Excess vitamin a might contribute to submucous clefting by inhibiting WNT-mediated bone formation.

OBJECTIVES: Cleft lip and/or palate (CLP) is a common craniofacial birth defect caused by genetic as well as environmental factors. The phenotypic spectrum of CLP also includes submucous clefts with a defect in the palatal bone. To elucidate the contribution of vitamin A, we evaluated the effects of the vitamin A metabolite all-trans retinoic acid (ATRA) on the osteogenic differentiation and mineralization of mouse embryonic palatal mesenchymal cells (MEPM). SETTING AND SAMPLE POPULATION: MEPM cells were isolated from the prefusion palates of E13 mouse embryos from three different litters. MATERIALS AND METHODS: MEPM cells were cultured with and without 0.5 µM ATRA in osteogenic medium. Differentiation was analysed by the expression of osteogenic marker genes and alkaline phosphatase (ALP) activity after 1, 2, and 7 days. The expression of Wnt marker genes was also analysed. Mineralization was assessed by alizarin red staining after 7, 14, 21, and 28 days. RESULTS: The bone marker genes Sp7, Runx2, Alpl, and Col1a1 were inhibited 10% ± 2%, 59% ± 7%, 79% ± 12% and 57% ± 20% (P < .05) at day 7. ALP activity was inhibited at days 1 and 7 by 35 ± 0% (P < .05) and 23 ± 6% (P < .001). ATRA also inhibited mineralization at 3 and 4 weeks. Finally, expression of the universal Wnt marker gene Axin2 was strongly reduced, by 31 ± 18% (P < .001), at day 7. CONCLUSION: Our data indicate that ATRA (vitamin A) inhibits bone formation by reducing Wnt signalling. This might contribute to the molecular aetiology of submucous clefting.

A potential osteogenic role for microRNA-181a-5p during palatogenesis.

BACKGROUND: In a previous study, we found that the highly conserved hsa-miR-181a-5p is downregulated in palatal fibroblasts of non-syndromic cleft palate-only infants. OBJECTIVES: To analyze the spatiotemporal expression pattern of mmu-miR-181a-5p during palatogenesis and identify possible mRNA targets and their involved molecular pathways. MATERIAL AND METHODS: The expression of mmu-miR-181a-5p was analyzed in the developing palates of mouse embryos from E11 to E18 using qPCR and ISH. Mouse embryonic palatal mesenchyme cells from E13 were used to analyze mmu-miR-181a-5p expression during osteogenic differentiation. Differential mRNA expression and target identification were analyzed using whole transcriptome RNA sequencing after transfection with a mmu-miR-181a-5p mimic. Differentially expressed genes were linked with underlying pathways using gene set enrichment analysis. RESULTS: The expression of mmm-miR-181a-5p in the palatal shelves increased from E15 and overlapped with palatal osteogenesis. During early osteogenic differentiation, mmu-miR-181a-5p was upregulated. Transient overexpression resulted in 49 upregulated mRNAs and 108 downregulated mRNAs (adjusted P-value < 0.05 and fold change > ± 1.2). Ossification (Stc1, Mmp13) and cell-cycle-related GO terms were significantly enriched for upregulated mRNAs. Analysis of possible mRNA targets indicated significant enrichment of Hippo signaling (Ywhag, Amot, Frmd6 and Serpine1) and GO terms related to cell migration and angiogenesis. LIMITATIONS: Transient overexpression of mmu-miR-181a-5p in mouse embryonic palatal mesenchyme cells limited its analysis to early osteogenesis. CONCLUSION: Mmu-miR-181-5p expression is increased in the developing palatal shelves in areas of bone formation and targets regulators of the Hippo signaling pathway.

Effect of niche components on masseter satellite cell differentiation on fibrin coatings.

In skeletal muscles, niche factors stimulate satellite cells to activate and induce muscle regeneration after injury. In vitro, matrigel is widely used for myoblast differentiation, however, is unsuitable for clinical applications. Therefore, this study aimed to analyze attachment and differentiation of satellite cells into myotubes on fibrin coatings with selected niche components. The attachment of satellite cells to fibrin alone and fibrin with niche components (laminin, collagen-IV, laminin-entactin complex [LEC]) were compared to matrigel. Only on matrigel and fibrin with LEC, Pax7-positive cells attached well. Then, LEC was selected to analyze proliferation, differentiation, and fusion indices. The proliferation index at day 1 on fibrin-LEC (22.5%, SD 9.1%) was similar to that on matrigel (30.8% [SD 11.1%]). The differentiation index on fibrin-LEC (28.7% [SD 6.1%] at day 5 and 32.8% [SD 6.7%] at day 7) was similar to that on matrigel (40.1% [5.1%] at day 5 and 27.1% [SD 4.3%] at day 7). On fibrin-LEC, the fusion index at day 9 (26.9% [SD 11.5%]) was similar to that on matrigel (25.5% [SD 4.7%]). Our results showed that the addition of LEC enhances the formation of myotubes on fibrin. Fibrin with LEC might be suitable to enhance muscle regeneration after surgery such as cleft palate repair and other muscle defects.

Retinoic acid effects on in vitro palatal fusion and WNT signaling.

Retinoic acid is the main active vitamin A derivate and a key regulator of embryonic development. Excess of retinoic acid can disturb palate development in mice leading to cleft palate. WNT signaling is one of the main pathways in palate development. We evaluated the effects of retinoic acid on palate fusion and WNT signaling in in vitro explant cultures. Unfused palates from E13.5 mouse embryos were cultured for 4 days with 0.5 µM, 2 µM or without retinoic acid. Apoptosis, proliferation, WNT signaling and bone formation were analyzed by histology and quantitative PCR. Retinoic acid treatment with 0.5 and 2.0 µM reduced palate fusion from 84% (SD 6.8%) in the controls to 56% (SD 26%) and 16% (SD 19%), respectively. Additionally, 2 µM retinoic acid treatment increased Axin2 expression. Retinoic acid also increased the proliferation marker Pcna as well as the number of Ki-67-positive cells in the palate epithelium. At the same time, the WNT inhibitors Dkk1, Dkk3, Wif1 and Sfrp1 were downregulated at least two-fold. Retinoic acid also down-regulated Alpl and Col1a2 gene expression. Alkaline phosphatase (ALP) activity was notably reduced in the osteogenic areas of the retinoic acid- treated palates. Our data suggest that retinoic acid impairs palate fusion and bone formation by upregulation of WNT signaling.

Highly variable rate of orthodontic tooth movement measured by a novel 3D method correlates with gingival inflammation.

OBJECTIVES: Individual orthodontic treatment duration is hard to predict. Individual biological factors are amongst factors influencing individual rate of orthodontically induced tooth movement (OTM). The study aim is to determine the rate of OTM by a novel 3D method and investigate parameters that may predict the rate of tooth movement. MATERIALS AND METHODS: In this prospective cohort study, rate of OTM was determined from 90 three-dimensional intra-oral scans in 15 patients (aged 12-15) undergoing orthodontic treatment. For each patient, intra-oral scans were taken every week for up to 6 weeks (T0-T5). The teeth were segmented from the scans and the scans were superimposed on the palatal rugae. The rate of OTM was calculated for each tooth. Other parameters were gingival inflammation, contact-point displacement and the biological markers, matrix metalloproteinases (MMP), MMP-9 and MMP-2 in gingival crevicular fluid (GCF). RESULTS: Our study showed a high variation in the rate of OTM, varying from 0.15 to 1.24 mm/week. Teeth in the anterior segment tended to move more compared with the posterior segment. The contact point displacement and gingival inflammation varied greatly amongst the patients. The MMPs measured did not correlate with tooth movement. However, the gingival inflammation index showed a significant correlation with OTM. Future studies should include other biological markers related to bone-remodeling. CONCLUSION: This novel and efficient 3D method is suitable for measuring OTM and showed large individual variation in rate of OTM. CLINICAL RELEVANCE: Patients show different rates of OTM. The rate of OTM in an individual patient can provide guidance in timing of follow-up appointments.

Tissue engineering strategies combining molecular targets against inflammation and fibrosis, and umbilical cord blood stem cells to improve hampered muscle and skin regeneration following cleft repair.

Cleft lip with or without cleft palate is a congenital deformity that occurs in about 1 of 700 newborns, affecting the dentition, bone, skin, muscles and mucosa in the orofacial region. A cleft can give rise to problems with maxillofacial growth, dental development, speech, and eating, and can also cause hearing impairment. Surgical repair of the lip may lead to impaired regeneration of muscle and skin, fibrosis, and scar formation. This may result in hampered facial growth and dental development affecting oral function and lip and nose esthetics. Therefore, secondary surgery to correct the scar is often indicated. We will discuss the molecular and cellular pathways involved in facial and lip myogenesis, muscle anatomy in the normal and cleft lip, and complications following surgery. The aim of this review is to outline a novel molecular and cellular strategy to improve musculature and skin regeneration and to reduce scar formation following cleft repair. Orofacial clefting can be diagnosed in the fetus through prenatal ultrasound screening and allows planning for the harvesting of umbilical cord blood stem cells upon birth. Tissue engineering techniques using these cord blood stem cells and molecular targeting of inflammation and fibrosis during surgery may promote tissue regeneration. We expect that this novel strategy improves both muscle and skin regeneration, resulting in better function and esthetics after cleft repair.

Differential microRNA expression in cultured palatal fibroblasts from infants with cleft palate and controls.

Background: The role of microRNAs (miRNAs) in animal models of palatogenesis has been shown, but only limited research has been carried out in humans. To date, no miRNA expression study on tissues or cells from cleft palate patients has been published. We compared miRNA expression in palatal fibroblasts from cleft palate patients and age-matched controls. Material and Methods: Cultured palatal fibroblasts from 10 non-syndromic cleft lip and palate patients (nsCLP; mean age: 18 ± 2 months), 5 non-syndromic cleft palate only patients (nsCPO; mean age: 17 ± 2 months), and 10 controls (mean age: 24 ± 5 months) were analysed with next-generation small RNA sequencing. All subjects are from Western European descent. Sequence reads were bioinformatically processed and the differentially expressed miRNAs were technically validated using quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Results: Using RNA sequencing, three miRNAs (hsa-miR-93-5p, hsa-miR-18a-5p, and hsa-miR-92a-3p) were up-regulated and six (hsa-miR-29c-5p, hsa-miR-549a, hsa-miR-3182, hsa-miR-181a-5p, hsa-miR-451a, and hsa-miR-92b-5p) were down-regulated in nsCPO fibroblasts. One miRNA (hsa-miR-505-3p) was down-regulated in nsCLP fibroblasts. Of these, hsa-miR-505-3p, hsa-miR-92a, hsa-miR-181a, and hsa-miR-451a were also differentially expressed using RT-PCR with a higher fold change than in RNAseq. Limitations: The small sample size may limit the value of the data. In addition, interpretation of the data is complicated by the fact that biopsy samples are taken after birth, while the origin of the cleft lies in the embryonic period. This, together with possible effects of the culture medium, implies that only cell-autonomous genetic and epigenetic differences might be detected. Conclusions: For the first time, we have shown that several miRNAs appear to be dysregulated in palatal fibroblasts from patients with nsCLP and nsCPO. Furthermore, large-scale genomic and expression studies are needed to validate these findings.

Retinoic acid signalling in the development of the epidermis, the limbs and the secondary palate.

Retinoic acid (RA), the active derivative of vitamin A, is one of the major regulators of embryonic development, including the development of the epidermis, the limbs and the secondary palate. In the embryo, RA levels are tightly regulated by the activity of RA synthesizing and degrading enzymes. Aberrant RA levels due to genetic variations in RA metabolism pathways contribute to congenital malformations in these structures. In vitro and in vivo studies provide considerable evidence on the effects of RA and its possible role in the development of the epidermis, the limbs and the secondary palate. In conjunction with other regulatory factors, RA seems to stimulate the development of the epidermis by inducing proliferation and differentiation of ectodermal cells into epidermal cells. In the limbs, the exact timing of RA location and level is crucial to initiate limb bud formation and to allow chondrogenesis and subsequent osteogenesis. In the secondary palate, the correct RA concentration is a key factor for mesenchymal cell proliferation during palatal shelf outgrowth, elevation and adhesion, and finally to allow bone formation in the hard palate. These findings are highly relevant to understanding the mechanism of RA signalling in development and in the aetiology of specific congenital diseases.

Novel mutations in LRP6 highlight the role of WNT signaling in tooth agenesis.

PURPOSE: We aimed to identify a novel genetic cause of tooth agenesis (TA) and/or orofacial clefting (OFC) by combining whole-exome sequencing (WES) and targeted resequencing in a large cohort of TA and OFC patients. METHODS: WES was performed in two unrelated patients: one with severe TA and OFC and another with severe TA only. After deleterious mutations were identified in a gene encoding low-density lipoprotein receptor-related protein 6 (LRP6), all its exons were resequenced with molecular inversion probes in 67 patients with TA, 1,072 patients with OFC, and 706 controls. RESULTS: We identified a frameshift (c.4594delG, p.Cys1532fs) and a canonical splice-site mutation (c.3398-2A>C, p.?) in LRP6, respectively, in the patient with TA and OFC and in the patient with severe TA only. The targeted resequencing showed significant enrichment of unique LRP6 variants in TA patients but not in nonsyndromic OFC patients. Of the five variants in patients with TA, two affected the canonical splice site and three were missense variants; all variants segregated with the dominant phenotype, and in one case the missense mutation occurred de novo. CONCLUSION: Mutations in LRP6 cause TA in humans.Genet Med 18 11, 1158-1162.

Curcumin induces differential expression of cytoprotective enzymes but similar apoptotic responses in fibroblasts and myofibroblasts.

Excessive extracellular matrix (ECM) deposition and tissue contraction after injury can lead to esthetic and functional problems. Fibroblasts and myofibroblasts activated by transforming growth factor (TGF)-ß1 play a key role in these processes. The persistence of (myo)fibroblasts and their excessive ECM production and continuous wound contraction have been linked to pathological scarring. The identification of compounds reducing myofibroblast survival and function may thus offer promising therapeutic strategies to optimize impaired wound healing. The plant-derived polyphenol curcumin has shown promising results as a wound healing therapeutic in vivo; however, the exact mechanism is still unclear. In vitro, curcumin induces apoptosis in various cell types via a reactive oxygen species (ROS)-dependent mechanism. Here we treated human dermal fibroblasts with TGF-ß1 to induce myofibroblast differentiation, and compared the responses of fibroblasts and myofibroblasts to 25 µM curcumin. Curcumin induced caspase-independent apoptosis in both fibroblasts and myofibroblasts in a ROS-dependent manner. Oxidative stress leads to the induction of several antioxidant systems to regain cellular homeostasis. We detected stress-induced induction of heme oxygenase (HO)-1 in fibroblasts but not in myofibroblasts following curcumin exposure. Instead, myofibroblasts expressed higher levels of heat shock protein (HSP)72 compared to fibroblasts in response to curcumin, suggesting that TGF-ß1 treatment alters the stress-responses of the cells. However, we did not detect any differences in curcumin toxicity between the two populations. The differential stress responses in fibroblasts and myofibroblasts may open new therapeutic approaches to reduce myofibroblasts and scarring.

Cytoprotective responses in HaCaT keratinocytes exposed to high doses of curcumin.

Wound healing is a complex process that involves the well-coordinated interactions of different cell types. Topical application of high doses of curcumin, a plant-derived polyphenol, enhances both normal and diabetic cutaneous wound healing in rodents. For optimal tissue repair interactions between epidermal keratinocytes and dermal fibroblasts are essential. We previously demonstrated that curcumin increased reactive oxygen species (ROS) formation and apoptosis in dermal fibroblasts, which could be prevented by pre-induction of the cytoprotective enzyme heme oxygenase (HO)-1. To better understand the effects of curcumin on wound repair, we now assessed the effects of high doses of curcumin on the survival of HaCaT keratinocytes and the role of the HO system. We exposed HaCaT keratinocytes to curcumin in the presence or absence of the HO-1 inducers heme (FePP) and cobalt protoporphyrin (CoPP). We then assessed cell survival, ROS formation, and caspase activation. Curcumin induced caspase-dependent apoptosis in HaCaT keratinocytes via a ROS-dependent mechanism. Both FePP and CoPP induced HO-1 expression, but only FePP protected against curcumin-induced ROS formation and caspase-mediated apoptosis. In the presence of curcumin, FePP but not CoPP induced the expression of the iron scavenger ferritin. Together, our data show that the induction of ferritin, but not HO, protects HaCaT keratinocytes against cytotoxic doses of curcumin. The differential response of fibroblasts and keratinocytes to high curcumin doses may provide the basis for improving curcumin-based wound healing therapies.

Fibrosis impairs the formation of new myofibers in the soft palate after injury.

Muscle repair is a crucial component of palatoplasty but little is known about muscle regeneration after cleft palate repair. We hypothesized that the formation of new myofibers is hampered by collagen accumulation after experimental injury of the soft palate of rats. One-millimeter excisional defects were made in the soft palates of 32 rats. The wound area was evaluated after 3, 7, 28, and 56 days using azocarmine G and aniline blue to stain for collagen and immunohistochemistry to identify myofibroblasts and to monitor skeletal muscle differentiation. To evaluate age effects, 16 unwounded animals were evaluated at 3 and 56 days. Staining was quantified by image analysis, and one-way ANOVA was used for the statistical analysis. At day 56, the area percentage of collagen-rich tissue was higher in the injured palatal muscles (46.7 ± 6.9%) than in nonwounded controls (15.9 ± 1.0%, p < 0.05). Myofibroblasts were present in the injured muscles at days 3 and 7 only. The numbers of proliferating and differentiating myoblasts within the wound area were greater at day 7 (p < 0.05), but only a few new myofibers had formed by 56 days. No age effects were found. The results indicate that surgical wounding of the soft palate results in muscle fibrosis. Although activated satellite cells migrated into the wound area, no new myofibers formed. Thus, regeneration and function of the soft palate muscles after injury may be improved by regenerative medicine approaches.

Mechanical cues in orofacial tissue engineering and regenerative medicine.

Cleft lip and palate patients suffer from functional, aesthetical, and psychosocial problems due to suboptimal regeneration of skin, mucosa, and skeletal muscle after restorative cleft surgery. The field of tissue engineering and regenerative medicine (TE/RM) aims to restore the normal physiology of tissues and organs in conditions such as birth defects or after injury. A crucial factor in cell differentiation, tissue formation, and tissue function is mechanical strain. Regardless of this, mechanical cues are not yet widely used in TE/RM. The effects of mechanical stimulation on cells are not straight-forward in vitro as cellular responses may differ with cell type and loading regime, complicating the translation to a therapeutic protocol. We here give an overview of the different types of mechanical strain that act on cells and tissues and discuss the effects on muscle, and skin and mucosa. We conclude that presently, sufficient knowledge is lacking to reproducibly implement external mechanical loading in TE/RM approaches. Mechanical cues can be applied in TE/RM by fine-tuning the stiffness and architecture of the constructs to guide the differentiation of the seeded cells or the invading surrounding cells. This may already improve the treatment of orofacial clefts and other disorders affecting soft tissues.

Wnt16 is involved in intramembranous ossification and suppresses osteoblast differentiation through the Wnt/ß-catenin pathway.

In the course of embryonic development skeletal elements form either through intramembranous or endochondral ossification. Wnt proteins play diverse roles during vertebrate skeletal development. Wnt16 is a key factor in developing long bones, but its exact role in craniofacial bone formation remains unclear. This study was initially undertaken to investigate the expression of Wnt16 during craniofacial bone development in mouse embryos. Wnt16 expression in the osteoid of calvaria, maxilla, and mandible started later than that of ALP and osteocalcin (OCN), but before mineralization of the craniofacial bones, suggesting that Wnt16 is involved in intramembranous ossification in the head. To confirm this, MC3T3-E1 cells were transfected with an adenovirus containing Wnt16 (Ad-Wnt16). Ad-Wnt16 cells showed decreased ALP activity and less mineralized nodule formations compared with control cells. In addition, the mRNA levels of osteogenic markers were reduced. Moreover, Wnt16 activated ß-catenin signaling in MC3T3-E1 cells at both transcription and protein levels as shown by a TOPflash luciferase reporter gene assay and western blot analysis. On the other hand, Wnt/ß-catenin pathway blockade by Dickkopf 1 abrogated the suppression of mineralization by Wnt16. Our findings suggest that Wnt16 is involved in intramembranous ossification and suppresses osteoblast differentiation through the Wnt/ß-catenin pathway.

Effects of retinoic acid on proliferation and gene expression of cleft and non-cleft palatal keratinocytes.

BACKGROUND: Retinoic acid (RA) is a key regulator of embryonic development and linked to several birth defects including cleft lip and palate (CLP). The aim was to investigate the effects of RA on proliferation and gene expression of human palatal keratinocytes (KCs) in vitro. METHODS: KCs from children with and without CLP were cultured with 2 and 5 µM RA. Proliferation was measured by quantification of DNA after 2, 4, 6, and 8 days. In addition, we analysed the effects of RA on messenger RNA expression of genes for proliferation, differentiation, apoptosis, and RA receptors. RESULTS: RA similarly inhibited proliferation of palatal KC from cleft and non-cleft subjects. The proliferation of KCs from cleft subjects was reduced to 59.8±13.4% (2 µM) and 41.5±14.0% (5 µM, Day 6), while that of cells from age-matched non-cleft subjects was reduced to 66.9±12.1% (2 µM) and 33.9±10.1% (5 µM). RA treatment reduced the expression of several of the investigated genes; the proliferating cell nuclear antigen (PCNA) was reduced in CLP KCs only. Keratins 10 and 16 were downregulated in keratinocytes from both cleft and non-cleft subjects. P63, a master regulator for epithelial differentiation, was only downregulated in KCs from cleft subjects, as was the RXRa receptor. Two P63 target genes (GJB6 and DLX5) were strongly downregulated by RA in all cell lines. None of the apoptosis genes was affected. CONCLUSION: Overall, RA similarly inhibits proliferation of palatal KCs from cleft and non-cleft subjects and reduces the expression of specific genes.

Stem cells and extracellular vesicles to improve preclinical orofacial soft tissue healing.

Orofacial soft tissue wounds caused by surgery for congenital defects, trauma, or disease frequently occur leading to complications affecting patients' quality of life. Scarring and fibrosis prevent proper skin, mucosa and muscle regeneration during wound repair. This may hamper maxillofacial growth and speech development. To promote the regeneration of injured orofacial soft tissue and attenuate scarring and fibrosis, intraoral and extraoral stem cells have been studied for their properties of facilitating maintenance and repair processes. In addition, the administration of stem cell-derived extracellular vesicles (EVs) may prevent fibrosis and promote the regeneration of orofacial soft tissues. Applying stem cells and EVs to treat orofacial defects forms a challenging but promising strategy to optimize treatment. This review provides an overview of the putative pitfalls, promises and the future of stem cells and EV therapy, focused on orofacial soft tissue regeneration.

Disruption of the foxe1 gene in zebrafish reveals conserved functions in development of the craniofacial skeleton and the thyroid.

Introduction: Mutations in the FOXE1 gene are implicated in cleft palate and thyroid dysgenesis in humans. Methods: To investigate whether zebrafish could provide meaningful insights into the etiology of developmental defects in humans related to FOXE1, we generated a zebrafish mutant that has a disruption in the nuclear localization signal in the foxe1 gene, thereby restraining nuclear access of the transcription factor. We characterized skeletal development and thyroidogenesis in these mutants, focusing on embryonic and larval stages. Results: Mutant larvae showed aberrant skeletal phenotypes in the ceratohyal cartilage and had reduced whole body levels of Ca, Mg and P, indicating a critical role for foxe1 in early skeletal development. Markers of bone and cartilage (precursor) cells were differentially expressed in mutants in post-migratory cranial neural crest cells in the pharyngeal arch at 1 dpf, at induction of chondrogenesis at 3 dpf and at the start of endochondral bone formation at 6 dpf. Foxe1 protein was detected in differentiated thyroid follicles, suggesting a role for the transcription factor in thyroidogenesis, but thyroid follicle morphology or differentiation were unaffected in mutants. Discussion: Taken together, our findings highlight the conserved role of Foxe1 in skeletal development and thyroidogenesis, and show differential signaling of osteogenic and chondrogenic genes related to foxe1 mutation.

Myogenic capacity of muscle progenitor cells from head and limb muscles.

The restoration of muscles in the soft palate of patients with cleft lip and/or palate is accompanied by fibrosis, which leads to speech and feeding problems. Treatment strategies that improve muscle regeneration have only been tested in limb muscles. Therefore, in the present study the myogenic potential of muscle progenitor cells (MPCs) isolated from head muscles was compared with that of limb muscles. Muscle progenitor cells were isolated from the head muscles and limb muscles of rats and cultured. The proliferation of MPCs was analysed by DNA quantification. The differentiation capacity was analysed by quantifying the numbers of fused cells, and by measuring the mRNA levels of differentiation markers. Muscle progenitor cells were stained to quantify the expression of paired box protein Pax 7 (Pax-7), myoblast determination protein 1 (MyoD), and myogenin. Proliferation was similar in the head MPCs and the limb MPCs. Differentiating head and limb MPCs showed a comparable number of fused cells and mRNA expression levels of myosin-1 (Myh1), myosin-3 (Myh3), and myosin-4 (Myh4). During proliferation and differentiation, the number of Pax-7(+), MyoD(+), and myogenin(+) cells in head and limb MPCs was equal. It was concluded that head and limb MPCs show similar myogenic capacities in vitro. Therefore, in vivo myogenic differences between those muscles might rely on the local microenvironment. Thus, regenerative strategies for limb muscles might also be used for head muscles.

Targeting fibroblast growth factor receptors causes severe craniofacial malformations in zebrafish larvae.

Background and Objective: A key pathway controlling skeletal development is fibroblast growth factor (FGF) and FGF receptor (FGFR) signaling. Major regulatory functions of FGF signaling are chondrogenesis, endochondral and intramembranous bone development. In this study we focus on fgfr2, as mutations in this gene are found in patients with craniofacial malformations. The high degree of conservation between FGF signaling of human and zebrafish (Danio rerio) tempted us to investigate effects of the mutated fgfr2 sa10729 allele in zebrafish on cartilage and bone formation. Methods: We stained cartilage and bone in 5 days post fertilization (dpf) zebrafish larvae and compared mutants with wildtypes. We also determined the expression of genes related to these processes. We further investigated whether pharmacological blocking of all FGFRs with the inhibitor BGJ398, during 0-12 and 24-36 h post fertilization (hpf), affected craniofacial structure development at 5 dpf. Results: We found only subtle differences in craniofacial morphology between wildtypes and mutants, likely because of receptor redundancy. After exposure to BGJ398, we found dose-dependent cartilage and bone malformations, with more severe defects in fish exposed during 0-12 hpf. These results suggest impairment of cranial neural crest cell survival and/or differentiation by FGFR inhibition. Compensatory reactions by upregulation of fgfr1a, fgfr1b, fgfr4, sp7 and dlx2a were found in the 0-12 hpf group, while in the 24-36 hpf group only upregulation of fgf3 was found together with downregulation of fgfr1a and fgfr2. Conclusions: Pharmacological targeting of FGFR1-4 kinase signaling causes severe craniofacial malformations, whereas abrogation of FGFR2 kinase signaling alone does not induce craniofacial skeletal abnormalities. These findings enhance our understanding of the role of FGFRs in the etiology of craniofacial malformations.

The recruitment of bone marrow-derived cells to skin wounds is independent of wound size.

Wounded skin recruits progenitor cells, which repair the tissue defect. These cells are derived from stem cells in several niches in the skin. In addition, bone marrow-derived cells (BMDCs) are recruited and contribute to wound repair. We hypothesized that larger wounds recruit more cells from the bone marrow. Wild-type rats were lethally irradiated and transplanted with bone marrow cells from green fluorescent protein (GFP)-transgenic rats. Seven weeks later, 4, 10, and 20 mm wounds were created. The wound tissue was harvested after 14 days. The density of GFP-positive cells in the wounds and the adjacent tissues was determined, as well as in normal skin from the flank. Bone marrow-derived myofibroblasts, activated fibroblasts, and macrophages were also quantified. After correction for cell density, the recruitment of BMDCs (23±11%) was found to be independent of wound size. Similar fractions of GFP-positive cells were also detected in nonwounded adjacent tissue (29±11%), and in normal skin (26±19%). The data indicate that BMDCs are not preferentially recruited to skin wounds. Furthermore, wound size does not seem to affect the recruitment of BMDCs.