BMP Signaling Mediated by BMPR1A in Osteoclasts Negatively Regulates Osteoblast Mineralization Through Suppression of Cx43

Osteoblasts and osteoclasts are well orchestrated through different mechanisms of communication during bone remodeling. Previously, we found that osteoclast‐specific disruption of one of the BMP receptors, Bmpr1a, results in increased osteoblastic bone formation in mice. We hypothesized that BMPR1A signaling in osteoclasts regulates production of either membrane bound proteins or secreted molecules that regulated osteoblast differentiation. In our current study, we co‐cultured wild‐type osteoblasts with either control osteoclasts or osteoclasts lacking BMPR1A signaling activity. We found that loss of Bmpr1a in osteoclasts promoted osteoblast mineralization in vitro. Further, we found that the expression of Cx43/Gja1 in the mutant osteoclasts was increased, which encoded for one of the gap junction proteins connexin 43/gap junction alpha 1. Knockdown of Gja1 in the mutant osteoclasts for Bmpr1a reduced osteoblastic mineralization when co‐cultured. Our findings suggest that GJA1 may be one of the downstream targets of BMPR1A signaling in osteoclasts that mediates osteoclast–osteoblast communication during bone remodeling. J. Cell. Biochem. 118: 605–614, 2017. © 2016 Wiley Periodicals, Inc.Disruption of Bmpr1a in osteoclasts promoted osteoblast mineralization when co‐cultured. Up‐regulation of gap junction Cx43/Gja1 in mutant osteoclasts is responsible for the enhanced osteoblast function.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135668/1/jcb25746_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135668/2/jcb25746.pd

The Role of Ellis‐Van Creveld 2(EVC2) in Mice During Cranial Bone Development

EvC syndrome is a type of autosomal‐recessive chondrodysplasia. Previous case studies in patients suggest abnormal craniofacial development, in addition to dwarfism and tooth abnormalities. To investigate how craniofacial development is affected in EvC patients, surface models were generated from micro‐CT scans of control mice, Evc2 global mutant mice and Evc2 neural crest‐specific mutant mice. The anatomic landmarks were placed on the surface model to assess the morphological abnormalities in the Evc2 mutants. Through analyzing the linear and angular measurements between landmarks, we identified a smaller overall skull, shorter nasal bone, shorter frontal bone, and shorter cranial base in the Evc2 global mutants. By comparing neural crest‐specific Evc2 mutants with control mice, we demonstrated that the abnormalities within the mid‐facial regions are not accounted for by the Evc2 mutation within these regions. Additionally, we also identified disproportionate length to width ratios in the Evc2 mutants at all levels from anterior to posterior of the skull. Overall, this study demonstrates a more comprehensive analysis on the craniofacial morphological abnormalities in EvC syndrome and provides the developmental insight to appreciate the impact of Evc2 mutation within the neural crest cells on multiple aspects of skull deformities. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 301:46–55, 2018. © 2017 Wiley Periodicals, Inc.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141635/1/ar23692_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141635/2/ar23692.pd

A Ciliary Protein EVC2/LIMBIN Plays a Critical Role in the Skull Base for Mid-Facial Development

Ellis-van Creveld (EvC) syndrome is an autosomal recessive chondrodysplastic disorder. Affected patients present a wide spectrum of symptoms including short stature, postaxial polydactyly, and dental abnormalities. We previously disrupted Evc2, one of the causative genes for EvC syndrome, in mice using a neural crest-specific, Cre-mediated approach (i.e., P0-Cre, referred to as Evc2 P0 mutants). Despite the fact that P0-Cre predominantly targets the mid-facial region, we reported that many mid-facial defects identified in Evc2 global mutants are not present in Evc2 P0 mutants at postnatal day 8 (P8). In the current study, we used multiple Cre lines (P0-Cre and Wnt1-Cre, respectively), to specifically delete Evc2 in neural crest-derived tissues and compared the resulting mid-facial defects at multiple time points (P8 and P28, respectively). While both Cre lines indistinguishably targeted the mid-facial region, they differentially targeted the anterior portion of the skull base. By comprehensively analyzing the shapes of conditional mutant skulls, we detected differentially affected mid-facial defects in Evc2 P0 mutants and Evc2 Wnt1 mutants. Micro-CT analysis of the skull base further revealed that the Evc2 mutation leads to a differentially affected skull base, caused by premature closure of the intersphenoid synchondrosis (presphenoidal synchondrosis), which limited the elongation of the anterior skull base during the postnatal development of the skull. Given the importance of the skull base in mid-facial bone development, our results suggest that loss of function of Evc2 within the skull base secondarily leads to many aspects of the mid-facial defects developed by the EvC syndrome

Molecular and Cellular Pathogenesis of Ellis-van Creveld Syndrome: Lessons from Targeted and Natural Mutations in Animal Models

Ellis-van Creveld syndrome (EVC; MIM ID #225500) is a rare congenital disease with an occurrence of 1 in 60,000. It is characterized by remarkable skeletal dysplasia, such as short limbs, ribs and polydactyly, and orofacial anomalies. With two of three patients first noted as being offspring of consanguineous marriage, this autosomal recessive disease results from mutations in one of two causative genes: EVC or EVC2/LIMBIN. The recent identification and manipulation of genetic homologs in animals has deepened our understanding beyond human case studies and provided critical insight into disease pathogenesis. This review highlights the utility of animal-based studies of EVC by summarizing: (1) molecular biology of EVC and EVC2/LIMBIN, (2) human disease signs, (3) dysplastic limb development, (4) craniofacial anomalies, (5) tooth anomalies, (6) tracheal cartilage abnormalities, and (7) EVC-like disorders in non-human species

Temporally distinct transcriptional regulation of myocyte dedifferentiation and Myofiber growth during muscle regeneration

Abstract Background Tissue regeneration requires a series of steps, beginning with generation of the necessary cell mass, followed by cell migration into damaged area, and ending with differentiation and integration with surrounding tissues. Temporal regulation of these steps lies at the heart of the regenerative process, yet its basis is not well understood. The ability of zebrafish to dedifferentiate mature “post-mitotic” myocytes into proliferating myoblasts that in turn regenerate lost muscle tissue provides an opportunity to probe the molecular mechanisms of regeneration. Results Following subtotal excision of adult zebrafish lateral rectus muscle, dedifferentiating residual myocytes were collected at two time points prior to cell cycle reentry and compared to uninjured muscles using RNA-seq. Functional annotation (GAGE or K-means clustering followed by GO enrichment) revealed a coordinated response encompassing epigenetic regulation of transcription, RNA processing, and DNA replication and repair, along with protein degradation and translation that would rewire the cellular proteome and metabolome. Selected candidate genes were phenotypically validated in vivo by morpholino knockdown. Rapidly induced gene products, such as the Polycomb group factors Ezh2 and Suz12a, were necessary for both efficient dedifferentiation (i.e. cell reprogramming leading to cell cycle reentry) and complete anatomic regeneration. In contrast, the late activated gene fibronectin was important for efficient anatomic muscle regeneration but not for the early step of myocyte cell cycle reentry. Conclusions Reprogramming of a “post-mitotic” myocyte into a dedifferentiated myoblast requires a complex coordinated effort that reshapes the cellular proteome and rewires metabolic pathways mediated by heritable yet nuanced epigenetic alterations and molecular switches, including transcription factors and non-coding RNAs. Our studies show that temporal regulation of gene expression is programmatically linked to distinct steps in the regeneration process, with immediate early expression driving dedifferentiation and reprogramming, and later expression facilitating anatomical regeneration

Quantification of three- dimensional morphology of craniofacial mineralized tissue defects in Tgfbr2/Osx- Cre mice

Craniofacial morphology is affected by the growth, development, and three- dimensional (3D) relationship of mineralized structures including the skull, jaws, and teeth. Despite fulfilling different purposes within this region, cranial bones and tooth dentin are derived from mesenchymal cells that are affected by perturbations within the TGF- β signaling pathway. TGFBR2 encodes a transmembrane receptor that is part of the canonical, SMAD- dependent TGF- β signaling pathway and mutations within this gene are associated with Loeys- Dietz syndrome, a condition which often presents with craniofacial signs including craniosynostosis and cleft palate. To investigate the role of Tgfbr2 in immature, but committed, mineralized tissue forming cells, we analyzed postnatal craniofacial morphology in mice with conditional Tgfbr2 deletion in Osx- expressing cells. Novel application of a 3D shape- based comparative technique revealed that Tgfbr2 in Osx- expressing cells results in impaired postnatal molar root and anterior cranial growth. These findings support those from studies using similar Tgfbr2 conditional knockout models, highlight the anomalous facial and dental regions/structures using tomographic imaging- based techniques, and provide insight into the role of Tgfbr2 during postnatal craniofacial development.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/170199/1/osi21099_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/170199/2/osi21099.pd

Extraocular muscle regeneration in zebrafish requires late signals from Insulin-like growth factors

<div><p>Insulin-like growth factors (Igfs) are key regulators of key biological processes such as embryonic development, growth, and tissue repair and regeneration. The role of Igf in myogenesis is well documented and, in zebrafish, promotes fin and heart regeneration. However, the mechanism of action of Igf in muscle repair and regeneration is not well understood. Using adult zebrafish extraocular muscle (EOM) regeneration as an experimental model, we show that Igf1 receptor blockage using either chemical inhibitors (BMS754807 and NVP-AEW541) or translation-blocking morpholino oligonucleotides (MOs) reduced EOM regeneration. Zebrafish EOMs regeneration depends on myocyte dedifferentiation, which is driven by early epigenetic reprogramming and requires autophagy activation and cell cycle reentry. Inhibition of Igf signaling had no effect on either autophagy activation or cell proliferation, indicating that Igf signaling was not involved in the early reprogramming steps of regeneration. Instead, blocking Igf signaling produced hypercellularity of regenerating EOMs and diminished myosin expression, resulting in lack of mature differentiated muscle fibers even many days after injury, indicating that Igf was involved in late re-differentiation steps. Although it is considered the main mediator of myogenic Igf actions, Akt activation decreased in regenerating EOMs, suggesting that alternative signaling pathways mediate Igf activity in muscle regeneration. In conclusion, Igf signaling is critical for re-differentiation of reprogrammed myoblasts during late steps of zebrafish EOM regeneration, suggesting a regulatory mechanism for determining regenerated muscle size and timing of differentiation, and a potential target for regenerative therapy.</p></div

Role of Akt in the regenerating muscle.

<p>The activation of Akt in injured muscles (non-BMS754807 treated fish) was assessed by western blot in a time course experiment (A). Immunoblotting was performed with anti-phosphorylated Akt antibody. Total amounts of Akt were monitored by reprobing membranes with anti-Akt antibody. Note that phosphorylated Akt (pAkt) was rapidly and persistently reduced in the injured muscle. Tubulin was used as a loading control. The densitometric quantification of the Akt bands is shown (B, C). The intensity of pAkt (B) and tAkt bands (C) was normalized to the tubulin content. The ratio between pAkt and tAkt was used to represent the fraction of active Akt (D). For comparative purposes, the pAkt/tAkt ratio of the injured muscle was divided by the pAkt/tAkt ratio of the uninjured muscle at each time point (E). U, uninjured muscle; I, injured muscle; R.U., relative units.</p

Inhibition of Igf1r impairs muscle regeneration.

<p>Myectomized α-actin-EGFP fish treated with the Igf1r inhibitor BMS754807 (B) or DMSO (A) for 5 days. At selected time points (3, 5, and 7 dpi), the length of the regenerating muscle was measured as described (C), values are averages ± SD (n = 5–6). For each group (DMSO or BMS754807), differences among time points were analyzed by ANOVA. Different letters (lower case over DMSO group, there was no statistically significant difference for the BMS754807 group) indicate significant differences among time points (P < 0.05, Newman-Keuls multiple comparisons test). For each time point, differences between DMSO and BMS754807 treated fish were analyzed by Student’s <i>t</i>-test (*p < 0.05; **p < 0.01; ***p <0.001). To confirm our findings, α-actin-EGFP were treated with the unrelated NVP-AEW541 Igf1R inhibitor. At 4 dpi the regenerating muscle was measured as before showing similar results (D); values are averages ± SD (Student's <i>t</i>-test, **p < 0.01, n = 5). To knock down Igf1r, lissamine-tagged MOs (red) against both Igf1r paralogs (a and b) were microinjected into α-actin-EGFP (green) fish muscles prior myectomy. MOs were detected through the whole regenerating muscle, including the distal ends (arrowhead). Control MO (up) and <i>Igfra/b</i> MO (down) injected fish are shown (E). The length of the regenerating muscle was measured as described (F); values are averages ± SD (Student’s <i>t</i>-test, *p < 0.05, n = 10). Diagram of a craniectomized zebrafish head (G); muscles visualized by this technique are shown, and LR muscles are highlighted in green. Green and red boxes show approximately the picture used for regeneration or LysoTracker Red and GFP-Lc3 (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192214#pone.0192214.g002" target="_blank">Fig 2</a>) assessment, respectively. The white arrows mark the growing end of the regenerating muscle. P, pituitary; e, eye. Gray box in panels C, D and F represent the 50% muscle length as baseline following myectomy.</p

Inhibition of Igf signaling does not affect autophagy activation in the regenerating muscle.

<p>GFP-LC3 (A and D) fish were myectomyzed and LysoTracker Red (B and E) was used to label autophagy in the regenerating LR in fish treated with DMSO (A-C) or BMS754807 (D-F). C and F show the merging of A-B and D-E, respectively. GFP-LC3 (G) and LysoTracker (H) fluorescence intensity of the regenerating muscle were measured and no statistically significant difference between DMSO and BMS754807 treated fish was found. Values represent average ± SD (Student’s <i>t</i>-test, significance set at P < 0.05, n = 5). P, pituitary.</p