Generation and molecular characteristics of a highly attenuated GPV strain through adaptation in GEF cells.

BACKGROUND: Goose parvoviruses (GPVs) spread globally and cause a huge economic loss to the poultry industry. Although the attenuated GPV vaccines play a key role in preventing the disease caused by GPV, the molecular basis for the attenuation of GPV is barely known. RESULTS: A highly attenuated GPV strain, GPV-CZM-142, was generated through blindly passaging of the highly pathogenic strain, GPV-CZM, in goose embryonic fibroblasts (GEF) for 142 generations. The GEF-adapted GPV strain's virulence was 10,000 times weaker than its wild type counterpart, GPV-CZM, based on the ELD50 (50% Embryo Lethal Dose). By comparing with the wild type strain, genome sequencing analysis identified adapted mutations either in ITR or in NS and VP1 of GPV-CZM-142. CONCLUSIONS: The highly attenuated GPV strain, GPV-CZM-142, provides a GPV vaccine candidate, and the identified virulence-related mutations give a novel insight into the molecular determinants of GPV virulence.

Combined deletions of IHH and NHEJ1 cause chondrodystrophy and embryonic lethality in the Creeper chicken.

The Creeper (Cp) chicken is characterized by chondrodystrophy in Cp/+ heterozygotes and embryonic lethality in Cp/Cp homozygotes. However, the genes underlying the phenotypes have not been fully known. Here, we show that a 25 kb deletion on chromosome 7, which contains the Indian hedgehog (IHH) and non-homologous end-joining factor 1 (NHEJ1) genes, is responsible for the Cp trait in Japanese bantam chickens. IHH is essential for chondrocyte maturation and is downregulated in the Cp/+ embryos and completely lost in the Cp/Cp embryos. This indicates that chondrodystrophy is caused by the loss of IHH and that chondrocyte maturation is delayed in Cp/+ heterozygotes. The Cp/Cp homozygotes exhibit impaired DNA double-strand break (DSB) repair due to the loss of NHEJ1, resulting in DSB accumulation in the vascular and nervous systems, which leads to apoptosis and early embryonic death.

Analysis of differentially expressed proteins in Muscovy duck embryo fibroblasts infected with virulent and attenuated Muscovy duck reovirus by two-dimensional polyacrylamide gel electrophoresis.

Muscovy duck reovirus (MDRV) belongs to the Orthoreovirus genus of the Reoviridae family, which is a significant poultry pathogen leading to high morbidity and mortality in ducklings. However, the pathogenesis of the virus is not well understood. In the present study, two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) combined with LC-MS-MS was used to identify differentially expressed proteins between Muscovy duck embryo fibroblasts (MDEF) infected with virulent (MV9710 strain) and attenuated (CA strain) MDRV and non-infected MDEFs. A total of 115 abundant protein spots were identified. Of these, 59 of differentially expressed proteins were detected, with functions in metabolism and utilization of carbohydrates and nucleotides, anti-stress, and regulation of immune and cellular process. GO analysis of the identified proteins showed that they belonged to the classes molecular function (141 proteins), cellular component (62 proteins), and biological process (146 proteins). The results were validated by qRT-PCR, which suggests that the analysis method of 2D PAGE combined with LC-MS-MS used in this study is reliable. This study lays a foundation for further investigation of the biology of MDRV infection in MDEF.

The relationship between liver-kidney impairment and viral load after nephropathogenic infectious bronchitis virus infection in embryonic chickens.

To examine the relationship of impairments of the liver and kidney with viral load after nephropathogenic infectious bronchitis virus (NIBV) infection in embryonic chickens, 120 specific-pathogen-free Leghorn embryonated chicken eggs were randomly divided into two groups (infected and control), with three replicates per group and 20 eggs in each replicate. The eggs in the infected and control groups were challenged with 0.2 mL of 105.5 ELD50 NIBV and sterile saline solution, respectively. The embryonic chickens' plasma and liver and kidney tissues were collected at 1, 3, and 5 days post-inoculation (dpi), the liver and kidney functional parameters were quantified, and the tissue viral loads were determined with real-time PCR. The results showed that plasma potassium, sodium, chlorine, magnesium, calcium, and phosphorus levels were increased. The infected group exhibited significantly higher plasma uric acid, blood urea nitrogen, and creatinine levels than the control group at 3 dpi. The plasma concentrations of aspartate aminotransferase and alanine aminotransferase were significantly increased in the infected group. The total protein, albumin, and globulin levels in the infected group were significantly lower than those in the control group. The liver-kidney viral load in the infected group peaked at 3 dpi, at which time the kidney viral load was significantly higher than that of the liver. Our results indicated that NIBV infection caused liver and kidney damage in the embryonic chickens, and the results also demonstrated that the liver and kidney damage was strongly related to the tissue viral load following NIBV infection in embryonic chickens.

Pathogeny of Fatigued Walking Condition in Pekin Ducks.

Fatigued walking condition (FWC) in ducks is an important welfare and processing issue during the loading/unloading to the processing plant that can be related to heart and bone development. An experiment was conducted to evaluate the effects of incubation conditions on duck embryo bone and heart development and their subsequent effects on ducks showing FWC at market age. Four groups of 2500 Pekin duck eggs were subjected to combinations of two incubation temperature profiles (elevated [E] and normal [N]) and two eggshell conductance profiles (G) (reduced [GR] and normal [GN]). At hatch 10 ducklings from each treatment combination were sampled for heart, liver, residual yolk, and total body weight as well as relative weights (organ percentage of whole body weight). Femur, tibia, and tarsus length and weight were also obtained, and relative asymmetry (RA) was calculated for each leg section. At 35 days of age during unloading of the truck at the slaughter plant, five hens and five drakes demonstrating normal walking and FWC were sampled. Body, heart, and ventricular weights were obtained along with femur, tibia and tarsus length, weight, and RA. Bone strength was evaluated using a three-point bending test, and tibia ash content was assessed. At hatch duckling bone characteristics and organ weights were found to be primarily affected by GR conditions, while heart development in older ducks was mainly impacted by E incubation temperatures. Tibia and relative weight at 35 days were also increased by GR and E. Fatigued ducks presented heavier tibias with more RA and cortical thickness but lower ash percentage. In conclusion, the changes in bone development during incubation and posthatch life were related to duck FWC presence during transportation to the processing plant.

Construction and sequencing of an infectious clone of the goose embryo-adapted Muscovy duck parvovirus vaccine strain FZ91-30.

BACKGROUND: Muscovy duck parvovirus (MDPV) is the etiological agent of Muscovy duckling parvoviral disease, which is characterized by diarrhea, locomotive dysfunction, stunting, and death in young ducklings, and causes substantial economic losses in the Muscovy duck industry worldwide. FZ91-30 is an attenuated vaccine strain that is safe and immunogenic to ducklings, but the genomic information and molecular mechanism underlining the attenuation are not understood. METHODS: The FZ91-30 strain was propagated in 11-day-old embryonated goose eggs, and viral particles were purified from the pooled allantoic fluid by differential centrifugation and ultracentrifugation. Single-stranded genomic DNA was extracted and annealed to form double-stranded DNA. The dsDNA digested with NcoI resulted two sub-genomic fragments, which were then cloned into the modified plasmid pBluescript II SK, respectively, generating plasmid pBSKNL and pBSKNR. The sub-genomic plasmid clones were sequenced and further combined to construct the plasmid pFZ that contained the entire genome of strain FZ91-30. The complete genome sequences of strain FM and YY and partial genome sequences of other strains were retrieved from GenBank for sequence comparison. The plasmid pFZ containing the entire genome of FZ91-30 was transfected in 11-day-old embryonated goose eggs via the chorioallantoic membranes route to rescue infectious virus. A genetic marker was introduced into the rescued virus to discriminate from its parental virus. RESULTS: The genome of FZ91-30 consists of 5,131 nucleotides and has 98.9 % similarity to the FM strain. The inverted terminal repeats (ITR) are 456 nucleotides in length, 14 nucleotides longer than that of Goose parvovirus (GPV). The exterior 415 nucleotides of the ITR form a hairpin structure, and the interior 41 nucleotides constitute the D sequence, a reverse complement of the D' sequence at the 3' ITR. Amino acid sequence alignment of the VP1 proteins between FZ91-30 and five pathogenic MDPV strains revealed that FZ91-30 had five mutations; two in the unique region of the VP1 protein (VP1u) and three in VP3. Sequence alignment of the Rep1 proteins revealed two amino acid alterations for FZ91-30, both of which were conserved for two pathogenic strains YY and P. Transfection of the plasmid pFZ in 11-day-old embryonated goose eggs resulted in generation of infectious virus with similar biological properties as compared with the parental strain. CONCLUSIONS: The amino acid mutations identified in the VP1 and Rep1 protein may contribute to the attenuation of FZ91-30 in Muscovy ducklings. Plasmid transfection in embryonated goose eggs was suitable for rescue of infectious MDPV.

Replication cycle of duck hepatitis A virus type 1 in duck embryonic hepatocytes.

Duck hepatitis A virus type 1 (DHAV-1) is an important agent of duck viral hepatitis. Until recently, the replication cycle of DHAV-1 is still unknown. Here duck embryonic hepatocytes infected with DHAV-1 were collected at different time points, and dynamic changes of the relative DHAV-1 gene expression during replication were detected by real-time PCR. And the morphology of hepatocytes infected with DHAV was evaluated by electron microscope. The result suggested that the adsorption of DHAV-1 saturated at 90 min post-infection, and the virus particles with size of about 50 nm including more than 20 nm of vacuum drying gold were observed on the infected cells surface. What's more, the replication lasted around 13 h after the early protein synthesis for about 5h, and the release of DHAV-1 was in steady state after 32 h. The replication cycle will enrich the data for DVH control and provide the foundation for future studies.

Utilizing the chicken as an animal model for human craniofacial ciliopathies.

The chicken has been a particularly useful model for the study of craniofacial development and disease for over a century due to their relatively large size, accessibility, and amenability for classical bead implantation and transplant experiments. Several naturally occurring mutant lines with craniofacial anomalies also exist and have been heavily utilized by developmental biologist for several decades. Two of the most well known lines, talpid(2) (ta(2)) and talpid(3) (ta(3)), represent the first spontaneous mutants to have the causative genes identified. Despite having distinct genetic causes, both mutants have recently been identified as ciliopathic. Excitingly, both of these mutants have been classified as models for human craniofacial ciliopathies: Oral-facial-digital syndrome (ta(2)) and Joubert syndrome (ta(3)). Herein, we review and compare these two models of craniofacial disease and highlight what they have revealed about the molecular and cellular etiology of ciliopathies. Furthermore, we outline how applying classical avian experiments and new technological advances (transgenics and genome editing) with naturally occurring avian mutants can add a tremendous amount to what we currently know about craniofacial ciliopathies.

Dynamics of blood viscosity regulation during hypoxic challenges in the chicken embryo (Gallus gallus domesticus).

Hypoxia in chicken embryos increases hematocrit (Hct), blood O2 content, and blood viscosity. The latter may limit O2 transport capacity (OTC) via increased peripheral resistance. Hct increase may result from increased nucleated red blood cell concentration ([RBC]) and mean corpuscular volume (MCV) or reduced plasma volume. We hypothesized changes in Hct, hemoglobin concentration ([Hb]), [RBC] and MCV and their effects on viscosity would reduce OTC. Five experimental treatments that increase Hct were conducted on day 15 embryos: 60min water submergence with 60min recovery in air; exposure to 15% O2 with or without 5% CO2 for 24 h with 6 h recovery; or exposure to 10% O2 with or without 5% CO2 for 120 min with 120 min recovery. Control Hct, [Hb], [RBC], MCV, and viscosity were approximately 26%, 9g%, 2.0 10(6)µL(-1), 130µm(3), and 1.6mPas, respectively. All manipulations increased Hct and blood viscosity without changing blood osmolality (276mmolkg(-1)). Increased viscosity was attributed to increased [RBC] and MCV in submerged embryos, but solely MCV in embryos experiencing 10% O2 regardless of CO2. Blood viscosity in embryos exposed to 15% O2 increased via increased MCV alone, and viscosity was constant during recovery despite increased [RBC]. Consequently, blood viscosity was governed by MCV and [RBC] during submergence, while MCV was the strongest determinant of blood viscosity in extrinsic hypoxia with or without hypercapnia. Increased Hct and blood O2 content did not compensate for the effect of increased viscosity on OTC during these challenges.

Autophagy inhibitors reduce avian-reovirus-mediated apoptosis in cultured cells and in chicken embryos.

Avian reovirus (ARV)-induced apoptosis contributes to the pathogenesis of reovirus in infected chickens. However, methods for effectively reducing ARV-triggered apoptosis remain to be explored. Here, we show that pretreatment with chloroquine (CQ) or E64d plus pepstatin A decreases ARV-mediated apoptosis in chicken DF-1 cells. By acting as autophagy inhibitors, CQ and E64d plus pepstatin A increase microtubule-associated protein 1 light chain 3-II (LC3II) accumulation in ARV-infected cells, which results in decreased ARV protein synthesis and virus yield and thereby contributes to the reduction of apoptosis. Furthermore, ARV-mediated apoptosis in the bursa, heart and intestines of chicken embryos is attenuated by CQ and E64d plus pepstatin A treatment. Importantly, treatment with these autophagy inhibitors increases the survival of infected chicken embryos. Together, our data suggest that pharmacological inhibition of autophagy might represent a novel strategy for reducing ARV-mediated apoptosis.

Prenatal tolbutamide treatment alters plasma glucose and insulin concentrations and negatively affects the postnatal performance of chickens.

To examine the relationship of insulin and glucose, broiler embryos were subjected to acute or prolonged hypoglycemia during the late embryonic phase by, respectively, injecting once (at embryonic day [ED] 16 or 17) or on 3 consecutive days (ED 16, 17, and 18) with tolbutamide (80 µg/g embryo weight), a substance that stimulates insulin secretion from the pancreas. After 1 tolbutamide injection, a prolonged (32 h) decrease of plasma glucose and a profound acute increase in plasma insulin were observed. The 3 consecutive tolbutamide injections induced hypoglycemia for 4 days (from ED 16 to ED 19). The postnatal performance after 3 consecutive tolbutamide injections in broiler embryos was also investigated. Body weight was lower in tolbutamide-treated chickens from hatch to 42 d compared with sham (P = 0.001) and control (P < 0.001) chickens. Feed intake was lower in the tolbutamide group from hatch to 42 d as compared with sham (P = 0.007) and control (P = 0.017) animals. In addition, at 42 d, plasma glucose concentrations, after an insulin injection challenge (50 µg/kg body weight), were higher in tolbutamide-treated chickens compared with the sham and the control group as were their basal glucose levels (P value of group effect <0.001). In conclusion, tolbutamide treatment during the late embryonic development in broilers resulted in prolonged hypoglycemia in this period and negatively influenced the posthatch performance.

Embryonic protein undernutrition by albumen removal programs the hepatic amino acid and glucose metabolism during the perinatal period in an avian model.

Different animal models have been used to study the effects of prenatal protein undernutrition and the mechanisms by which these occur. In mammals, the maternal diet is manipulated, exerting both direct nutritional and indirect hormonal effects. Chicken embryos develop independent from the hen in the egg. Therefore, in the chicken, the direct effects of protein deficiency by albumen removal early during incubation can be examined. Prenatal protein undernutrition was established in layer-type eggs by the partial replacement of albumen by saline at embryonic day 1 (albumen-deprived group), compared to a mock-treated sham and a non-treated control group. At hatch, survival of the albumen-deprived group was lower compared to the control and sham group due to increased early mortality by the manipulation. No treatment differences in yolk-free body weight or yolk weight could be detected. The water content of the yolk was reduced, whereas the water content of the carcass was increased in the albumen-deprived group, compared to the control group, indicating less uptake of nutrients from the yolk. At embryonic day 16, 20 and at hatch, plasma triiodothyronine (T3), corticosterone, lactate or glucose concentrations and hepatic glycogen content were not affected by treatment. At embryonic day 20, the plasma thyroxine (T4) concentrations of the albumen-deprived embryos was reduced compared to the control group, indicating a decreased metabolic rate. Screening for differential protein expression in the liver at hatch using two-dimensional difference gel electrophoresis revealed not only changed abundance of proteins important for amino acid metabolism, but also of enzymes related to energy and glucose metabolism. Interestingly, GLUT1, a glucose transporter, and PCK2 and FBP1, two out of three regulatory enzymes of the gluconeogenesis were dysregulated. No parallel differences in gene expressions causing the differences in protein abundance could be detected pointing to post-transcriptional or post-translational regulation of the observed differences.

Astroviruses associated with stunting and pre-hatching mortality in duck and goose embryos.

The first detection of avian nephritis virus (ANV) in goose embryos and of turkey astrovirus-1 (TAstV-1) in duck embryos is described. Intestinal samples from duck and goose embryos from five duck and four goose flocks in Croatia were tested by polymerase chain reaction for the presence of ANV, TAstV-1, turkey astrovirus-2, chicken astrovirus, duck astrovirus and also for the presence of avian reovirus, Derzsy's disease virus and duck enteritis virus. The kidneys from duck and goose embryos were also tested for ANV, while liver samples were tested for duck astrovirus. Duck embryos were also tested to detect duck circovirus and goose embryos for the presence of goose circovirus and goose haemorrhagic polyomavirus. All embryos were in the final stage of incubation and were characterized by moderate to markedly retarded growth. ANV was confirmed in the intestines and kidneys of embryos from two duck and two goose flocks and TAstV-1 was found in embryos from two duck flocks. One duck flock was positive for both ANV and TAstV-1. No other viruses were found in tested flocks. Phylogenetic analysis based on the ANV polymerase gene fragment of ANV sequences detected in duck and goose embryos revealed greatest similarity (88.1 to 97.2%) with ANV isolates from chickens. Further, the existence of at least two types of ANV circulating in Croatian duck and goose flocks was confirmed. Based on the phylogenetic analysis of the portion of TAstV-1 polymerase gene, two detected TAstV-1 nucleotide sequences were 99.5% similar. Compared with six TAstV-1 sequences, Croatian sequences showed one unique nucleotide change. In addition to other possible causes of stunted growth and late embryonic death, these findings suggest that ANV and/or TAstV-1 infection may be a contributing factor in the pre-hatching mortality of ducklings and goslings.

Regulatory capacities of a broiler and layer strain exposed to high CO2 levels during the second half of incubation.

It has been shown that during embryonic chicken (Gallus gallus) development, the metabolism of broiler embryos differs from that of layers in terms of embryonic growth, pCO2/pO2 blood levels, heat production, and heart rate. Therefore, these strains might adapt differently on extreme environmental factors such as exposure to high CO2. The aim of this study was to compare broiler and layer embryos in their adaptation to 4% CO2 from embryonic days (ED) 12 to 18. Due to hypercapnia, blood pCO2 increased in both strains. Blood bicarbonate concentration was ~10 mmol/L higher in embryos exposed to high CO2 of both strains, while the bicarbonates of broilers had ~5 mmol/L higher values than layer embryos. In addition, the pH increased when embryos of both strains were exposed to CO2. Moreover, under CO2 conditions, the blood potassium concentration increased in both strains significantly, reaching a plateau at ED14. At ED12, the layer strain had a higher increase in CAII protein in red blood cells due to incubation under high CO2 compared to the broiler strain, whereas at ED14, the broiler strain had the highest increase. In conclusion, the most striking observation was the similar mechanism of broiler and layer embryos to cope with high CO2 levels.

Mycoplasma meleagridis-induced lesions in the tarsometatarsal joints of turkey embryos.

Mycoplasma meleagridis (MM) has the ability to cause bone deformity in turkey poults. However, few pathological lesions have been described and no evidence of MM-induced damage to the bones has been shown. In this study, 17-day-old turkey embryos were inoculated with MM into the allantoic cavity. On the 27th day, eight of the 22 embryos presented with curved toes. Scanning electron microscopy of the tarsometatarsal joints showed fissures in the cartilage. Histological sections of the joints revealed only the infiltration of cells with eosinophilic granules. Immunohistochemical staining (IHS) showed the presence of MM in the aggregates of the bone marrow cells and the cells with eosinophilic granules. Some of these cells were harvested by laser capture microdissection (LCM), lysed, and used as template DNA. With a pair of MM-specific primers in a conventional polymerase chain reaction (PCR), a gene product was amplified, and it comigrated with the MM DNA, which indicates that these captured cells contained MM DNA. Thus, this research shows that inoculation of MM into the turkey embryos produced joint lesions and caused cellular infiltration within the bones.

Effect of chronic hypoxia during embryonic development on physiological functioning and on hatching and post-hatching parameters related to ascites syndrome in broiler chickens.

The present study was designed to investigate the effect of different atmospheric pressure on the endogenous functions of broiler chickens during embryonic, hatching and growing periods related to ascites. Eggs from a commercial broiler line were incubated in two similar commercial incubators at high and low altitudes. The effect on embryonic development and physiological functions including hatching parameters, incidence of ascites and growth performance were examined. Embryos incubated at high altitude had higher plasma tri-iodothyronine, thyroxine, corticosteroid and lactic acid levels, and hatched earlier than those incubated at low altitude. Embryonic mortality was higher at high altitude. Chickens that had been incubated at high altitude showed less right ventricular hypertrophy and ascites mortality than those incubated at low altitude. It was concluded that different atmospheric pressure during incubation interacts with the endocrine functions of the embryo and hence affects hatching parameters, thereby influencing ascites susceptibility.

Viral pathogenesis in chicken embryos and tumor induction in chickens after in ovo exposure to serotype 1 Marek's disease virus.

We examined the susceptibility of late-stage chicken embryos to infection with oncogenic serotype 1 Marek's disease virus (MDV 1). Intravenous inoculation of MDV 1 at embryonic day (ED) 16 resulted in significant replication of the virus in embryonic tissues. Within 5 days of virus exposure, pp38 viral antigen (pp38) was detected in embryonic bursae and MDV 1 was isolated by plaque assay from the spleens, thymuses, and bursae of embryos. The pathogenesis of MDV 1 after intravenous inoculation at ED 16 was similar to that in chicks exposed to MDV 1 after hatching. In contrast to the response of the embryo to intravenous inoculation, embryos exposed to MDV 1 by the amniotic route did not develop detectable pp38, nor could the virus be isolated from the embryonic tissues by plaque assay. These results show that the route of inoculation of MDV 1 in the embryos is critical for allowing the virus to come in contact with target cells.

Oral famciclovir against duck hepatitis B virus replication in hepatic and nonhepatic tissues of ducklings infected in ovo.

Detection of hepadnaviral DNA in extrahepatic tissues of human and animal models of hepatitis B virus (HBV) has raised the question of whether virus replication in organs other than the liver could be targeted for the treatment of chronic hepatitis B. Since duck hepatitis B virus (DHBV) replication is dynamic in the liver, kidney, pancreas, and spleen of newly hatched ducklings infected in ovo, we used the duck model and the new antiherpesvirus agent, famciclovir (FCV), to determine whether antiviral effect of nucleoside analogues on DHBV replication is pluripotential. Day-old ducklings hatched from eggs laid by a DHBV-carrier duck were bled and administered FCV (25 mg/kg/bd) orally for periods of 1, 2, 3, 6, 9, and 12 days. Seventeen (17) hours after the last dose of each regimen the duckling(s) was bled and postmortem samples of liver, kidney, pancreas, and spleen were snap-frozen and stored at -70 degrees C. Analysis of plasma samples of ducklings treated for 2 days and longer by dot-blot hybridisation showed that levels of DHBV DNA were reduced significantly compared to levels in samples collected before treatment begun. Southern blot hybridisation of tissue DNA corroborated these results and showed that DHBV DNA replicative intermediates in all the tissues examined were reduced to levels that reflected the amount of virus released into the blood of each treated duckling. It is concluded from these results that if antiviral agents could be transformed to active metabolites in any infected tissues including the liver, replication of hepadnaviruses would be inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Failure to make normal alpha ryanodine receptor is an early event associated with the crooked neck dwarf (cn) mutation in chicken.

We have investigated the molecular basis of the Crooked Neck Dwarf (cn) mutation in embryonic chickens. Using biochemical and pharmacological techniques we are unable to detect normal alpha ryanodine receptor (RyR) protein in intact cn/cn skeletal muscle. Extremely low levels of alpha RyR immunoreactivity can be observed in mutant muscles, but the distribution of this staining differs from that in normal muscle and colocalizes with the rough endoplasmic reticulum immunoglobulin binding protein, BiP. This suggests the existence of an abnormal alpha RyR protein in mutant muscle. In day E12 cn/cn muscle the levels of RyR mRNA are reduced by approximately 80%, while the levels of other muscle proteins, including the alpha 1 subunit of the dihydropyridine receptor, the SR Ca(2+)-ATPase, calsequestrin, and glyceraldehyde-3-phosphate dehydrogenase, and their associated mRNAs are essentially normal in cn/cn muscle. There is also a failure to express alpha RyR in cn/cn cerebellar Purkinje neurons. Expression of the beta RyR, a second RyR isoform, is not initiated in normal skeletal muscle until day E18. In cn/cn skeletal muscle significant muscle degeneration has occurred by this time and the beta RyR is found at low levels in only a subset of fibers suggesting the reduced levels of this isoform are a secondary consequence of the mutation. The cardiac RyR isoform is found in cn/cn cardiac muscle, which contracts in a vigorous manner. In summary, a failure to make normal alpha RyR receptor appears to be an event closely associated with the cn mutation and one which may be largely responsible for development of the cn/cn phenotype in embryonic skeletal muscle.