Neonatal Enthesis Healing Involves Noninflammatory Acellular Scar Formation through Extracellular Matrix Secretion by Resident Cells.

Wound healing typically recruits the immune and vascular systems to restore tissue structure and function. However, injuries to the enthesis, a hypocellular and avascular tissue, often result in fibrotic scar formation and loss of mechanical properties, severely affecting musculoskeletal function and life quality. This raises questions about the healing capabilities of the enthesis. Herein, this study established an injury model to the Achilles entheses of neonatal mice to study the effectiveness of early-age enthesis healing. Histology and immunohistochemistry analyses revealed an atypical process that did not involve inflammation or angiogenesis. Instead, healing was mediated by secretion of collagen types I and II by resident cells, which formed a permanent hypocellular and avascular scar. Transmission electron microscopy showed that the cellular response to injury, including endoplasmic reticulum stress, autophagy, and cell death, varied between the tendon and cartilage ends of the enthesis. Single-molecule in situ hybridization, immunostaining, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays verified these differences. Finally, gait analysis showed that these processes effectively restored function of the injured leg. These findings reveal a novel healing mechanism in neonatal entheses, whereby local extracellular matrix secretion by resident cells forms an acellular extracellular matrix deposit without inflammation, allowing gait restoration. These insights into the healing mechanism of a complex transitional tissue may lead to new therapeutic strategies for adult enthesis injuries.

Vasorin: a newly identified regulator of ovarian folliculogenesis.

Members of the TGF-ß superfamily take part in the control of folliculogenesis. Vasorin (Vasn) is a newly identified negative regulator of TGF-ß signaling whose possible involvement in ovarian physiology has never been studied. Here, we demonstrate that Vasn is expressed in the ovary by somatic cells of follicles, and that its expression is up-regulated by LH. We established a conditional knockout (cKO) mouse model in which Vasn is deleted specifically in granulosa cells of growing follicles from the secondary stage onwards. Using this model, we show that, upon hormonal stimulation, follicle ovulation size is almost 2-fold higher. This enhanced ovulatory response is associated with overactivation of the TGF-ß signaling pathway and a lower number of atretic antral follicles. Of importance, we demonstrate that the number of primordial follicles is reduced in prepubertal cKO mouse ovaries, which suggests that the production of VASN by growing follicles protects the ovarian reserve. Finally, analysis of systemic KO mice revealed that the ovarian reserve is almost 2.5-fold higher, which implies that Vasn may also play a role in primordial follicle formation. Overall, our findings reveal that Vasn is a new regulator that exerts an effect on several key ovarian functions, including folliculogenesis, maintenance of the ovarian reserve, and ovulation.-Rimon-Dahari, N., Heinemann-Yerushalmi, L., Hadas, R., Kalich-Philosoph, L., Ketter, D., Nevo, N., Galiani, D., Dekel, N. Vasorin: a newly identified regulator of ovarian folliculogenesis.

Molecular characterization of the intact mouse muscle spindle using a multi-omics approach.

The proprioceptive system is essential for the control of coordinated movement, posture, and skeletal integrity. The sense of proprioception is produced in the brain using peripheral sensory input from receptors such as the muscle spindle, which detects changes in the length of skeletal muscles. Despite its importance, the molecular composition of the muscle spindle is largely unknown. In this study, we generated comprehensive transcriptomic and proteomic datasets of the entire muscle spindle isolated from the murine deep masseter muscle. We then associated differentially expressed genes with the various tissues composing the spindle using bioinformatic analysis. Immunostaining verified these predictions, thus establishing new markers for the different spindle tissues. Utilizing these markers, we identified the differentiation stages the spindle capsule cells undergo during development. Together, these findings provide comprehensive molecular characterization of the intact spindle as well as new tools to study its development and function in health and disease.

BCKDK regulates the TCA cycle through PDC in the absence of PDK family during embryonic development.

Pyruvate dehydrogenase kinases (PDK1-4) inhibit the TCA cycle by phosphorylating pyruvate dehydrogenase complex (PDC). Here, we show that PDK family is dispensable for murine embryonic development and that BCKDK serves as a compensatory mechanism by inactivating PDC. First, we knocked out all four Pdk genes one by one. Surprisingly, Pdk total KO embryos developed and were born in expected ratios but died by postnatal day 4 because of hypoglycemia or ketoacidosis. Moreover, PDC was phosphorylated in these embryos, suggesting that another kinase compensates for PDK family. Bioinformatic analysis implicated branched-chain ketoacid dehydrogenase kinase (Bckdk), a key regulator of branched-chain amino acids (BCAAs) catabolism. Indeed, knockout of Bckdk and Pdk family led to the loss of PDC phosphorylation, an increase in PDC activity and pyruvate entry into the TCA cycle, and embryonic lethality. These findings reveal a regulatory crosstalk hardwiring BCAA and glucose catabolic pathways, which feed the TCA cycle.

Piezo2 expressed in proprioceptive neurons is essential for skeletal integrity.

In humans, mutations in the PIEZO2 gene, which encodes for a mechanosensitive ion channel, were found to result in skeletal abnormalities including scoliosis and hip dysplasia. Here, we show in mice that loss of Piezo2 expression in the proprioceptive system recapitulates several human skeletal abnormalities. While loss of Piezo2 in chondrogenic or osteogenic lineages does not lead to human-like skeletal abnormalities, its loss in proprioceptive neurons leads to spine malalignment and hip dysplasia. To validate the non-autonomous role of proprioception in hip joint morphogenesis, we studied this process in mice mutant for proprioceptive system regulators Runx3 or Egr3. Loss of Runx3 in the peripheral nervous system, but not in skeletal lineages, leads to similar joint abnormalities, as does Egr3 loss of function. These findings expand the range of known regulatory roles of the proprioception system on the skeleton and provide a central component of the underlying molecular mechanism, namely Piezo2.

New functions for the proprioceptive system in skeletal biology.

Muscle spindles and Golgi tendon organs (GTOs) are two types of sensory receptors that respond to changes in length or tension of skeletal muscles. These mechanosensors have long been known to participate in both proprioception and stretch reflex. Here, we present recent findings implicating these organs in maintenance of spine alignment as well as in realignment of fractured bones. These discoveries have been made in several mouse lines lacking functional mechanosensors in part or completely. In both studies, the absence of functional spindles and GTOs produced a more severe phenotype than that of spindles alone. Interestingly, the spinal curve phenotype, which appeared during peripubertal development, bears resemblance to the human condition adolescent idiopathic scoliosis. This similarity may contribute to the study of the disease by offering both an animal model and a clue as to its aetiology. Moreover, it raises the possibility that impaired proprioceptive signalling may be involved in the aetiology of other conditions. Overall, these new findings expand considerably the scope of involvement of proprioception in musculoskeletal development and function.This article is part of the Theo Murphy meeting issue 'Mechanics of development'.