Losartan attenuates progression of osteoarthritis in the synovial temporomandibular and knee joints of a chondrodysplasia mouse model through inhibition of TGF-ß1 signaling pathway.

OBJECTIVE: Transforming growth factor beta 1 (TGF-ß1) is implicated in osteoarthritis (OA). The purpose of this study was to explore the ability of Losartan to inhibit the inflammatory signaling pathway of TGF-ß1 observed during osteoarthritic progression in the temporomandibular joint (TMJ) and knee joint using a genetic mouse model. METHODS: A murine OA model displaying the heterozygous chondrodysplasia gene (cho/+), a col11a1 mutation, was used to test this hypothesis. Following a 7-month treatment period with Losartan, the synovial joints were analyzed for histopathological improvement comparing two experimental groups. Tissues were fixed in paraformaldehyde, processed to paraffin section, and stained with Safranin O and Fast Green to visualize proteoglycans and collagen proteins in cartilage. Using the Modified Mankin scoring system, the degree of staining and OA progression were evaluated. RESULTS: Results show heterozygous animals receiving Losartan having diminished degeneration of TMJ condylar and knee joint articular cartilage. This was confirmed in the TMJ and knee by a statistically significant decrease in the Mankin histopathology score. Decreased expression of HtrA1, a key regulator to the TGF-ß1 signaling pathway, was demonstrated in vitro as well as in vivo, via Losartan inhibition. CONCLUSION: Using a genetic mouse model of OA, this study demonstrated the utility of Losartan to improve treatment of human OA in the TMJ and knee joint through inhibition of the TGF-ß1 signaling cascade. We further demonstrated inhibition of HtrA1, the lowering of Mankin scores to wild type control levels, and the limiting of OA progressive damage with treatment of Losartan.

Osteoarthritis in temporomandibular joint of Col2a1 mutant mice.

OBJECTIVE: Col2a1 gene mutations cause premature degeneration of knee articular cartilage in disproportionate micromelia (Dmm) and spondyloepiphesial dysplasia congenita (sedc) mice. The present study analyses the temporomandibular joint (TMJ) in Col2a1 mutant mice in order to provide an animal model of TMJ osteoarthritis (OA) that may offer better understanding of the progression of this disease in humans. DESIGN: Dmm/+ mice and controls were compared at two, six, nine and 12 months. Craniums were fixed, processed to paraffin sections, stained with Safranin-O/Fast Green, and analysed with light microscopy. OA was quantified using a Mankin scoring procedure. Unfolded protein response (UPR) assay was performed and immunohistochemistry (IHC) was used to assay for known OA biomarkers. RESULTS: Dmm/+ TMJs showed fissuring of condylar cartilage as early as 6 months of age. Chondrocytes were clustered, leaving acellular regions in the matrix. Significant staining of HtrA1, Ddr2 and Mmp-13 was observed in Dmm/+ mice (p<0.01). We detected upregulation of the UPR in knee but not TMJ. CONCLUSIONS: Dmm/+ mice are subject to early-onset OA in the TMJ. We observed upregulation of biomarkers and condylar cartilage degradation concomitant with OA. An upregulated UPR may exacerbate the onset of OA. The Dmm/+ mouse TMJ is a viable model for the study of the progression of OA in humans.

Osteoarthritis-like changes in the heterozygous sedc mouse associated with the HtrA1-Ddr2-Mmp-13 degradative pathway: a new model of osteoarthritis.

OBJECTIVE: To test the hypothesis that the spondyloepiphyseal dysplasia congenita (sedc) heterozygous (sedc/+) mouse, a COL2A1 mutant, is a model for the study of osteoarthritis (OA) in the absence of dwarfism and to investigate the presence of HtrA1, Ddr2, and Mmp-13 and their possible involvement in a universal mechanism leading to OA. DESIGN: Whole mount skeletons of adult animals were analyzed to determine whether sedc/+ mice exhibit dwarfism. To characterize progression of osteoarthritic degeneration over time, knee and temporomandibular joints from sedc/+ and wild-type mice were analyzed histologically, and severity of articular cartilage degradation was graded using the Osteoarthritis Research Society International (OARSI) scoring system. Immunohistochemistry was used to detect changes in expression of HtrA1, Ddr2, and Mmp-13 in articular cartilage of knees. RESULTS: As previously reported, the sedc/+ skeleton morphology was indistinguishable from wild type, and skeletal measurements revealed no significant differences. The sedc/+ mouse did, however, show significantly higher OARSI scores in knee (9, 12 and 18 months) and temporomandibular joints at all ages examined. Histological staining showed regions of proteoglycan degradation as early as 2 months in both temporomandibular and knee joints of the mutant. Cartilage fissuring and erosion were observed to begin between 2 and 6 months in temporomandibular joints and 9 months in knee joints from sedc/+ mice. Immunohistochemistry of mutant knee articular cartilage showed increased expression of HtrA1, Ddr2, and Mmp-13 compared to wild type, which upregulation preceded fibrillation and fissuring of the articular surfaces. CONCLUSIONS: With regard to skeletal morphology, the sedc/+ mouse appears phenotypically normal but develops premature OA as hypothesized. We conclude that the sedc/+ mouse is a useful model for the study of OA in individuals with overtly normal skeletal structure and a predisposition for articular cartilage degeneration.

Premature osteoarthritis in the Disproportionate micromelia (Dmm) mouse.

OBJECTIVE: Degeneration of articular cartilage leads to the development of osteoarthritis (OA), but the molecular pathology of the disease is poorly understood. The Disproportionate micromelia (Dmm) mouse has a deletion mutation in the C-propeptide encoding region of Col2a1, which leads to a defective cartilage matrix. The objective of this study was to determine whether heterozygous (Dmm/+) mice develop premature OA, and could therefore serve as an animal model for studying the molecular pathways leading to OA. DESIGN: Histological analysis was utilized to determine the state of articular cartilage degeneration in Dmm/+ mice at 3, 6, 9, 12, 15, and 22 months of age. Severity of OA was quantified with a modified Mankin scoring system. In addition, articular cartilage thickness, cell density, and the extracellular matrix (ECM) fraction of articular cartilage were quantified. RESULTS: Articular cartilage erosion was significantly more severe in Dmm/+ than in wild-type (+/+) mice beginning at 9 months, and modified Mankin scoring revealed Dmm/+ articular cartilage to be in a more severe osteoarthritic state as early as 3 months. In addition, Dmm/+ articular cartilage was thinner than +/+ cartilage and showed increased cell density and decreased matrix fraction compared with +/+ from the earliest time points measured. CONCLUSIONS: The present study demonstrates that Dmm/+ mice develop premature OA. The observed degenerative changes of Dmm/+ articular cartilage closely resemble those of human OA patients, with or without Col2a1 mutations, suggesting that Dmm/+ mice are a useful model for investigating mechanisms involved in OA.

A type XI collagen mutation leads to increased degradation of type II collagen in articular cartilage.

OBJECTIVE: To determine in articular cartilage whether degraded type II collagen is more abundant in Col11a1 mutant cho/+ than in age-matched +/+ mice and whether collagen degradation occurs in a generalized or localized fashion. DESIGN: Knee joints from cho/+ and +/+ mice at 6, 9, 12 and 15 months of age were dissected, fixed, cryosectioned, and stained with antibody COL2-3/4m against denatured type II collagen using a FITC-conjugated secondary antibody. Sections were viewed and photographed under a fluorescence microscope and areas of staining were quantified. RESULTS: Before 12 months of age, little degraded collagen staining was detectable in +/+ or cho/+ mice. By 15 months, however, cho/+ mice showed significantly more degraded type II collagen than age-matched controls. Degraded collagen staining was localized at the articular surface, not distributed generally throughout the articular cartilage. CONCLUSIONS: The results suggest a model in which cumulative biomechanical stresses trigger increased collagen synthesis and degradation in both +/+ and cho/+ mice at around 12 months of age. Cho/+ mice, however, are less able to synthesize and assemble normal replacement collagen fibrils because of the Col11a1 mutation. Degradation is further activated, resulting in the accumulation of degraded type II collagen in the articular cartilage extracellular matrix. Similar mutations that do not overtly affect skeletal development may likewise predispose humans to increased collagen degradation and resultant osteoarthritis.

The mechanism of palatal clefting in the Col11a1 mutant mouse.

The occurrence of cleft palate in mutant mice offers an opportunity to understand the possible role of specific genes in palatogenesis. Here, cleft palate in mice carrying the chondrodysplasia (cho) defect, which consists of an autosomal-recessive mutation in the collagen gene Col11a1, was investigated. The proposed cause of cleft palate in cho homozygous mice is failure of the palatal shelves to adhere and make contact due to mandibular growth abnormalities. Another cause of cleft palate that has recently been demonstrated in other animal models is failure of the midline epithelial seam forming between the shelves to undergo epithelial-mesenchymal transformation (EMT). The present strategy to test the likelihood of this second possibility was to culture the unfused cho/cho palatal shelves at different stages of development to see if they were capable of adhering and undergoing EMT in vitro. By using carboxydichlorofluorescein succinimidyl ester to trace the fate of the medial-edge epithelium (MEE), it was shown that cho/cho palates have full potential for MEE adherence and EMT up to embryonic day 17.5/18.5, when epithelia keratinize before birth, preventing the adherence of both the normal and homozygous palatal shelves. Thus, the major effect of the mutant collagen gene on the palate is likely to be via mandibular growth disruption. The possibility that unfused palatal shelves in other clinical syndromes can adhere and undergo EMT if brought into contact at appropriate times before birth has important therapeutic implications.

Developmental toxicity of carbon black oil in mice.

BACKGROUND: Carbon black oil (CBO) is a refinery side-stream product used to produce asphalt and other commercial products. CBO contains several classes of hydrocarbons, several of which are known to exhibit systemic and gestational toxicities, making this mixture a candidate for causing reproductive toxicity. METHODS: Swiss-Webster mice were administered CBO (300, 350, 400 mg/kg/day) via oral gavage in a dosage volume of 10 microl/g body weight on gestation days (GD) 6-15. Uterine contents were evaluated on GD 18. RESULTS: Treatment with CBO at all dosage levels resulted in a high frequency of maternal clinical symptoms and a decrease in maternal weight gain. Decreased fetal viability was observed, manifested as a decrease in viable implants and, in a high percentage of treated dams, as early resorption of the entire litter. A significant reduction in fetal weight was also observed. However, neither structural malformations nor developmental delays in ossification were observed in any of the living offspring. To minimize maternal toxicity, the dosage range was lowered (100, 200, 300 mg/kg/day), and the concentration was adjusted such that the volume administered to each dam was decreased by 20%. In this trial, the only maternal effect observed was an increase in maternal liver weight at 200 and 300 mg/kg. The fetal lethality effects observed previously were reduced substantially. Nevertheless, the frequency of resorption among all treatment groups was higher statistically than in controls. CONCLUSIONS: These data support the hypothesis that CBO is reproductively toxic in Swiss-Webster mice at oral doses of >/=100 mg/kg/day.

A developmental toxicity study of tretinoin administered topically and orally to pregnant Wistar rats.

BACKGROUND: Although it is well established that oral tretinoin produces embryofetal developmental toxicity in various laboratory animals, the toxic potential of topical tretinoin has not been clearly established. OBJECTIVE: This study of tretinoin administration to pregnant Wistar rats was conducted to determine whether topical tretinoin is associated with adverse effects on reproductive function or embryofetal growth and development and to compare outcomes with topical and oral tretinoin. METHODS: Topical and oral tretinoin (1 to 20 mg/kg and 1 to 10 mg/kg, respectively) or vehicles alone were administered on gestational days 6 through 16 and 15, respectively. RESULTS: Topical tretinoin: After topical treatment, dams receiving 10 mg/kg daily or greater had severe local and systemic toxicity prompting discontinuation of tretinoin. At doses of 2.5 mg/kg or greater, dam weight gain and food consumption were significantly less than those of control dams. Offspring of dams receiving 5 mg/kg weighed significantly less, and offspring of dams receiving 2.5 mg/kg or greater had a significantly greater occurrence of supernumerary ribs compared with control offspring. Oral tretinoin: After oral treatment, in the absence of maternal toxicity, significantly more offspring of dams receiving 5 mg/kg or greater had supernumerary ribs, and offspring of the 10 mg/kg treatment group had a greater incidence of cleft palate than had control offspring. CONCLUSION: The local and systemic maternal toxicity found in association with supernumerary ribs and low weights in the offspring at topical tretinoin doses of 2.5 and 5 mg/kg suggests that these developmental effects may be nonspecific or maternally mediated. Oral tretinoin at doses of 10 mg/kg, however, is clearly associated with embryofetal alterations in the Wistar rat.

Disproportionate micromelia (Dmm) in mice caused by a mutation in the C-propeptide coding region of Col2a1.

Mice that are homozygous for the autosomal semidominant disproportionate micromelia (Dmm) mutation are characterized by disproportionate micromelia, thoracic dysplasia, and cleft palate. Chondrocytes of the epiphyseal growth plates are not organized into columns, and ultrastructural analysis reveals excessive dilation of the endoplasmic reticulum and a paucity of collagen fibrils in the extracellular matrix. To map the Dmm locus, Dmm mice were crossed with the multiple ecotropic viral (MEV) linkage testing stock. Significant linkage of Dmm to the fourteen MEV linkage markers was not observed, thereby excluding approximately 50% of the genome as candidate regions encoding Dmm. Subsequently, microsatellite markers were used to assess linkage to the nonexcluded regions of the genome, revealing tight linkage to the locus of Col2a1, the gene encoding the alpha-chains of type II collagen. alpha 1(II) collagen cDNA, synthesized with RNA from homozygotes, was cloned and sequenced, revealing a three-nucleotide deletion in the region encoding the C-propeptide globular domain. The deletion leads to the substitution of one amino acid, Asn, in the mutant for two amino acids, Lys and Thr, in the wild type. Several human chondrodysplasias with similar phenotypes to that of Dmm are associated with defects in type II collagen. Thus, mice bearing the Dmm mutation serve as a model for studying the pathogenesis of these disorders while revealing novel insights into normal skeletal morphogenesis.

Pulmonary hypoplasia associated with reduced thoracic space in mice with disproportionate micromelia (DMM).

BACKGROUND: Fetal mice homozygous for the Disproportionate micromelia (Dmm) gene were studied as a model for pulmonary hypoplasia in chondrodystrophy. METHODS: Wet weight, dry weight, and biochemical content were determined in excised whole lungs, terminal sac morphology and presence of multilamellar bodies were determined by electron microscopy, and volume of the thoracic space was estimated from paraffin casts. Lung development of the mutant was further assessed in whole organ culture. RESULTS. Compared with normal littermates, the mutant showed a significant decrease (28%) in lung wet weight without showing altered lung dry weight or tissue content of DNA and protein. The terminal sacs of lungs fixed by intratracheal instillation were significantly smaller than normal. However, the lungs appeared to have undergone maturation on schedule since the surfactant precursors, multilamellar bodies, were observed and normal tissue-levels of phospholipid were detected. The volume of the mutant's thorax was markedly reduced. Finally, the mutant's lungs when removed from the fetus prior to the onset of thoracic dystrophy (day 15) and cultured for three days demonstrated that, without the confining influence of a reduced thoracic space, they are capable of development comparable to normal. CONCLUSIONS: These findings support the hypothesis that the Dmm mutant can be further studied as a model for human pulmonary hypoplasia associated with chondrodystrophy, and that the relationship between the reduced thorax and the lung disorder is cause-and-effect.

Morphological differences elicited by two weak acids, retinoic and valproic, in rat embryos grown in vitro.

We compared in rat whole-embryo culture the morphological changes elicited by valproic acid (VPA) with those elicited by trans-retinoic acid (RA). Rat embryos explanted on day 9.5 of gestation were treated on day 10 with RA or VPA at concentrations producing equivalent reductions in embryonic protein. The concentrations selected for morphological assessment by scanning and transmission electron microscopy, 2.3 and 800 microM, respectively, for RA and VPA, produced approximately a 50% incidence of abnormally open anterior neuropores in initial range-finding experiments in the culture system. Protein and DNA analyses were also performed on corresponding groups of embryos at three different doses. With concurrent control groups used as reference standards, the two treatment groups were compared for differences in external and internal morphology, protein and DNA contents, and growth indices. While certain variables responded similarly in the two treatment groups, e.g., the growth variables, protein and DNA contents, each drug produced selective morphological effects. Whereas treatment with RA produced underdeveloped branchial arches, symmetrically cleft cranial defects resulting in openings in rhombencephalic and prosencephalic regions, and exteriorized neural tissue in the caudal neuropore region, VPA produced irregular clefts with wavy margins along the entire length of the neural tube, and an open caudal neuropore without eversion of the neuroepithelium, while producing no detectable effect on the branchial arches. The similar effects of these two drugs on protein and DNA contents suggest comparable degrees of overall toxicity; however, the dissimilar effects on neural tube and branchial arches, coupled with the large difference in concentration of the drug required to produce the effects, add to the evidence that their mechanisms for elicitation of abnormal development are qualitatively different.

Human hair morphology: a scanning electron microscopy study on a male Caucasoid and a computerized classification of regional differences.

The present study was performed to provide a better understanding of the morphological variations of mammalian hair. Terminal hair samples were obtained from different regions of the body of the same Caucasian male. All hair samples were either cleaned or treated before being examined with scanning electron microscopy. As human scalp hair grew it appeared small like lanugo hair, but the increase in diameter appeared to have been relatively rapid. As hair increased in diameter the appearance of the scales changed. Neck hair was slightly smaller in diameter than scalp hair, and axillary hair was slightly smaller in diameter than neck hair. Nostril hair was larger than scalp or axillary hair. Eyelash hair was much smaller and much shorter than eyebrow hair. Neck hair, forearm hair, and shin hair were smaller than hair from most other regions of the body. Chest hair was similar in size to scalp hair, and pubic and sideburn hair were larger than scalp hair. A morphological feature called "steak-boning" was more characteristically present in whiskers of Caucasoids than Orientals or Blacks. "Steak-boning" occurred most frequently in hair of the mustache, followed by that of the chin, sideburn, cheek and under the chin. Cut surfaces of whiskers were different for electric as compared with straightedge razors. Hair morphology varied relative to the body region. Computer analysis of resin-embedded hair made it possible to classify arm, mustache, cheek, chin, head, shin, and pubic hair, and to quantify cross-sectioned differences.

Induction of maternal toxicity in the rat by dermal application of retinoic acid and its effect on fetal outcome.

Time-mated Sprague-Dawley rats were administered all-trans-retinoic acid (RA) dermally on gestational days 11 through 14 at three dosage levels (25, 100, and 250 mg/kg body weight). Dams administered ethylenethiourea (ETU) dermally on gestational days 11 to 12 or RA orally on day 12 were used to indicate the strain's sensitivity to teratogenesis. The chemicals were dissolved in dimethylsulfoxide (DMSO) for dermal application or suspended in corn oil for treatment by gavage. The maternal weight gain, pup weight, number of resorptions and number of fetuses with gross malformations, and skeletal/organ-level anomalies were determined. Beginning with day 15, dams dermally treated with RA exhibited dermal lesions at the site of application, most dams showed vaginal bleeding by day 16, and approximately 20% did not survive to day 19. Relative to the DMSO control group, maternal weight gain in the dermal RA groups was decreased by approximately 50% at the lowest dose, with essentially no weight gain at the intermediate- and high-dose levels. The decrease in average fetal weight at the two higher doses was significant, whereas the resorption and malformation frequencies were not significantly increased by dermal treatment with RA. Without significantly affecting fetal weight or resorption frequency, dermal application of ETU significantly increased the frequency of skeletal anomalies, primarily tail defects. Oral administration of RA did not increase the malformation frequency nor produce significant maternal or fetotoxic effects. In summary, treatment of pregnant Sprague-Dawley rats by dermal application of RA dissolved in DMSO resulted in significant toxicity to the dam.(ABSTRACT TRUNCATED AT 250 WORDS)

Thoracic volume reduction as a mechanism for pulmonary hypoplasia in chondrodystrophic mice.

Day-18 fetal mice with chondrodysplasia (CHO) have been shown to have pulmonary hypoplasia and thoracic volume reduction, and it has been hypothesized that the former is a secondary effect of the latter. To establish a pathogenetic relationship between thoracic volume reduction and pulmonary hypoplasia, it must first be demonstrated that the decrease in thoracic volume precedes or coincides with the morphologic effect on the lungs. At 24-h intervals through gestation days 14 to 18, the thoracic volume and lung histopathology for normal and chondrodystrophic fetuses were estimated by image analysis of frontal histologic sections. Relative to normal littermates, the average thoracic volumes of day-16, day-17, and day-18 mutants were significantly decreased by 18%, 34%, and 49%, respectively. In the mutants' lungs the histology was normal through day 16, but on days 17 and 18 the frequency of the more distended primary saccules and the amount of potential airspace were decreased relative to normal. These results support the hypothesis that the relative decrease in thoracic volume of the mutant precedes the departure from normal for primary saccule development. The reduction effect on thoracic volume therefore is pathogenetically related to the hypoplastic lungs of fetal mice with lethal chondrodystrophy.

A stereoscopic scanning electron microscope study of pulmonary hypoplasia in chondrodystrophic mice.

Pulmonary hypoplasia is a life threatening condition in newborns resulting from a generalized underdevelopment of the lungs. The lung disorder is usually secondary to conditions outside the lung such as thoracic volume reduction. The precise mechanism by which thoracic volume reduction prevents normal lung development and growth is unknown. As a model for human pulmonary hypoplasia associated with lethal skeletal dysplasia, a stereoscopic SEM study of chondrodystrophic (cho) fetal mouse lungs fixed by intratracheal instillation with 3% glutaraldehyde was conducted. In comparison with lungs of phenotypically normal littermates, the mutant's lungs appeared unaffected with respect to structure of major bronchiolar airways and in the morphology and amount of surfactant precursors (multilamellar bodies). The primary saccules within the mutant's lungs were significantly smaller and more numerous relative to those of normal littermates. These observations provide evidence that the lungs for this type of pulmonary hypoplasia are ultrastructurally normal with respect to upper airways, but that the primary saccules, or units of function in neonatal breathing in the rodent, are significantly smaller. This effect, however, does not appear to inhibit differentiation of type II pneumocytes or production of surfactant.

Technique for estimating fetal mouse thoracic volumes through image analysis of histological sections.

In a previous study we estimated fetal mouse thoracic volume by use of paraffin casts. While this procedure provided useful information, it did not allow histologic examination of thoracic viscera. In the present study the thoracic volumes of day 14-18 fetal mice were determined through serial histological sections. The thoracic cavity was traced from the sections and the area of each tracing was determined by computer image analysis. These areas were summed and then multiplied by the thickness of each section to derive the thoracic volume. This procedure thus permitted both volumetric determinations and histological inspection of the thoracic viscera. In addition, two randomized sampling methods designed to increase the utility of such volumetric estimates were compared for reliability. The method best suited for this study was a random stratified sampling method because it reproduced estimates with minimal standard deviation.

Evidence for embryotoxicity of gossypol in mice and chicks with no evidence of mutagenic activity in the Ames test.

Effects of gossypol treatment were studied in pregnant mice and chick embryos. Pregnant Balb C mice were treated orally with 60 or 120 mg/kg of gossypol acetic acid on days 6-13 of pregnancy and killed on day 18. The uteri were removed, the number of resorptions and late fetal deaths were recorded, and the fetuses were weighed and assessed for malformations. Fertilized hen eggs were injected with 0.25 mg gossypol/egg at 24, 48, 72, or 96 h of incubation. The embryos were examined at day 9 of incubation. The mutagenic potential of gossypol was determined by the Ames test. Treatment of mice with gossypol produced significant adverse effects on the dam and offspring including decreased pregnancy weight gain of the dam and growth retardation of the offspring. There were increased resorptions and late fetal deaths in mice and high mortality in chick embryos. Exencephalic fetuses were observed in one of four litters exposed to the higher dose of gossypol, micromelia was observed in one of 26 chick embryos treated at 24 h, and gastroschisis was observed in one of 21 chick embryos treated at 72 h. No malformations were observed in the controls. The number of revertants per plate in the gossypol treatment group (100 or 500 mg gossypol/plate) did not differ significantly from that of control. This study provides evidence that gossypol has embryotoxic and possibly teratogenic activity in mouse and chick embryos but no mutagenic activity according to the Ames assay.

Pulmonary hypoplasia in mice homozygous for the cartilage matrix deficiency (cmd) gene: a model for human congenital disorder.

As a potential model for the study of pulmonary hypoplasia in the lethal chondrodystrophies in man, lungs of day 18 mouse fetuses homozygous for cartilage matrix deficiency (cmd) were studied by biochemical, histological, and morphometric techniques. Wet and dry mutant lung weights were 30% less than corresponding normal lung weights. Total DNA and protein contents per whole mutant lung were decreased by 23% and 29%, respectively. An increased number of smaller-than-normal primary saccules were observed in histological sections of mutant lungs, which correspond to the difference in lung wet weight. The thoracic volume of mutants was decreased by an average of 38%. Amniotic fluid volume measurements indicated polyhydramnios in the mutant. The smaller-than-normal thoracic cavity observed in the cmd mutant, imposing a significant restriction on the developing lungs, is the most likely mechanism of pulmonary hypoplasia in this form of chondrodystrophy-confirming that reported for the cho mouse mutant.

Chondrodystrophic mice with coincidental agnathia: evidence for the tongue obstruction hypothesis in cleft palate.

Mice homozygous for either of two mutations, chondrodysplasia (cho) or cartilage matrix deficiency (cmd), have short-limbed chondrodystrophy. This phenotype includes retrognathia, relative macroglossia, and cleft palate. It has been postulated that the cleft palate in these mice is the result of tongue obstruction during palatogenesis. Agnathia associated with microglossia is an independent spontaneously occurring defect in the strains bearing these mutations. The coincidental occurrence of agnathia-microglossia with chondrodystrophy lends itself to the study of the mechanism of cleft palate formation. We examined approximate midsagittal histological sections of normal and chondrodystrophic newborn mice, both with and without agnathia. Mandibular measurements and examinations of palate closure and tongue structure were made from photographic prints. Typical chondrodystrophic mutants with cleft palates had a mean mandibular length that was 66% of normal and a tongue that appeared large relative to the shortened mandible. Chondrodystrophic mutants with agnathia and microglossia had a mean mandibular length that was further reduced to 30% of normal, yet had a closed palate. We also observed two nonagnathic chondrodystrophic mutants that had slightly decreased mandibular lengths, microglossia, and closed palates. These observations suggest that tongue obstruction during palatogenesis is the pathogenetic mechanism of cleft palate in chondrodystrophic mice. A similar tongue obstruction hypothesis has been proposed as the mechanism of cleft palate formation in the human Pierre Robin sequence, which consists of retrognathia, glossoptosis, and cleft palate. This mechanistic hypothesis has been challenged, but our findings support the tongue obstruction hypothesis in the Robin cleft.