Scleraxis-lineage cells are required for correct muscle patterning.

Movement of the vertebrate body is supported by the connection of muscle, tendon and bone. Each skeletal muscle in the vertebrate body has a unique shape and attachment site; however, the mechanism that ensures reproducible muscle patterning is incompletely understood. In this study, we conducted targeted cell ablation using scleraxis (Scx)-Cre to examine the role of Scx-lineage cells in muscle morphogenesis and attachment in mouse embryos. We found that muscle bundle shapes and attachment sites were significantly altered in embryos with Scx-lineage cell ablation. Muscles in the forelimb showed impaired bundle separation and limb girdle muscles distally dislocated from their insertion sites. Scx-lineage cells were required for post-fusion myofiber morphology, but not for the initial segregation of myoblasts in the limb bud. Furthermore, muscles could change their attachment site, even after formation of the insertion. Lineage tracing suggested that the muscle patterning defect was primarily attributed to the reduction of tendon/ligament cells. Our study demonstrates an essential role of Scx-lineage cells in the reproducibility of skeletal muscle attachment, in turn revealing a previously unappreciated tissue-tissue interaction in musculoskeletal morphogenesis.

Digoxigenin-labeled RNA probes for untranslated regions enable the isoform-specific gene expression analysis of myosin heavy chains in whole-mount in situ hybridization.

Myosin heavy chains (MyHCs), which are encoded by myosin heavy chain (Myh) genes, are the most abundant proteins in myofiber. Among the 11 sarcomeric Myh isoform genes in the mammalian genome, seven are mainly expressed in skeletal muscle. Myh genes/MyHC proteins share a common role as force producing units with highly conserved sequences, but have distinct spatio-temporal expression patterns. As such, the expression patterns of Myh genes/MyHC proteins are considered as molecular signatures of specific fiber types or the regenerative status of mammalian skeletal muscles. Immunohistochemistry is widely used for identifying MyHC expression patterns; however, this method is costly and is not ideal for whole-mount samples, such as embryos. In situ hybridization (ISH) is another versatile method for the analysis of gene expression, but is not commonly applied for Myh genes, partly because of the highly homologous sequences of Myh genes. Here we demonstrate that an ISH analysis with the untranslated region (UTR) sequence of Myh genes is cost-effective and specific method for analyzing the Myh gene expression in whole-mount samples. Digoxigenin (DIG)-labeled antisense probes for UTR sequences, but not for protein coding sequences, specifically detected the expression patterns of respective Myh isoform genes in both embryo and adult skeletal muscle tissues. UTR probes also revealed the isoform gene-specific polarized localization of Myh mRNAs in embryonic myofibers, which implied a novel mRNA distribution mechanism. Our data suggested that the DIG-labeled UTR probe is a cost-effective and versatile method to specifically detect skeletal muscle Myh genes in a whole-mount analysis.

Simultaneous loss of skeletal muscle myosin heavy chain IIx and IIb causes severe skeletal muscle hypoplasia in postnatal mice.

The skeletal muscle myosin heavy chain (MyHC) is a fundamental component of the sarcomere structure and muscle contraction. Two of the three adult fast MyHCs, MyHC-IIx and MyHC-IIb, are encoded by Myh1 and Myh4, respectively. However, skeletal muscle disorders have not yet been linked to these genes in humans. MyHC-IIb is barely detectable in human skeletal muscles. Thus, to characterize the molecular function of skeletal muscle MyHCs in humans, investigation of the effect of simultaneous loss of MyHC-IIb and other MyHCs on skeletal muscle in mice is essential. Here, we generated double knockout (dKO) mice with simultaneous loss of adult fast MyHCs by introducing nonsense frameshift mutations into the Myh1 and Myh4 genes. The dKO mice appeared normal after birth and until 2 weeks of age but showed severe skeletal muscle hypoplasia after 2 weeks. In 3-week-old dKO mice, increased expression of other skeletal muscle MyHCs, such as MyHC-I, MyHC-IIa, MyHC-neo, and MyHC-emb, was observed. However, these expressions were not sufficient to compensate for the loss of MyHC-IIb and MyHC-IIx. Moreover, the aberrant sarcomere structure with altered expression of sarcomere components was observed in dKO mice. Our findings imply that the simultaneous loss of MyHC-IIb and MyHC-IIx is substantially detrimental to postnatal skeletal muscle function and will contribute to elucidating the molecular mechanisms of skeletal muscle wasting disorders caused by the loss of skeletal muscle MyHCs.

Protocadherin-1 is expressed in the notochord of mouse embryo but is dispensable for its formation.

Notochord is an embryonic midline structure that serves as mechanical support for axis elongation and the signaling center for the surrounding tissues. Precursors of notochord are initially induced in the dorsal most mesoderm region in gastrulating embryo and separate from the surrounding mesoderm/endoderm tissue to form an elongated rod-like structure, suggesting that cell adhesion molecules may play an important role in this step. In Xenopus embryo, axial protocadherin (AXPC), an orthologue of mammalian Protocadherin-1 (PCDH1), is indispensable for the assembly and separation from the surrounding tissue of the notochord cells. However, the role of PCDH1 in mammalian notochord remains unknown. We herein report that PCDH1 is expressed in the notochord of mouse embryo and that PCDH1-deficient mice form notochord normally. First, we examined the temporal expression pattern of pcdh1 and found that pcdh1 mRNA was expressed from embryonic day (E) 7.5, prior to the stage when notochord cells detach from the surrounding endoderm tissue. Second, we found that PCDH1 protein is expressed in the notochord of mouse embryos in addition to the previously reported expression in endothelial cells. To further investigate the role of PCDH1 in embryonic development, we generated PCDH1-deficient mice using the CRISPR-Cas9 system. In PCDH1-deficient embryos, notochord formation and separation from the surrounding tissue were normal. Structure and marker gene expression of notochord were also unaffected by loss of PCDH1. Major vascular patterns in PCDH1-deficient embryo were essentially normal. These results suggest that PCDH1 is dispensable for notochord formation, including the tissue separation process, in mammalian embryos. We successfully identified the evolutionary conserved expression of PCDH1 in notochord, but its function may differ among species.

Lin28a/let-7 pathway modulates the Hox code via Polycomb regulation during axial patterning in vertebrates.

The body plan along the anteroposterior axis and regional identities are specified by the spatiotemporal expression of Hox genes. Multistep controls are required for their unique expression patterns; however, the molecular mechanisms behind the tight control of Hox genes are not fully understood. In this study, we demonstrated that the Lin28a/let-7 pathway is critical for axial elongation. Lin28a-/- mice exhibited axial shortening with mild skeletal transformations of vertebrae, which were consistent with results in mice with tail bud-specific mutants of Lin28a. The accumulation of let-7 in Lin28a-/- mice resulted in the reduction of PRC1 occupancy at the Hox cluster loci by targeting Cbx2. Consistently, Lin28a loss in embryonic stem-like cells led to aberrant induction of posterior Hox genes, which was rescued by the knockdown of let-7. These results suggest that the Lin28/let-7 pathway is involved in the modulation of the 'Hox code' via Polycomb regulation during axial patterning.

Generation of a Quantitative Luciferase Reporter for Sox9 SUMOylation.

Sox9 is a master transcription factor for chondrogenesis, which is essential for chondrocyte proliferation, differentiation, and maintenance. Sox9 activity is regulated by multiple layers, including post-translational modifications, such as SUMOylation. A detection method for visualizing the SUMOylation in live cells is required to fully understand the role of Sox9 SUMOylation. In this study, we generated a quantitative reporter for Sox9 SUMOylation that is based on the NanoBiT system. The simultaneous expression of Sox9 and SUMO1 constructs that are conjugated with NanoBiT fragments in HEK293T cells induced luciferase activity in SUMOylation target residue of Sox9-dependent manner. Furthermore, the reporter signal could be detected from both cell lysates and live cells. The signal level of our reporter responded to the co-expression of SUMOylation or deSUMOylation enzymes by several fold, showing dynamic potency of the reporter. The reporter was active in multiple cell types, including ATDC5 cells, which have chondrogenic potential. Finally, using this reporter, we revealed a extracellular signal conditions that can increase the amount of SUMOylated Sox9. In summary, we generated a novel reporter that was capable of quantitatively visualizing the Sox9-SUMOylation level in live cells. This reporter will be useful for understanding the dynamism of Sox9 regulation during chondrogenesis.

Deletion of a Seminal Gene Cluster Reinforces a Crucial Role of SVS2 in Male Fertility.

Multiple genes, whose functions or expression are overlapping, compensate for the loss of one gene. A gene cluster in the mouse genome encodes five seminal vesicle proteins (SVS2, SVS3, SVS4, SVS5, and SVS6). These proteins are produced by male rodents and function in formation of the copulatory plug following mating. SVS2 plays an essential role in the successful internal fertilization by protecting the sperm membrane against a uterine immune attack. We hypothesized that the four remaining seminal vesicle proteins (SVPs) of this gene cluster may partially/completely compensate for the deficiency of SVS2. For confirming our hypothesis, we generated mice lacking the entire SVP-encoding gene cluster and compared their fecundity with Svs2-deficient (Svs2-/-) mice; that is, mice deficient in Svs2 alone. A single loxP site remained after the deletion of the Svs2 gene. Therefore, we inserted another loxP site by combining the CRISPR/Cas9 system with single-stranded oligodeoxynucleotides (ssODN). Male mice lacking the entire SVP-encoding gene cluster (Svs2-6-/- mice) and thereby all five SVP proteins, generated by the deletion of 100kbp genomic DNA, showed low fecundity. However, the fecundity level was comparable with that from Svs2-/- male mice. Our results demonstrate that SVS3, SVS4, SVS5, and SVS6 do not function in the protection of sperm against a uterine immune attack in the absence of SVS2. Thus, Svs2 is the critical gene in the SVP gene cluster.

Comparative analysis demonstrates cell type-specific conservation of SOX9 targets between mouse and chicken.

SRY (sex-determining region Y)-box 9 (SOX9) is a transcription factor regulating both chondrogenesis and sex determination. Among vertebrates, SOX9's functions in chondrogenesis are well conserved, while they vary in sex determination. To investigate the conservation of SOX9's regulatory functions in chondrogenesis and gonad development among species, we performed chromatin immunoprecipitation sequencing (ChIP-seq) using developing limb buds and male gonads from embryos of two vertebrates, mouse and chicken. In both mouse and chicken, SOX9 bound to intronic and distal regions of genes more frequently in limb buds than in male gonads, while SOX9 bound to the proximal upstream regions of genes more frequently in male gonads than in limb buds. In both species, SOX palindromic repeats were identified more frequently in SOX9 binding regions in limb bud genes compared with those in male gonad genes. The conservation of SOX9 binding regions was significantly higher in limb bud genes. In addition, we combined RNA expression analysis (RNA sequencing) with the ChIP-seq results at the same stage in developing chondrocytes and Sertoli cells and determined SOX9 target genes in these cells of the two species and disclosed that SOX9 targets showed high similarity of targets in chondrocytes, but not in Sertoli cells.

Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD.

Methods to create genetically engineered mice involve three major steps: harvesting embryos from one set of females, microinjection of reagents into embryos ex vivo and their surgical transfer to another set of females. Although tedious, these methods have been used for more than three decades to create mouse models. We recently developed a method named GONAD (genome editing via oviductal nucleic acids delivery), which bypasses these steps. GONAD involves injection of CRISPR components (Cas9 mRNA and guide RNA (gRNA)) into the oviducts of pregnant females 1.5 d post conception, followed by in vivo electroporation to deliver the components into the zygotes in situ. Using GONAD, we demonstrated that target genes can be disrupted and analyzed at different stages of mouse embryonic development. Subsequently, we developed improved GONAD (i-GONAD) by delivering CRISPR ribonucleoproteins (RNPs; Cas9 protein or Cpf1 protein and gRNA) into day-0.7 pregnant mice, which made it suitable for routine generation of knockout and large-deletion mouse models. i-GONAD can also generate knock-in models containing up to 1-kb inserts when single-stranded DNA (ssDNA) repair templates are supplied. i-GONAD offers other advantages: it does not require vasectomized males and pseudo-pregnant females, the females used for i-GONAD are not sacrificed and can be used for other experiments, it can be easily adopted in laboratories lacking sophisticated microinjection equipment, and can be implemented by researchers skilled in small-animal surgery but lacking embryo-handling skills. Here, we provide a step-by-step protocol for establishing the i-GONAD method. The protocol takes ∼6 weeks to generate the founder mice.

Wwp2 maintains cartilage homeostasis through regulation of Adamts5.

The WW domain-containing protein 2 (Wwp2) gene, the host gene of miR-140, codes for the Wwp2 protein, which is an HECT-type E3 ubiquitin ligases abundantly expressed in articular cartilage. However, its function remains unclear. Here, we show that mice lacking Wwp2 and mice in which the Wwp2 E3 enzyme is inactivated (Wwp2-C838A) exhibit aggravated spontaneous and surgically induced osteoarthritis (OA). Consistent with this phenotype, WWP2 expression level is downregulated in human OA cartilage. We also identify Runx2 as a Wwp2 substrate and Adamts5 as a target gene, as similar as miR-140. Analysis of Wwp2-C838A mice shows that loss of Wwp2 E3 ligase activity results in upregulation of Runx2-Adamts5 signaling in articular cartilage. Furthermore, in vitro transcribed Wwp2 mRNA injection into mouse joints reduces the severity of experimental OA. We propose that Wwp2 has a role in protecting cartilage from OA by suppressing Runx2-induced Adamts5 via Runx2 poly-ubiquitination and degradation.

CRISPR/Cas9-mediated simultaneous knockout of Dmrt1 and Dmrt3 does not recapitulate the 46,XY gonadal dysgenesis observed in 9p24.3 deletion patients.

DM domain transcription factors play important roles in sexual development in a wide variety of species from invertebrate to humans. Among seven mammalian family members of DM domain transcription factors, DMRT1 has been studied in mouse and human for its conserved role in male gonadal identity. Chromosomal deletion of 9p24.3, the region in which DMRT1 is located, is associated with 46,XY gonadal dysgenesis. Dmrt1 knockout (KO) mice also showed male-to-female gonadal reprogramming. However, the phenotype of Dmrt1 KO mouse appears only after birth while 46,XY gonadal dysgenesis occurs during the developmental phase, and the cause behind this difference remained unknown. We hypothesized that in human the function of other DMRT genes clustered with DMRT1, namely DMRT3, might also be impaired by the chromosomal deletion, which leads to the gonadal dysgenesis phenotype. Thus, simultaneous loss of multiple DM domain genes in mice could have a more severe impact on gonadal development. To address this issue, we generated double KO mice for Dmrt1 and Dmrt3 via the CRISPR/Cas9 system. Comparing adult and neonatal testes of single and double KO mice, we found that loss of Dmrt1 or Dmrt3, or both, does not have apparent effect on male gonadal formation during embryonic development. Our study demonstrated that the discrepancy between human with 9p24.3 deletion and Dmrt1 KO mouse could not be explained by the simultaneous loss of Dmrt3 gene. CRISPR/Cas9 is a versatile and straightforward approach to elucidate the questions that were otherwise difficult to address with conventional methods.

Tendons and Ligaments: Connecting Developmental Biology to Musculoskeletal Disease Pathogenesis.

Tendons and ligaments provide connections between muscle and bone or bone and bone to enable locomotion. Damage to tendons and ligaments caused by acute or chronic injury or associated with aging and arthritis is a prevalent cause of disability. Improvements in approaches for the treatment of these conditions depend on a better understanding of tendon and ligament development, cell biology, and pathophysiology. This review focuses on recent advances in the discovery of transcription factors that control ligament and tendon cell differentiation, how cell and extracellular matrix homeostasis are altered in disease, and how this new insight can lead to novel therapeutic approaches. © 2017 American Society for Bone and Mineral Research.

The p.R92W variant of NR5A1/Nr5a1 induces testicular development of 46,XX gonads in humans, but not in mice: phenotypic comparison of human patients and mutation-induced mice.

NR5A1 is the key regulator of adrenal and gonadal development in both humans and mice. Recently, a missense substitution in human NR5A1, p.R92W, was shown to underlie gonadal dysgenesis in genetic males and testicular formation in genetic females. Here, we investigated the phenotypic effects of the p.R92W mutation on murine development. Mice carrying the p.R92W mutation manifested a similar but milder phenotype than that of the previously described Nr5a1 knockout mice. Importantly, mutation-positive XX mice showed no signs of masculinization. These results, together with prior observations, indicate that the p.R92W mutation in NR5A1/Nr5a1 encodes unique molecules that disrupt male gonadal development in both humans and mice and induces testicular formation specifically in human females. Our findings provide novel insights into the conservation and divergence in the molecular networks underlying mammalian sexual development.

Mohawk promotes the maintenance and regeneration of the outer annulus fibrosus of intervertebral discs.

The main pathogenesis of intervertebral disc (IVD) herniation involves disruption of the annulus fibrosus (AF) caused by ageing or excessive mechanical stress and the resulting prolapse of the nucleus pulposus. Owing to the avascular nature of the IVD and lack of understanding the mechanisms that maintain the IVD, current therapies do not lead to tissue regeneration. Here we show that homeobox protein Mohawk (Mkx) is a key transcription factor that regulates AF development, maintenance and regeneration. Mkx is mainly expressed in the outer AF (OAF) of humans and mice. In Mkx(-/-) mice, the OAF displays a deficiency of multiple tendon/ligament-related genes, a smaller OAF collagen fibril diameter and a more rapid progression of IVD degeneration compared with the wild type. Mesenchymal stem cells overexpressing Mkx promote functional AF regeneration in a mouse AF defect model, with abundant collagen fibril formation. Our results indicate a therapeutic strategy for AF regeneration.

Generation of mutant mice via the CRISPR/Cas9 system using FokI-dCas9.

Genome editing, which introduces mutations in genes of interest using artificial DNA nucleases such as the ZFN, TALEN, and CRISPR/Cas9 systems in living cells, is a useful tool for generating mutant animals. Although CRISPR/Cas9 provides advantages over the two other systems, such as an easier vector construction and high efficiency of genome editing, it raises concerns of off-target effects when single guide RNA (gRNA) is used. Recently, FokI-dCas9 (fCas9), a fusion protein comprised of the inactivated mutant form of Cas9 and the DNA nuclease domain of FokI, has been developed. It enables genome editing with reduced risks of off-target effects in mammalian cultured cell lines, as fCas9 requires gRNAs to bind opposite strands with an appropriate distance between them. Here, we demonstrated that fCas9 efficiently generates living mutant mice through microinjection of its mRNA and gRNAs into zygotes. A comparison of the relative efficiencies of genome editing using fCas9 and other modified Cas9s showed that these mutagenesis efficiencies are similar when the targets of two gRNAs are separated by an appropriate distance, suggesting that in addition to the ease of vector construction, fCas9 exhibit high efficiency in producing mutant mice and in reducing risks of off-target effects.

Transcription factor Mohawk controls tenogenic differentiation of bone marrow mesenchymal stem cells in vitro and in vivo.

Mohawk homeobox (MKX) has been demonstrated as a tendon/ligament specific transcription factor. The aim of this study was to investigate the role of MKX in ligament/tenogenic differentiation of bone marrow derived mesenchymal stem cells (BMMSCs). Human BMMSCs were treated with 50 ng/ml BMP-12 or transduced with MKX or scleraxis (SCX) adenoviral vector. Gene expression analysis was performed by quantitative reverse transcribed polymerase chain reaction (qRT-PCR). Rat BMMSCs were seeded in a collagen scaffold and transplanted into a rat Achilles tendon defect model. Tenogenesis related gene expressions and histological features were analyzed. BMP-12 induced tenogenesis in BMMSCs as indicated by increased COL1a1, TNXB, DCN and SCX mRNA, and MKX expression increased simultaneously. Rat BMMSCs enhanced defect repair and were still detectable 3 weeks after transplantation. Increased expressions of COL1a1, TNC and TNMD in vivo were also correlated with upregulated MKX. Adenoviral MKX promoted expression of COL1a1, TNXB, and TNMD in BMMSCs. This study demonstrated that MKX gene expression is enhanced during the tenogenic differentiation of BMMSCs in vitro and in vivo, and the adenoviral overexpression of MKX increases tendon extracellular matrix gene expression and protein production. Thus, MKX is a key factor for tenogenic differentiation of MSCs.

Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system.

Introducing a point mutation is a fundamental method used to demonstrate the roles of particular nucleotides or amino acids in the genetic elements or proteins, and is widely used in in vitro experiments based on cultured cells and exogenously provided DNA. However, the in vivo application of this approach by modifying genomic loci is uncommon, partly due to its technical and temporal demands. This leaves many in vitro findings un-validated under in vivo conditions. We herein applied the CRISPR/Cas9 system to generate mice with point mutations in their genomes, which led to single amino acid substitutions in proteins of interest. By microinjecting gRNA, hCas9 mRNA and single-stranded donor oligonucleotides (ssODN) into mouse zygotes, we introduced defined genomic modifications in their genome with a low cost and in a short time. Both single gRNA/WT hCas9 and double nicking set-ups were effective. We also found that the distance between the modification site and gRNA target site was a significant parameter affecting the efficiency of the substitution. We believe that this is a powerful technique that can be used to examine the relevance of in vitro findings, as well as the mutations found in patients with genetic disorders, in an in vivo system.

Production of Sry knockout mouse using TALEN via oocyte injection.

Recently developed transcription activator-like effector nuclease (TALEN) technology has enabled the creation of knockout mice, even for genes on the Y chromosome. In this study, we generated a knockout mouse for Sry, a sex-determining gene on the Y chromosome, using microinjection of TALEN RNA into pronuclear stage oocytes. As expected, the knockout mouse had female external and internal genitalia, a female level of blood testosterone and a female sexually dimorphic nucleus in the brain. The knockout mouse exhibited an estrous cycle and performed copulatory behavior as females, although it was infertile or had reduced fertility. A histological analysis showed that the ovary of the knockout mouse displayed a reduced number of oocytes and luteinized unruptured follicles, implying that a reduced number of ovulated oocytes is a possible reason for infertility and/or reduced fertility in the KO mouse.

Self-regulation of the head-inducing properties of the Spemann organizer.

The Spemann organizer stands out from other signaling centers of the embryo because of its broad patterning effects. It defines development along the anteroposterior and dorsoventral axes of the vertebrate body, mainly by secreting antagonists of growth factors. Qualitative models proposed more than a decade ago explain the organizer's region-specific inductions (i.e., head and trunk) as the result of different combinations of antagonists. For example, head induction is mediated by extracellular inhibition of Wnt, BMP, and Nodal ligands. However, little is known about how the levels of these antagonists become harmonized with those of their targets and with the factors initially responsible for germ layers and organizer formation, including Nodal itself. Here we show that key ingredients of the head-organizer development, namely Nodal ligands, Nodal antagonists, and ADMP ligands reciprocally adjust each other's strength and range of activity by a self-regulating network of interlocked feedback and feedforward loops. A key element in this cross-talk is the limited availability of ACVR2a, for which Nodal and ADMP must compete. By trapping Nodal extracellularly, the Nodal antagonists Cerberus and Lefty are permissive for ADMP activity. The system self-regulates because ADMP/ACVR2a/Smad1 signaling in turn represses the expression of the Nodal antagonists, reestablishing the equilibrium. In sum, this work reveals an unprecedented set of interactions operating within the organizer that is critical for embryonic patterning.