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.

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.

Cubam receptor-mediated endocytosis in hindgut-derived pseudoplacenta of a viviparous teleost (Xenotoca eiseni).

Nutrient transfer from mother to embryo is essential for reproduction in viviparous animals. In the viviparous teleost Xenotoca eiseni (family Goodeidae), the intraovarian embryo intakes the maternal component secreted into the ovarian fluid via the trophotaenia. Our previous study reported that the epithelial layer cells of the trophotaenia incorporate a maternal protein via vesicle trafficking. However, the molecules responsible for the absorption were still elusive. Here, we focused on Cubam (Cubilin-Amnionless) as a receptor involved in the absorption, and cathepsin L as a functional protease in the vesicles. Our results indicated that the Cubam receptor is distributed in the apical surface of the trophotaenia epithelium and then is taken into the intracellular vesicles. The trophotaenia possesses acidic organelles in epithelial layer cells and cathepsin L-dependent proteolysis activity. This evidence does not conflict with our hypothesis that receptor-mediated endocytosis and proteolysis play roles in maternal macromolecule absorption via the trophotaenia in viviparous teleosts. Such nutrient absorption involving endocytosis is not a specific trait in viviparous fish. Similar processes have been reported in the larval stage of oviparous fish or the suckling stage of viviparous mammals. Our findings suggest that the viviparous teleost acquired trophotaenia-based viviparity from a modification of the intestinal absorption system common in vertebrates. This is a fundamental study to understand the strategic variation of the reproductive system in vertebrates.

Transient and lineage-restricted requirement of Ebf3 for sternum ossification.

Osteoblasts arise from bone-surrounding connective tissue containing tenocytes and fibroblasts. Lineages of these cell populations and mechanisms of their differentiation are not well understood. Screening enhancer-trap lines of zebrafish allowed us to identify Ebf3 as a transcription factor marking tenocytes and connective tissue cells in skeletal muscle of embryos. Knockout of Ebf3 in mice had no effect on chondrogenesis but led to sternum ossification defects as a result of defective generation of Runx2+ pre-osteoblasts. Conditional and temporal Ebf3 knockout mice revealed requirements of Ebf3 in the lateral plate mesenchyme cells (LPMs), especially in tendon/muscle connective tissue cells, and a stage-specific Ebf3 requirement at embryonic day 9.5-10.5. Upregulated expression of connective tissue markers, such as Egr1/2 and Osr1, increased number of Islet1+ mesenchyme cells, and downregulation of gene expression of the Runx2 regulator Shox2 in Ebf3-deleted thoracic LPMs suggest crucial roles of Ebf3 in the onset of lateral plate mesoderm differentiation towards osteoblasts forming sternum tissues.

Giantin is required for coordinated production of aggrecan, link protein and type XI collagen during chondrogenesis.

Extracellular matrix (ECM) constitutes a proper micro-environment for cell proliferation, migration and differentiation, as well as playing pivotal roles in developmental processes including endochondral ossification. Cartilage ECM is mainly composed of fibrous proteins, including collagen, proteoglycan, and hyaluronan. Because almost all ECM components are transported by intracellular vesicular transport systems, molecules that mediate vesicle transport are also important for endochondral ossification. Giantin, encoded by the Golgb1 gene, is a tethering factor for coatomer 1 (COPI) vesicles and functions in the cis-medial Golgi compartments. An insertion mutation in the Golgb1 gene, resulting in a lack of giantin protein expression, has been detected in ocd/ocd rats that exhibit a pleiotropic phenotype including osteochondrodysplasia. To reveal the function of giantin in chondrogenesis, the present study assessed the effects of loss of giantin expression on cartilage ECM and Golgi morphology. Giantin was expressed in normal, but not in ocd/ocd, chondrocytes in the epiphyseal areas of embryonic femurs, whereas GM130 was expressed in both normal and ocd/ocd chondrocytes. The staining intensities of safranin O and azan (aniline blue) were reduced and enhanced, respectively, in epiphyseal cartilage of ocd/ocd femurs. Immunostaining showed that levels of type II collagen and fibronectin were comparable in normal and ocd/ocd cartilage. Levels of type XI collagen were higher, while levels of aggrecan, link protein and hyaluronan were lower, in ocd/ocd than in normal cartilage, although semi-quantitative RT-PCR showed similar levels of type XI collagen, aggrecan and link protein mRNAs in normal and ocd/ocd cartilage. Isolated chondrocytes of ocd/ocd and normal rats showed similar immunostaining patterns for cis-, medial-, and trans-Golgi marker proteins, whereas monolayers of ocd/ocd chondrocytes showed reduced levels of aggrecan and link protein and increased level of type XI collagen in spite of similar transcripts levels. These findings suggest that giantin plays a pivotal role in coordinated production of aggrecan, link protein and type XI collagen in chondrocytes, and that loss of giantin causes osteochondrodysplasia with disturbance of these ECM components.