where they arise from cranial mesoderm. Here, using multiple lineage-tracing strategies combined with single cell RNAseq and in situ analyses, we identify bipotent progenitors expressing Myf5 (an upstream regulator of myogenic fate) that give rise to both muscle and juxtaposed connective tissue. Following this bifurcation, muscle and connective tissue cells retain complementary signalling features and maintain spatial proximity. Disrupting myogenic identity shifts muscle progenitors to a connective tissue fate. The emergence of Myf5-derived connective tissue is associated with the activity of several transcription factors, including Foxp2. Interestingly, this unexpected bifurcation in cell fate was not observed in craniofacial regions that are colonised by neural crest cells. Therefore, we propose that an ancestral bi-fated program gives rise to muscle and connective tissue cells in skeletal muscles that are deprived of neural crest cells.

Expression patterns of the Wtx/Amer gene family during mouse embryonic development.

WTX/AMER1 is a novel negative regulator of the WNT/beta-catenin pathway with mutations detected in Wilms' tumors and an X-linked sclerosing bone dysplasia. WTX/AMER1 (Fam123b) shares several domains of homology with two other recently identified proteins: AMER2 (Fam123a) and AMER3 (Fam123c). Here, we describe an in-depth expression analysis of all three members of this gene family during mouse embryonic development. All genes were strongly expressed in the central as well as the peripheral nervous system, thus suggesting important roles of this gene family during neurogenesis. Specific expression was found in the retina, inner ear, and nasal epithelium. Outside of the nervous system Wtx/Amer1 showed the broadest expression domains including cephalic and limb mesenchyme, skeletal muscle, bladder, gonads, lung bud, salivary glands, and the kidneys. The widespread expression pattern of Wtx/Amer1, together with its role as a modulator of the Wnt signaling pathway, suggest that Wtx/Amer1 serves pleiotropic roles during mammalian organogenesis.

Dynamics of myogenic differentiation using a novel Myogenin knock-in reporter mouse.

BACKGROUND: Myogenin is a transcription factor that is expressed during terminal myoblast differentiation in embryonic development and adult muscle regeneration. Investigation of this cell state transition has been hampered by the lack of a sensitive reporter to dynamically track cells during differentiation. RESULTS: Here, we report a knock-in mouse line expressing the tdTOMATO fluorescent protein from the endogenous Myogenin locus. Expression of tdTOMATO in MyogntdTom mice recapitulated endogenous Myogenin expression during embryonic muscle formation and adult regeneration and enabled the isolation of the MYOGENIN+ cell population. We also show that tdTOMATO fluorescence allows tracking of differentiating myoblasts in vitro and by intravital imaging in vivo. Lastly, we monitored by live imaging the cell division dynamics of differentiating myoblasts in vitro and showed that a fraction of the MYOGENIN+ population can undergo one round of cell division, albeit at a much lower frequency than MYOGENIN- myoblasts. CONCLUSIONS: We expect that this reporter mouse will be a valuable resource for researchers investigating skeletal muscle biology in developmental and adult contexts.

Unexpected contribution of fibroblasts to muscle lineage as a mechanism for limb muscle patterning.

Positional information driving limb muscle patterning is contained in connective tissue fibroblasts but not in myogenic cells. Limb muscles originate from somites, while connective tissues originate from lateral plate mesoderm. With cell and genetic lineage tracing we challenge this model and identify an unexpected contribution of lateral plate-derived fibroblasts to the myogenic lineage, preferentially at the myotendinous junction. Analysis of single-cell RNA-sequencing data from whole limbs at successive developmental stages identifies a population displaying a dual muscle and connective tissue signature. BMP signalling is active in this dual population and at the tendon/muscle interface. In vivo and in vitro gain- and loss-of-function experiments show that BMP signalling regulates a fibroblast-to-myoblast conversion. These results suggest a scenario in which BMP signalling converts a subset of lateral plate mesoderm-derived cells to a myogenic fate in order to create a boundary of fibroblast-derived myonuclei at the myotendinous junction that controls limb muscle patterning.

The WTX/AMER1 gene family: evolution, signature and function.

BACKGROUND: WTX is a novel gene mutated in a proportion of Wilms' tumors and in patients suffering from sclerosing bone dysplasia. On the molecular level WTX has been shown to act as an antagonist of canonical Wnt/ß-catenin signaling in fish and mammals thus linking it to an essential pathway involved in normal development and cancer formation. Interestingly, WTX seems to also localize to an intranuclear component called paraspeckles. In spite of the growing interest of molecular biologists in WTX, little is known about its paralogs and its phylogenetic history. RESULTS: Using the amino-acid sequence of WTX/AMER1 as a tool for the assignment of orthology and paralogy, we here identify two novel proteins, AMER2 and AMER3, as "WTX" related. This Amer gene family is present in all currently available vertebrate genome sequences, but not invertebrate genomes and is characterized by six conserved blocks of sequences. The phylogenetic analysis suggests that the protoAmer gene originated early in the vertebrate lineage and was then duplicated due to whole genome duplications (WGD) giving rise to the three different Amer genes. CONCLUSION: Our study represents the first phylogenetic analysis of Amer genes and reveals a new vertebrate specific gene family that is likely to have played an important role in the evolution of this subphylum. Divergent and conserved molecular functions of Wtx/Amer1, Amer2 and Amer3 are discussed.

Dullard-mediated Smad1/5/8 inhibition controls mouse cardiac neural crest cells condensation and outflow tract septation.

The establishment of separated pulmonary and systemic circulation in vertebrates, via cardiac outflow tract (OFT) septation, is a sensitive developmental process accounting for 10% of all congenital anomalies. Neural Crest Cells (NCC) colonising the heart condensate along the primitive endocardial tube and force its scission into two tubes. Here, we show that NCC aggregation progressively decreases along the OFT distal-proximal axis following a BMP signalling gradient. Dullard, a nuclear phosphatase, tunes the BMP gradient amplitude and prevents NCC premature condensation. Dullard maintains transcriptional programs providing NCC with mesenchymal traits. It attenuates the expression of the aggregation factor Sema3c and conversely promotes that of the epithelial-mesenchymal transition driver Twist1. Altogether, Dullard-mediated fine-tuning of BMP signalling ensures the timed and progressive zipper-like closure of the OFT by the NCC and prevents the formation of a heart carrying the congenital abnormalities defining the tetralogy of Fallot.

A distinct cardiopharyngeal mesoderm genetic hierarchy establishes antero-posterior patterning of esophagus striated muscle.

In most vertebrates, the upper digestive tract is composed of muscularized jaws linked to the esophagus that permits food ingestion and swallowing. Masticatory and esophagus striated muscles (ESM) share a common cardiopharyngeal mesoderm (CPM) origin, however ESM are unusual among striated muscles as they are established in the absence of a primary skeletal muscle scaffold. Using mouse chimeras, we show that the transcription factors Tbx1 and Isl1 are required cell-autonomously for myogenic specification of ESM progenitors. Further, genetic loss-of-function and pharmacological studies point to MET/HGF signaling for antero-posterior migration of esophagus muscle progenitors, where Hgf ligand is expressed in adjacent smooth muscle cells. These observations highlight the functional relevance of a smooth and striated muscle progenitor dialogue for ESM patterning. Our findings establish a Tbx1-Isl1-Met genetic hierarchy that uniquely regulates esophagus myogenesis and identify distinct genetic signatures that can be used as framework to interpret pathologies arising within CPM derivatives.

An interactive and intuitive visualisation method for X-ray computed tomography data of biological samples in 3D Portable Document Format.

3D imaging approaches based on X-ray microcomputed tomography (microCT) have become increasingly accessible with advancements in methods, instruments and expertise. The synergy of material and life sciences has impacted biomedical research by proposing new tools for investigation. However, data sharing remains challenging as microCT files are usually in the range of gigabytes and require specific and expensive software for rendering and interpretation. Here, we provide an advanced method for visualisation and interpretation of microCT data with small file formats, readable on all operating systems, using freely available Portable Document Format (PDF) software. Our method is based on the conversion of volumetric data into interactive 3D PDF, allowing rotation, movement, magnification and setting modifications of objects, thus providing an intuitive approach to analyse structures in a 3D context. We describe the complete pipeline from data acquisition, data processing and compression, to 3D PDF formatting on an example of craniofacial anatomical morphology in the mouse embryo. Our procedure is widely applicable in biological research and can be used as a framework to analyse volumetric data from any research field relying on 3D rendering and CT-biomedical imaging.

A cell fitness selection model for neuronal survival during development.

Developmental cell death plays an important role in the construction of functional neural circuits. In vertebrates, the canonical view proposes a selection of the surviving neurons through stochastic competition for target-derived neurotrophic signals, implying an equal potential for neurons to compete. Here we show an alternative cell fitness selection of neurons that is defined by a specific neuronal heterogeneity code. Proprioceptive sensory neurons that will undergo cell death and those that will survive exhibit different molecular signatures that are regulated by retinoic acid and transcription factors, and are independent of the target and neurotrophins. These molecular features are genetically encoded, representing two distinct subgroups of neurons with contrasted functional maturation states and survival outcome. Thus, in this model, a heterogeneous code of intrinsic cell fitness in neighboring neurons provides differential competitive advantage resulting in the selection of cells with higher capacity to survive and functionally integrate into neural networks.

Glutathione produced by Rhizobium tropici is important to prevent early senescence in common bean nodules.

In this paper, we examine the importance of glutathione in symbiosis, using a glutathione biosynthetic gshB mutant derived from Rhizobium tropici CIAT899, a common bean (Phaseolus vulgaris) endosymbiont. Plants infected with the mutant strain presented a delayed nodulation phenotype and a reduction in the dry weight of aerial part of plants, suggesting diminished nitrogen-fixation activity. In addition, bacterial gshB expression was assayed in wild-type infected nodules, during the different steps of nodulation, and found to increase in mature and early senescent nodules. Conspicuously, nodules induced by gshB mutant bacteria presented an early senescent pattern, which was associated with increased levels of superoxide accumulation. These results provide a direct evidence of the role of bacterial glutathione in protecting nodules from reactive oxygen species, which may determine nodule senescence.