Effects of age and diet consistency on the expression of myosin heavy-chain isoforms on jaw-closing and jaw-opening muscles in a rat model.

BACKGROUND: Skeletal craniofacial morphology can be influenced by changes in masticatory muscle function, which may also change the functional profile of the muscles. OBJECTIVES: To investigate the effects of age and functional demands on the expression of Myosin Heavy-Chain (MyHC) isoforms in representative jaw-closing and jaw-opening muscles, namely the masseter and digastric muscles respectively. METHODS: Eighty-four male Wistar rats were divided into four age groups, namely an immature (n = 12; 4-week-old), early adult (n = 24; 16-week-old), adult (n = 24; 26-week-old) and mature adult (n = 24; 38-week-old) group. The three adult groups were divided into two subgroups each based on diet consistency; a control group fed a standard (hard) diet, and an experimental group fed a soft diet. Rats were sacrificed, and masseter and digastric muscles dissected. Real-time quantitative polymerase chain reaction was used to compare the mRNA transcripts of the MyHC isoforms-Myh7 (MyHC-I), Myh2 (MyHC-IIa), Myh4 (MyHC-IIb) and Myh1 (MyHC-IIx)-of deep masseter and digastric muscles. RESULTS: In the masseter muscle, hypofunction increases Myh1 (26, 38 weeks; p < .0001) but decreases Myh4 (26 weeks; p = .046) and Myh2 (26 weeks; p < .0001) expression in adult rats. In the digastric muscle, hypofunction increases Myh1 expression in the mature adult rats (38 weeks; p < .0001), while Myh2 expression decreases in adult rats (26 weeks; p = .021) as does Myh4 (26 weeks; p = .001). Myh7 expression is increased in the digastric muscle of mature adult rats subjected to hypofunction (38 weeks; p = <.0001), while it is very weakly expressed in the masseter. CONCLUSION: In jaw-opening and jaw-closing muscles, differences in myosin expression between hard- and soft-diet-fed rats become evident in adulthood, suggesting that long-term alteration of jaw function is associated with changes in the expression of MyHC isoforms and potential fibre remodelling. This may give insight into the role of function on masticatory muscles and the resultant craniofacial morphology.

Myosin Heavy-Chain Messenger Ribonucleic Acid (mRNA) Expression and Fibre Cross-Sectional Area in Masseter, Digastric, Gastrocnemius and Soleus Muscles of Young and Adult Rats.

Different demands on the muscles of mastication may influence their functional profile (size and distribution of muscle fibre types), which may change during growth and maturation, potentially influencing craniofacial growth. The aim of this study was to evaluate mRNA expression and cross-sectional area of masticatory muscle fibres compared with limb muscles in young and adult rats. Twenty-four rats were sacrificed at two different ages, namely 12 at 4 weeks (young) and 12 at 26 weeks (adult). The masseter, digastric, gastrocnemius and soleus muscles were dissected. Gene expression of myosin heavy-chain isoforms Myh7 (MyHC-I), Myh2 (MyHC-IIa), Myh4 (MyHC-IIb) and Myh1 (MyHC-IIx) in the muscles was measured using qRT-PCR RNA analysis, and immunofluorescence staining was performed to measure the cross-sectional area of different muscle fibre types. Different muscle types and ages were compared. Significant differences were found in the functional profile between masticatory and limb muscles. For the masticatory muscles, there was an increase in Myh4 expression with age, and this change was more intense for the masseter muscles, which also presented an increase in Myh1 expression, similarly to limb muscles. The fibre cross-sectional area of the masticatory muscles was generally smaller in young rats; however, this difference was less pronounced than in limb muscles.

Interplay of RFX transcription factors 1, 2 and 3 in motile ciliogenesis.

Cilia assembly is under strict transcriptional control during animal development. In vertebrates, a hierarchy of transcription factors (TFs) are involved in controlling the specification, differentiation and function of multiciliated epithelia. RFX TFs play key functions in the control of ciliogenesis in animals. Whereas only one RFX factor regulates ciliogenesis in C. elegans, several distinct RFX factors have been implicated in this process in vertebrates. However, a clear understanding of the specific and redundant functions of different RFX factors in ciliated cells remains lacking. Using RNA-seq and ChIP-seq approaches we identified genes regulated directly and indirectly by RFX1, RFX2 and RFX3 in mouse ependymal cells. We show that these three TFs have both redundant and specific functions in ependymal cells. Whereas RFX1, RFX2 and RFX3 occupy many shared genomic loci, only RFX2 and RFX3 play a prominent and redundant function in the control of motile ciliogenesis in mice. Our results provide a valuable list of candidate ciliary genes. They also reveal stunning differences between compensatory processes operating in vivo and ex vivo.

Core Matrisome Protein Signature During Periodontal Ligament Maturation From Pre-occlusal Eruption to Occlusal Function.

The pre-occlusal eruption brings the molars into functional occlusion and initiates tensional strains during mastication. We hypothesized that upon establishment of occlusal contact, the periodontal ligament (PDL) undergoes cell and extracellular matrix maturation to adapt to this mechanical function. The PDL of 12 Wistar male rats were laser microdissected to observe the proteomic changes between stages of pre-occlusal eruption, initial occlusal contact and 1-week after occlusion. The proteome was screened by mass spectrometry and confirmed by immunofluorescence. The PDL underwent maturation upon establishment of occlusion. Downregulation of alpha-fetoprotein stem cell marker and protein synthesis markers indicate cell differentiation. Upregulated proteins were components of the extracellular matrix (ECM) and were characterized with the matrisome project database. In particular, periostin, a major protein of the PDL, was induced following occlusal contact and localized around collagen α-1 (III) bundles. This co-localization coincided with organization of collagen fibers in direction of the occlusal forces. Establishment of occlusion coincides with cellular differentiation and the maturation of the PDL. Co-localization of periostin and collagen with subsequent fiber organization may help counteract tensional forces and reinforce the ECM structure. This may be a key mechanism of the PDL to adapt to occlusal forces and maintain structural integrity.

TNF-α antagonist improves oxidative stress and lipid disorders induced by scorpion venom in the intestinal tissue.

We previously reported that Androctonus australis hector (Aah) venom induces inflammation in several tissues, however limited information is available on its role in gastrointestinal tract. Here we evaluate the involvement of TNF-α in lipid metabolism in the small intestine after Aah envenomation. To address these issues, NMRI mice (3-month-old) were pre-treated with a TNF-α antagonist, 30 min prior to Aah venom injection. Redox status, cytotoxicity and histopathological changes were analyzed in small intestine 3 and 24 h after Aah injection. Lipid metabolism was evaluated through lipid tolerance test in sera. Lipid content in small intestine was also evaluated at different times after envenomation. Obtained results showed that Aah venom affects the intestinal integrity. This cytotoxicity could be associated with lipid peroxidation and altered or insufficient antioxidant system. These results also highlight the perturbation of lipid absorption in intestine tissue of envenomed mice. The use of TNF-α antagonist prior to Aah venom injection seems to be able to improve lipid profile, oxidative stress and antioxidant activity. These findings suggest that Aah venom induces lipid alterations in the intestinal tissue mechanisms involving of TNF- α.

RFX transcription factors are essential for hearing in mice.

Sensorineural hearing loss is a common and currently irreversible disorder, because mammalian hair cells (HCs) do not regenerate and current stem cell and gene delivery protocols result only in immature HC-like cells. Importantly, although the transcriptional regulators of embryonic HC development have been described, little is known about the postnatal regulators of maturating HCs. Here we apply a cell type-specific functional genomic analysis to the transcriptomes of auditory and vestibular sensory epithelia from early postnatal mice. We identify RFX transcription factors as essential and evolutionarily conserved regulators of the HC-specific transcriptomes, and detect Rfx1,2,3,5 and 7 in the developing HCs. To understand the role of RFX in hearing, we generate Rfx1/3 conditional knockout mice. We show that these mice are deaf secondary to rapid loss of initially well-formed outer HCs. These data identify an essential role for RFX in hearing and survival of the terminally differentiating outer HCs.

TNF-alpha modulates adipose macrophage polarization to M1 phenotype in response to scorpion venom.

OBJECTIVE: We previously reported that Androctonus australis hector (Aah) venom and its toxic fraction affect adipose tissue metabolism. However, the contribution of immune system and the role of adipose tissue macrophages (ATMs) in the progression of inflammation induced by scorpion venom remain largely unknown. METHODS: Here we evaluate the capacity of the toxic fraction of Aah venom (FTox-G50) to induce the expression of M1 and M2 markers genes on adipose tissue and isolated stromal vascular cells (SVC). Quantitative real-time PCR was performed on the SVC 24 h after FTox-G50 venom injection to assess the gene expressions of IL12p40, IL23, and other macrophages-associated markers. RESULTS: We found that ATM from FTox-G50-venom-injected mice markedly increased the expressions of IL-12p40 and IL-23. Furthermore, the expression of nitric oxide synthase 2 (an M1 marker) was up-regulated, but the expression of Arginase1 (an M2 marker) was not. Systemic injection of a chemical inhibitor directed against TNF-α binding reduced the expression of inflammatory M1 macrophage markers and the MAPKpk2 gene, a key mediator of inflammatory signaling. CONCLUSION: These results indicate that TNF-α is a physiological regulator of inflammation and macrophage activation induced by scorpion venom.

HEATR2 plays a conserved role in assembly of the ciliary motile apparatus.

Cilia are highly conserved microtubule-based structures that perform a variety of sensory and motility functions during development and adult homeostasis. In humans, defects specifically affecting motile cilia lead to chronic airway infections, infertility and laterality defects in the genetically heterogeneous disorder Primary Ciliary Dyskinesia (PCD). Using the comparatively simple Drosophila system, in which mechanosensory neurons possess modified motile cilia, we employed a recently elucidated cilia transcriptional RFX-FOX code to identify novel PCD candidate genes. Here, we report characterization of CG31320/HEATR2, which plays a conserved critical role in forming the axonemal dynein arms required for ciliary motility in both flies and humans. Inner and outer arm dyneins are absent from axonemes of CG31320 mutant flies and from PCD individuals with a novel splice-acceptor HEATR2 mutation. Functional conservation of closely arranged RFX-FOX binding sites upstream of HEATR2 orthologues may drive higher cytoplasmic expression of HEATR2 during early motile ciliogenesis. Immunoprecipitation reveals HEATR2 interacts with DNAI2, but not HSP70 or HSP90, distinguishing it from the client/chaperone functions described for other cytoplasmic proteins required for dynein arm assembly such as DNAAF1-4. These data implicate CG31320/HEATR2 in a growing intracellular pre-assembly and transport network that is necessary to deliver functional dynein machinery to the ciliary compartment for integration into the motile axoneme.

TNF-α involvement in insulin resistance induced by experimental scorpion envenomation.

BACKGROUND: Scorpion venom induces systemic inflammation characterized by an increase in cytokine release and chemokine production. There have been few experimental studies assessing the effects of scorpion venom on adipose tissue function in vivo. METHODOLOGY/PRINCIPAL FINDINGS: To study the adipose tissue inflammation (ATI) induced by Androctonus australis hector (Aah) venom and to assess possible mechanisms of ATI, mice (n = 6, aged 1 month) were injected with Aah (0.45 mg/kg), toxic fraction of Aah (FTox-G50; 0.2 mg/kg) or saline solution (control). Inflammatory responses were evaluated by ELISA and cell sorting analyses in adipose tissue 45 minutes and 24 hours after injection. Quantitative real-time PCR was used to assess the regulation of genes implicated in glucose uptake. The titers of selected inflammatory cytokines (IL-1ß, IL-6 and TNF-α) were also determined in sera and in insulin target tissues. The serum concentration of IL-1ß rose 45 minutes after envenomation and returned to basal level after 24 hours. The pathophysiological effects of the venom after 24 hours mainly involved M1-proinflammatory macrophage infiltration in adipose tissue combined with high titers of IL-1ß, IL-6 and TNF-α. Indeed, TNF-α was strongly induced in both adipose tissue and skeletal muscle. We studied the effects of Aah venom on genes implicated in insulin-stimulated glucose uptake. Insulin induced a significant increase in the expression of the mRNAs for hexokinase 2 and phosphatidylinositol 3-kinase in both skeletal muscle and adipose tissue in control mice; this upregulation was completely abolished after 24 hours in mice envenomed with Aah or FTox-G50. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that Aah venom induces insulin resistance by mechanisms involving TNF-α-dependent Map4k4 kinase activation in the adipose tissue.

Cx36 is a target of Beta2/NeuroD1, which associates with prenatal differentiation of insulin-producing ß cells.

The insulin-producing ß cells of pancreatic islets are coupled by connexin36 (Cx36) channels. To investigate what controls the expression of this connexin, we have investigated its pattern during mouse pancreas development, and the influence of three transcription factors that are critical for ß-cell development and differentiation. We show that (1) the Cx36 gene (Gjd2) is activated early in pancreas development and is markedly induced at the time of the surge of the transcription factors that determine ß-cell differentiation; (2) the cognate protein is detected about a week later and is selectively expressed by ß cells throughout the prenatal development of mouse pancreas; (3) a 2-kbp fragment of the Gjd2 promoter, which contains three E boxes for the binding of the bHLH factor Beta2/NeuroD1, ensures the expression of Cx36 by ß cells; and (4) Beta2/NeuroD1 binds to these E boxes and, in the presence of the E47 ubiquitous cofactor, transactivates the Gjd2 promoter. The data identify Cx36 as a novel early marker of ß cells and as a target of Beta2/NeuroD1, which is essential for ß-cell development and differentiation.

The ciliogenic transcription factor RFX3 regulates early midline distribution of guidepost neurons required for corpus callosum development.

The corpus callosum (CC) is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3) is a transcription factor involved in the control of ciliogenesis, and Rfx3-deficient mice show several hallmarks of ciliopathies including left-right asymmetry defects and hydrocephalus. Here we show that Rfx3-deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8) at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3) repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies.

The transcription factor Rfx3 regulates beta-cell differentiation, function, and glucokinase expression.

OBJECTIVE: Pancreatic islets of perinatal mice lacking the transcription factor Rfx3 exhibit a marked reduction in insulin-producing beta-cells. The objective of this work was to unravel the cellular and molecular mechanisms underlying this deficiency. RESEARCH DESIGN AND METHODS: Immunofluorescence studies and quantitative RT-PCR experiments were used to study the emergence of insulin-positive cells, the expression of transcription factors implicated in the differentiation of beta-cells from endocrine progenitors, and the expression of mature beta-cell markers during development in Rfx3(-/-) and pancreas-specific Rfx3-knockout mice. RNA interference experiments were performed to document the consequences of downregulating Rfx3 expression in Min6 beta-cells. Quantitative chromatin immunoprecipitation (ChIP), ChIP sequencing, and bandshift experiments were used to identify Rfx3 target genes. RESULTS: Reduced development of insulin-positive cells in Rfx3(-/-) mice was not due to deficiencies in endocrine progenitors or beta-lineage specification, but reflected the accumulation of insulin-positive beta-cell precursors and defective beta-cells exhibiting reduced insulin, Glut-2, and Gck expression. Similar incompletely differentiated beta-cells developed in pancreas-specific Rfx3-deficient embryos. Defective beta-cells lacking Glut-2 and Gck expression dominate in Rfx3-deficent adults, leading to glucose intolerance. Attenuated Glut-2 and glucokinase expression, and impaired glucose-stimulated insulin secretion, were also induced by RNA interference-mediated inhibition of Rfx3 expression in Min6 cells. Finally, Rfx3 was found to bind in Min6 cells and human islets to two well-known regulatory sequences, Pal-1 and Pal-2, in the neuroendocrine promoter of the glucokinase gene. CONCLUSIONS: Our results show that Rfx3 is required for the differentiation and function of mature beta-cells and regulates the beta-cell promoter of the glucokinase gene.

RFX3 governs growth and beating efficiency of motile cilia in mouse and controls the expression of genes involved in human ciliopathies.

Cilia are cellular organelles that play essential physiological and developmental functions in various organisms. They can be classified into two categories, primary cilia and motile cilia, on the basis of their axonemal architecture. Regulatory factor X (RFX) transcription factors have been shown to be involved in the assembly of primary cilia in Caenorhabditis elegans, Drosophila and mice. Here, we have taken advantage of a novel primary-cell culture system derived from mouse brain to show that RFX3 is also necessary for biogenesis of motile cilia. We found that the growth and beating efficiencies of motile cilia are impaired in multiciliated Rfx3(-/-) cells. RFX3 was required for optimal expression of the FOXJ1 transcription factor, a key player in the differentiation program of motile cilia. Furthermore, we demonstrate for the first time that RFX3 regulates the expression of axonemal dyneins involved in ciliary motility by binding directly to the promoters of their genes. In conclusion, RFX proteins not only regulate genes involved in ciliary assembly, but also genes that are involved in ciliary motility and that are associated with ciliopathies such as primary ciliary dyskinesia in humans.

Pancreatic inactivation of c-Myc decreases acinar mass and transdifferentiates acinar cells into adipocytes in mice.

BACKGROUND & AIMS: The pancreatic mass is determined by the coordinated expansion and differentiation of progenitor cells and is maintained via tight control of cell replacement rates. The basic helix-loop-helix transcription factor c-Myc is one of the main regulators of these processes in many organs. We studied the requirement of c-Myc in controlling the generation and maintenance of pancreatic mass. METHODS: We conditionally inactivated c-Myc in Pdx1+ pancreatic progenitor cells. Pancreata of mice lacking c-Myc (c-Myc(P-/-) mice) were analyzed during development and ageing. RESULTS: Pancreatic growth in c-Myc(P-/-) mice was impaired starting on E12.5, in early primordia, because of decreased proliferation and altered differentiation of exocrine progenitors; islet progenitors were spared. Acinar cell maturation was defective in the adult hypotrophic pancreas, which hampered exocrine mass maintenance in aged animals. From 2 to 10 months of age, the c-Myc(P-/-) pancreas was progressively remodeled without inflammatory injury. Loss of acinar cells increased with time, concomitantly with adipose tissue accumulation. Using a genetic cell lineage tracing analysis, we demonstrated that pancreatic adipose cells were derived directly from transdifferentiating acinar cells. This epithelial-to-mesenchyme transition was also observed in normal aged specimens and in pancreatitis. CONCLUSIONS: These results provide evidence indicating that c-Myc activity is required for growth and maturation of the exocrine pancreas, and sheds new light on the ontogeny of pancreatic adipose cells in processes of organ degenerescence and tissue involution.

Vaccination against Marek's disease reduces telomerase activity and viral gene transcription in peripheral blood leukocytes from challenged chickens.

We investigated whether telomerase activity and viral gene transcription were associated with protection against the RB-1B strain of Marek's disease virus (MDV) in chickens vaccinated with Rispens CVI988 or the herpes virus of turkey (HVT). Telomerase activity in peripheral blood leukocytes (PBLs) seemed to be an appropriate marker of lymphoma and levels of viral transcription were correlated with the virulence of MDV strains. Vaccinated protected birds had lower levels of telomerase activity and RB-1B viral gene transcription than unvaccinated chickens infected with RB-1B. The decrease in RB-1B viral transcription was more marked in chickens vaccinated with CVI988 than in those vaccinated with HVT. Indeed, RB-1B viral transcription was not detectable after 14 days post-challenge. In conclusion, telomerase activity and gene transcription in challenge MDV strains are potential new reliable criteria of protection in vaccinated chickens.

Novel function of the ciliogenic transcription factor RFX3 in development of the endocrine pancreas.

The transcription factor regulatory factor X (RFX)-3 regulates the expression of genes required for the growth and function of cilia. We show here that mouse RFX3 is expressed in developing and mature pancreatic endocrine cells during embryogenesis and in adults. RFX3 expression already is evident in early Ngn3-positive progenitors and is maintained in all major pancreatic endocrine cell lineages throughout their development. Primary cilia of hitherto unknown function present on these cells consequently are reduced in number and severely stunted in Rfx3(-/-) mice. This ciliary abnormality is associated with a developmental defect leading to a uniquely altered cellular composition of the islets of Langerhans. Just before birth, Rfx3(-/-) islets contain considerably less insulin-, glucagon-, and ghrelin-producing cells, whereas pancreatic polypeptide-positive cells are markedly increased in number. In adult mice, the defect leads to small and disorganized islets, reduced insulin production, and impaired glucose tolerance. These findings suggest that RFX3 participates in the mechanisms that govern pancreatic endocrine cell differentiation and that the presence of primary cilia on islet cells may play a key role in this process.