Development of a model to investigate the effects of prolonged ischaemia on the muscles of mastication of male Sprague Dawley rats.

OBJECTIVE: The aims of this study were to develop a novel rodent model of masticatory muscle ischaemia via unilateral ligation of the external carotid artery (ECA), and to undertake a preliminary investigation to characterize its downstream effects on mechanosensitivity and cellular features of the masseter and temporalis muscles. DESIGN: The right ECA of 18 male Sprague-Dawley rats was ligated under general anaesthesia. Mechanical detection thresholds (MDTs) at the masseter and temporalis bilaterally were measured immediately before ECA ligation and after euthanasia at 10-, 20-, and 35-days (n = 6 rats/timepoint). Tissue samples from both muscles and sides were harvested for histological analyses and for assessing changes in the expression of markers of hypoxia and muscle degeneration (Hif-1α, VegfA, and Fbxo32) via real time PCR. Data were analyzed using mixed effect models and non-parametric tests. Statistical significance was set at p < 0.05. RESULTS: MDTs were higher in the right than left hemiface (p = 0.009) after 20 days. Histological changes indicative of muscle degeneration and fibrosis were observed in the right muscles. Hif-1α, VegfA, and Fbxo32 were more highly expressed in the masseter than temporalis muscles (all p < 0.05). Hif-1α and, VegfA did not change significantly with time in all muscles (all p > 0.05). Fbxo32 expression gradually increased in the right masseter (p = 0.024) and left temporalis (p = 0.05). CONCLUSIONS: ECA ligation in rats induced hyposensitivity in the homolateral hemiface after 20 days accompanied by tissue degenerative changes. Our findings support the use of this model to study pathophysiologic mechanisms of masticatory muscle ischaemia in larger investigations.

[Peripheral small airway dysfunction differences between idiopathic pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension].

Objective: To investigate the peripheral small airway dysfunction differences between idiopathic pulmonary arterial hypertension (IPAH) and chronic thromboembolic pulmonary hypertension (CTEPH). Methods: Impulse oscillmetory system testing (IOS) and pulmonary function testing (PFT) were performed in IPAH and CTEPH patients and 30 healthy control group. We also carried out a subgroup analysis depending on their medical history of airway diseases. Results: We included 42 IPAH and 47 CTEPH patients (with or without airways disease: 8 vs. 34 and 17 vs. 34, respectively). Compared with CTEPH patients, IPAH patients were younger but had more serious pulmonary vessel resistance and mean pulmonary arterial resistance. Compared with IPAH patients, CTEPH patients had significant impaired peripheral small airway dysfunction with decreased of MEF(50) (% pred), MMEF(75/25) evaluated by PFT and R5-R20, Δ R5-R20 and AX measured by IOS [10.6(2.0, 33.0) vs. 2.5(-5.0, 16.5); 22.1(14.0, 32.6) vs. 15.5 (7.0, 23.2); 7.64(4, 18.6) vs. 6(3, 11) respectively, all P<0.05]. Subgroup analysis revealed there were no significant peripheral small dysfunction differences in IPAH patients with or without airway diseases. CTEPH patients had a higher proportion of airway diseases and more serious peripheral dysfunction than IPAH patients with airway diseases. Compared with control healthy group, peripheral airway dysfunction was more obvious even in IPAH and CTEPH patients without airway diseases. Conclusion: Compared with IPAH, CTEPH patients were older, but had better hemodynamics and a higher proportion of airway diseases. The peripheral airway dysfunction were more serious in CTEPH patients without airway diseases than IPAH patients without airway diseases and healthy controls group.

[Clinical and imaging features of pulmonary veno-occlusive disease and pulmonary capillary hemangioma].

Objective: To improve the diagnosis and treatment of the pulmonary veno-occlusive disease (PVOD) and pulmonary capillary hemangioma (PCH). Methods: The clinical features, radiological findings, laboratory testing and treatment in 8 cases of PVOD/PCH which was diagnosed from 2013 to 2017 were described. Results: PVOD/PCH was rare. The clinical symptoms were easily confused with IPAH, but the decrease of hypoxemia, clubbing, D(L)CO were more obvious, and the imaging features of HRCT were helpful for PVOD/PCH diagnosis. Combined with gene testing, it was helpful to diagnose PVOD/PCH and avoid the risk of surgical biopsy. Conclusion: PVOD and PCH are rare type of pulmonary vascular diseases. According to clinical manifestations, physical examination, pulmonary function test results, HRCT imaging, CPET and gene detection results, PVOD or PCH can be diagnosed.

[Clinical analysis of Behçet disease associated with pulmonary vascular disease].

Objective: To investigate the clinical features, therapy and prognosis of Behcet's disease(BD) complicated with pulmonary vascular diseases (PVD). Methods: The clinical manifestation, hemodynamics, pulmonary artery angiographic findings, therapy and prognosis of PVD in BD in Shanghai Pulmonary Hospital from January 2009 to August 2016 were analyzed retrospectively. Results: Seven patients with average of (37±20) years were included. The ratio of male vs. female was 6∶1. Vascular involvement included pulmonary hypertension (PH) (4/7), pulmonary embolism (PE) (3/7) and pulmonary artery aneurysms (PAA) (3/7). All patients received immunosuppressive agents and all PH patients had therapy of PH target drugs. Three out of 4 PE patients underwent anticoagulant therapy. Conclusion: PVD is a rare complication of BD. The common clinical manifestations are PH, PE and PAA. Management of immunosuppressive agents may improve the prognosis and should be recommended.

Effects of two wattages of low-level laser therapy on orthodontic tooth movement.

INTRODUCTION: Mixed outcomes have been found in animal and clinical studies with regard to the use of low-level laser therapy (LLLT) as a modality to accelerate orthodontic tooth movement (OTM). One major reason for the variable findings is the different methodologies and protocols for laser therapy use. OBJECTIVE: The aim of this study was to determine whether orthodontically moved molars exposed to two different wattages at the same energy density of LLLT exhibited differences in the amount of tooth movement and molecular and histological changes in the adjacent periodontal areas. METHODS: An orthodontic force was applied to rat upper first molars exposed to 500mW (EX-500) and 1000mW (EX-1000) of laser application, with a control group (CT) with no laser application. Gene expression in the periodontal ligament (PDL) and histology of the palatal gingiva of the molars were analyzed. RESULTS: There was a statistically significant difference for OTM between EX-500 but not between EX-1000 and CT groups. RANKL and MMP-13 expression levels in the PDL of orthodontically moved molars, however, were increased significantly in laser-exposed groups compared to CT. Early signs of dysplasia were observed in over half of the animals in the EX-1000 group. CONCLUSIONS: Our results provide evidence for molecular changes and the potential dysplastic effects of laser on the surrounding soft tissues. Further studies are needed to better identify an optimum laser protocol to maximize the desired effect.

[End tidal PCO(2) for evaluation of severity of disease in idiopathic pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension].

Objective: To explore the difference in end tidal PCO(2) (P(ET)CO(2)) between idiopathic pulmonary arterial hypertension (IPAH) and chronic thromboembolic pulmonary hypertension (CTEPH), and to analyze the correlation between P(ET)CO(2) and the indexes of disease severity in IPAH and CTEPH patients. Methods: Data were retrieved from 68 IPAH patients and 52 CTEPH patients who all had received right-heart catheterization, pulmonary function test and cardiopulmonary exercise testing at Shanghai Pulmonary Hospital from October 2011 to October 2014. In addition, other clinical parameters were also collected. Results: The IPAH group had a significantly higher mPAP, PVR [(60±16) mmHg (1 mmHg=0.133 kPa), (13±6) Wood U ] than the CTEPH group [(46±12) mmHg, (9±4) Wood U, t=4.90, 4.83, all P<0.01]. Meanwhile, the IPAH group had a lower percentage of predicted peakVO(2), oxygen pulse [(45±15)%, (60±22)%] compared with the CTEPH group [(53±16)%, (68±21)%, t=-2.42, -1.96, all P<0.05]. The value of P(ET)CO(2) at rest, AT, peak in the IPAH patients [(27±5), (28±7), (25±7) mmHg] were higher than those in the CTEPH patients [(24±4) mmHg, (23±6) mmHg, (21±6) mmHg, t=3.22-4.54, all P<0.01]. There was a significantly difference in P(ET)CO(2) at AT and peak between WHO-FC Ⅰ-Ⅱ and Ⅲ-Ⅳ subgroups in IPAH (t=2.55, 2.60, all P<0.05) and CTEPH (t=2.39, P<0.05), except for P(ET)CO(2) at peak in the CTEPH patients (t=1.71, P>0.05). A moderately inverse correlation was found between P(ET)CO(2) at AT and NT-proBNP in the IPAH group (r=-0.58, P<0.01), meanwhile P(ET)CO(2) at AT in the CTEPH group was weakly correlated with NT-proBNP (r=-0.34, P<0.05). Conclusions: Compared with the CTEPH patients, the IPAH patients had significantly decreased exercise capacity and increased P(ET)CO(2). P(ET)CO(2) could reflect the disease severity in both IPAH and CTEPH patients, being superior in IPAH than in CTEPH. Furthermore, P(ET)CO(2) at AT might be better than P(ET)CO(2) at peak in reflecting the ventilatory efficiency.

Nitric oxide in human gingival crevicular fluid after orthodontic force application.

Nitric oxide (NO) is involved in bone remodelling and has been shown to play a role in regulating the rate of orthodontic tooth movement (OTM) in rat models. In humans, however, the role of NO in OTM remains less clear. In this study, NO concentration in gingival crevicular fluid (GCF) was measured in patients undergoing orthodontic treatment. Thirteen male participants (ages 11-18 years) planned for non-extraction fixed orthodontic therapy were recruited. Samples of GCF were collected from each maxillary central incisor and first and second molar immediately before (T0), 1h after (T1), and 3-4 days after (T2) application of light orthodontic forces. The maxillary second molars were not included in the appliance and served as controls. Measureable NO levels were consistently obtained from all sampled sites. Total NO levels showed significantly higher NO levels (p<0.05) at T1 at the buccal surfaces of the central incisors when compared to the first and second molars. The results indicate a possible role for NO in OTM at the pressure sites of incisors at early time points. Further studies are required to determine whether NO levels in the periodontal ligament tissues of human teeth during OTM are affected by a force gradient and the magnitude of the applied force.

Development of a three-dimensional in vitro model system to study orthodontic tooth movement.

Few three-dimensional (3-D) models exist to study the cellular aspects and molecular regulation of orthodontic tooth movement (OTM). The aim of this study was to develop a 3-D in vitro model to study mechanical loading of human periodontal ligament (PDL) fibroblasts (hPDLF). hPDLF were seeded within collagen gels to form a PDLF analogue. Characterisation of the seeded collagen gels revealed that the gels supported cell proliferation, viability and the emergence of a possible contractile phenotype, replicating the constrained condition of the human PDL in vivo. We next developed a 3-D model that incorporated a seeded collagen gel interlocked mechanically at two ends to movable end plates. The movable end plates allowed for static tensile or compressive loading of the hPDLF-seeded collagen gels. Preliminary testings showed that this 3-D model mimicked PDL strains similar to those observed during OTM. Our 3-D model of OTM therefore offers promise for use as a model system in future studies to improve our understanding of the effects of OTM on PDLF.

The Fgfr2 W290R mouse model of Crouzon syndrome.

PURPOSE: This study aimed to review and discuss the utility of the Fgfr2 (W290R) mouse mutant as a model of human Crouzon syndrome. METHODS: A review of current and past scientific literature on Fibroblast Growth Factor Receptor-2 (FGFR2) protein domain structure, FGFR mutations associated with human Crouzon syndrome, and phenotypic and molecular changes combined with recent observations and experimental data of the Fgfr2 (W290R) mouse mutant was conducted. A comparison of the Fgfr2 (W290R) mouse mutant with another mouse model of Crouzon syndrome, Fgfr2 (C342R) mouse mutant, was also performed. Finally, possible future research directions using the Fgfr2 (W290R) mutant mice were discussed. RESULTS: The Fgfr2 (W290R) heterozygous mouse exhibits defects characteristic of human Crouzon syndrome. At the molecular level, the defects observed in the mouse mutant are due to the dysregulation of signaling of both the IIIb and IIIc isoforms of Fgfr2. The involvement of the IIIb isoform of FGFR2 in the etiopathology of Crouzon syndrome is a novel finding in the craniosynostosis literature field. Dysregulated signaling of both IIIb and IIIc isoforms causes a broad spectrum of changes that explain some of the defects observed clinically in humans. Several of the defects observed in the Fgfr2 (W290R) homozygous mouse mutant are attributable to a loss-of-function mechanism in contrast to the frequently reported gain-of-function receptor function associated with mutated FGF receptors in craniosynostosis. CONCLUSIONS: The Fgfr2 ( W290R ) mouse model can be used as a model system to further investigate the cellular, molecular, and biochemical mechanisms of Crouzon syndrome.

Mouse FLRT2 interacts with the extracellular and intracellular regions of FGFR2.

Fibroblast Growth Factor (FGF) signaling is known to be critical in mediating key developmental events during craniofacial development. Recent evidence suggests that members of the Fibronectin (F) Leucine (L) Rich (R) Transmembrane (T), FLRT, family modulate FGF signaling. FLRT2 has a highly specific pattern of expression during craniofacial development, in close relationship with FGFR2. We therefore characterized FLRT2/FGFR2 interactions in the context of craniofacial development and showed, by co-immunoprecipitation and GST pulldown assays with embryonic craniofacial tissue lysates, that FLRT2 interacted with FGFR2. Yeast two-hybrid assays further showed that the intracellular regions of both proteins interacted in addition to the interactions in the extracellular portions. The extracellular Leucine Rich Repeats domain of FLRT2 contributed to the interactions with the extracellular regions of FGFR2. Interactions in the intracellular regions of the 2 proteins were mediated by the C-tail domain in FLRT2. Furthermore, cells stably transfected with FLRT2 shRNAs or FLRT2 cDNA exhibited a concomitant decrease and increase, respectively, in FGFR2 protein, mRNA, and ERK phosphorylation levels, suggesting a positive feedback regulatory loop of FLRT2 on FGF signaling in craniofacial tissues. We propose that FLRT2-FGFR2 interactions represent a potential mechanism for regulation of FGF signaling by FLRT2 during craniofacial development.

FLRT2 promotes cellular proliferation and inhibits cell adhesion during chondrogenesis.

One of the earliest events during chondrogenesis is the formation of condensations, a necessary pre-requisite for subsequent differentiation of a chondrogenic phenotype. Members of the Fibronectin Lecucine Rich Transmembrane (FLRT) proteins have been shown to be involved in cell sorting and neurite outgrowth. Additionally, FLRT2 is highly expressed at putative sites of chondrogenic differentiation during craniofacial development. In this study, we demonstrate that FLRT2 plays a role in mediating cell proliferation and cell-cell interactions during early chondrogenesis. Clones of stable transfectants of a murine chondroprogenitor cell line, ATDC5, were established in which FLRT2 was knocked down or overexpressed. Cells in which FLRT2 was knocked down proliferated at a slower rate compared to control wild-type ATDC5 cells or those containing a non-coding shRNA. In addition, FLRT2 knockdown cells formed numerous lectin peanut agglutinin (PNA) stained aggregates and exhibited higher expression of the cell adhesion molecule, N-cadherin. In an in vitro wound healing assay, fewer FLRT2 knockdown cells appeared to migrate into the defect. Surprisingly, the FLRT2 knockdown cells demonstrated increased formation of Alcian blue-stainable extracellular matrix, suggesting that their reduced aggregate formation did not inhibit subsequent chondrogenic differentiation. The opposite trends were observed in ATDC5 clones that overexpressed FLRT2. Specifically, FLRT overexpressing cells proliferated faster, formed fewer PNA-positive aggregates, accumulated increased Alcian blue-positive matrix, and migrated faster to close a wound. Collectively, our findings provide evidence for a role of FLRT2 in enhancing cell proliferation and reducing intercellular adhesion during the early stages of chondrogenesis.

The missense mutation W290R in Fgfr2 causes developmental defects from aberrant IIIb and IIIc signaling.

Missense mutations in the Fibroblast Growth Factor Receptor 2 (FGFR2) have been identified in human craniosynostotic syndromes such as Crouzon (CS) and Pfeiffer (PS). FGFR2 has two major isoforms, IIIb and IIIc, generated through alternative splicing with their own temporal, spatial, and ligand-binding specificities. In this study, we report the identification and characterization of a missense mutation in codon 290 of murine Fgfr2 (W290R). The defects in W290R mutants are suggestive of disruption of signalling in both IIIb and IIIc isoforms of the Fgfr2 gene. Heterozygous mutants presented with features resembling those found in patients with CS. Fgfr2(W290R) homozygotes displayed constitutive FGFR2 activation with increased, but correct tissue-specific, expression of the IIIb and IIIc isoforms in many of the defective organs. Our Fgfr2(W290R) mouse model thus represents an excellent mouse model of CS to probe the many questions around the pathogenesis of craniosynostotic birth defects consequent to defects in FGF signaling.

Flrt2 and Flrt3 have overlapping and non-overlapping expression during craniofacial development.

Craniofacial morphogenesis is a complex multi-step process that involves numerous biological processes to coordinate the growth, proliferation, migration, and subsequent differentiation of the cranial neural crest cells. Members of the Fibronectin Leucine-Rich Transmembrane (Flrt) gene family have been previously reported to be widely expressed in the developing embryo. We mapped the expression of Flrt2 and Flrt3 at critical stages of craniofacial development and found that, during early craniofacial development, Flrt2 was highly expressed initially in the cranial neural crest cells and Flrt3 in the midbrain. Later both genes were expressed in the developing pharyngeal region. Flrt2 expression predominated in the neural crest-derived mesenchyme in the medial aspect of the developing frontonasal region in close relationships with the expression of Fgfr2, Shh, and Msx1, three genes shown previously to play critical roles in craniofacial development. Flrt2 was also present in the vomero-nasal organ, mandibular primodia, and the posterior aspects of the unfused and fused secondary palatal shelves. Flrt3, however, had a more restrictive expression, being present in the mesenchyme underlying the ectoderm of the medial nasal process and in the mandibular primordium and in regions undergoing outgrowth, in a pattern that overlapped with Bmp4 expression. Both Flrt2 and Flrt3 were later found to be present at sites of epithelial-mesenchymal interactions such as the developing tooth buds, hair follicles, and eye. Together the data suggested important roles for Flrt2 and Flrt3 in mediating events such as NCC migration, chondrogenesis and epithelial-mesenchymal interactions during craniofacial development.

Phenotypic variation in dentinogenesis imperfecta/dentin dysplasia linked to 4q21.

Dentinogenesis imperfecta (DGI) and dentin dysplasia (DD) are allelic disorders that primarily affect the formation of tooth dentin. Both conditions are autosomal-dominant and can be caused by mutations in the dentin sialophosphoprotein gene (DSPP, 4q21.3). We recruited 23 members of a four-generation kindred, including ten persons with dentin defects, and tested the hypothesis that these defects are linked to DSPP. The primary dentition showed amber discoloration, pulp obliteration, and severe attrition. The secondary dentition showed either pulp obliteration with bulbous crowns and gray discoloration or thistle-tube pulp configurations, normal crowns, and mild gray discoloration. Haplotype analyses showed no recombination between three 4q21-q24 markers and the disease locus. Mutational analyses identified no coding or intron junction sequence variations associated with affection status in DMP1, MEPE, or the DSP portion of DSPP. The defects in the permanent dentition were typically mild and consistent with a diagnosis of DD-II, but some dental features associated with DGI-II were also present. We conclude that DD-II and DGI-II are milder and more severe forms, respectively, of the same disease.

Identification of markers of the midface.

Currently, much remains unknown of the genes that mediate the biological events during growth and fusion of the midfacial region, and the possible pathways through which these genes function. We took advantage of high-throughput microarray analysis to search for genes that may play a critical role in the growth and fusion of the midfacial region to become the primary palate. We identified several genes that were potentially expressed at different levels between tail somite (TS) 6-8 (pre-fusion) and TS 12-14 (fusion) in the 3 midfacial processes. Expression of 4 of these genes (Tbx14/15, Dickkopf-1, Fibroblast Growth Factor 8, and Keratin-18) was further verified by reverse-transcription/quantitative PCR and in situ hybridization at the 2 stages of midfacial development. With the identification of these genes, and possibly others, functional analyses can be conducted to improve our understanding of the mechanisms and pathways by which the midface forms.

Palatal development in Twirler mice.

OBJECTIVE: The development of the secondary palate of Twirler (Tw) mice was characterized, and a quantitative coronal plane evaluation of the width and length of the craniofacial regions of homozygous mutant embryos was conducted. RESULTS AND CONCLUSIONS: The secondary palatal shelves were retarded in growth and the timing of elevation from a vertical to horizontal position. The homozygous Tw embryos, when compared with their wild-type littermates, also had a significantly wider midface. It is not surprising, therefore, that the short palatal shelves in a wide midface resulted in cleftings of the secondary palate observed in all homozygous Tw mice. These findings are consistent with similar studies in humans and other murine models.

Tumorhead, a Xenopus gene product that inhibits neural differentiation through regulation of proliferation.

Tumorhead (TH) is a novel maternal gene product from Xenopus laevis containing several basic domains and a weak coiled-coil. Overexpression of wild-type TH resulted in increased proliferation of neural plate cells, causing expansion of the neural field followed by neural tube and craniofacial abnormalities. Overexpressed TH protein repressed neural differentiation and neural crest markers, but did not inhibit the neural inducers, pan-neural markers or mesodermal markers. Loss of function by injection of anti-TH antibody inhibited cell proliferation. Our data are consistent with a model in which tumorhead functions in regulating differentiation of the neural tissues but not neural induction or determination through its effect on cell proliferation.

Phenotypic and molecular analyses of A/WySn mice.

OBJECTIVE: Since its first description, the A strain of mice have been utilized extensively as models to study the processes involved in clefting of the midfacial region. Of the A substrains, the A/WySn has a spontaneous rate of clefting of the lip of about 20% to 30%. The A/WySn mouse model was utilized in this study to analyze and compare the phenotypic and molecular changes in the midfacial region of embryos with and without cleft. RESULTS: Scanning electron microscopy and skeletal and cartilage preparations of newborn A/WySn pups showed the presence of bilateral and unilateral clefts of the lips and the disruption of the skeletal and cartilaginous components of the mice with clefts of the lip. The expression of the msx1 homeobox gene was analyzed by whole mount in situ hybridization of A/WySn embryos at different stages of midfacial development. The results showed that there was misregulation of the expression of the msx1 gene in embryos with cleft, with a persistence of expression in the distal growing tips of the midfacial processes and in areas that have fused in normal embryos without cleft. CONCLUSIONS: Although the genetic defect in A/WySn mice is not known, a possible candidate gene has been mapped to a corresponding human chromosome carrying retinoic acid receptor alpha, and there exists a possibility that msx1 is in the same genetic pathway affected by the mutation of the gene in A/WySn.

Characterization of olfactory nerve abnormalities in Twirler mice.

The sense of smell is perceived by olfactory receptor neurons (ORN) present in the olfactory epithelium located in the posterosuperior aspect of the nasal cavity. The axons of these ORN migrate to the olfactory bulb (OB), forming a nervous layer on the outermost part of the bulb, and finally synapse in glomerular structures in the OB. The ORN are unique in that they are constantly being renewed throughout life. We characterized the defects in the nasal cavity and olfactory nervous supply of Twirler (Tw) mice by histological and immunohistochemical means. Tw homozygotes have previously been shown to present with midfacial abnormalities in the form of clefts of the lip and palate (Lyon, 1958; Gong et al., 2000). We found that in the Tw homozygotes, the OB was abnormally shaped, the skeletal framework underlying the OB was disrupted, and the morphology of the nasal cavity was altered with poorly defined nasal turbinates. Immunohistochemical staining with antibodies that marked nerves in general (PGP 9.5) and mature ORN (omp) in the olfactory epithelium at two different embryonic stages and in newborn mice revealed the stratification of the olfactory epithelium in Tw homozygotes, albeit slightly thinner compared to wildtype. A striking difference in the olfactory epithelium was the lack of differentiation of the ORN in Tw homozygotes and the reduced axonal input to the OB. In Tw homozygotes at 14.5 days of embryonic development, the presence of many mature ORN found randomly in the mesenchyme suggests the loss of olfactory pathfinding cues to the OB. It is believed that the lack of appropriate pathfinding cues observed in the Tw homozygotes was responsible for the OB not having the appropriate trophic effect on the development and maturation of the ORN as had been observed in partially bulbectomized animals. The defects in the Twirler may prove to be a valuable system to analyze problems in olfactory pathfinding and maturation.

The Twirler mouse, a model for the study of cleft lip and palate.

Twirler (Tw) is a semidominant mutation in the mouse affecting the embryonic development of the midfacial region. Most heterozygous Tw mice, +/-, become obese at adulthood with a concomitant decrease in fertility. Homozygous mice have clefts of the midfacial region and a disrupted nasal cavity. Midfacial clefts included clefts of the palate combined with either unilateral or bilateral clefts of the lip. The clefts of the lip were either complete or incomplete. The palatal shelves in Tw/Tw were very much reduced. Apart from these defects, homozygous Tw looked normal, and were born alive, although they reportedly die within 24 h after birth. It is proposed that the Twirler model can be used to improve understanding of the genetic mechanisms involved in the normal development of the midfacial region.