Xingshen Sun

Disease phenotype of a ferret CFTR-knockout model of cystic fibrosis

Cystic fibrosis (CF) is a recessive disease that affects multiple organs. It is caused by mutations in CFTR. Animal modeling of this disease has been challenging, with species- and strain-specific differences in organ biology and CFTR function influencing the emergence of disease pathology. Here, we report the phenotype of a CFTR-knockout ferret model of CF. Neonatal CFTR-knockout ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport and submucosal gland fluid secretion; variably penetrant meconium ileus (MI); pancreatic, liver, and vas deferens disease; and a predisposition to lung infection in the early postnatal period. Severe malabsorption by the gastrointestinal (GI) tract was the primary cause of death in CFTR-knockout kits that escaped MI. Elevated liver function tests in CFTR-knockout kits were corrected by oral administration of ursodeoxycholic acid, and the addition of an oral proton-pump inhibitor improved weight gain and survival. To overcome the limitations imposed by the severe intestinal phenotype, we cloned 4 gut-corrected transgenic CFTR-knockout kits that expressed ferret CFTR specifically in the intestine. One clone passed feces normally and demonstrated no detectable ferret CFTR expression in the lung or liver. The animals described in this study are likely to be useful tools for dissecting CF disease pathogenesis and developing treatments.

Adeno-associated virus-targeted disruption of the CFTR gene in cloned ferrets.

Somatic cell gene targeting combined with nuclear transfer cloning presents tremendous potential for the creation of new, large-animal models of human diseases. Mouse disease models often fail to reproduce human phenotypes, underscoring the need for the generation and study of alternative disease models. Mice deficient for CFTR have been poor models for cystic fibrosis (CF), lacking many aspects of human CF lung disease. In this study, we describe the production of a CFTR gene-deficient model in the domestic ferret using recombinant adeno-associated virus-mediated gene targeting in fibroblasts, followed by nuclear transfer cloning. As part of this approach, we developed a somatic cell rejuvenation protocol using serial nuclear transfer to produce live CFTR-deficient clones from senescent gene-targeted fibroblasts. We transferred 472 reconstructed embryos into 11 recipient jills and obtained 8 healthy male ferret clones heterozygous for a disruption in exon 10 of the CFTR gene. To our knowledge, this study represents the first description of genetically engineered ferrets and describes an approach that may be of substantial utility in modeling not only CF, but also other genetic diseases.

Abnormal endocrine pancreas function at birth in cystic fibrosis ferrets

Diabetes is a common comorbidity in cystic fibrosis (CF) that worsens prognosis. The lack of an animal model for CF-related diabetes (CFRD) has made it difficult to dissect how the onset of pancreatic pathology influences the emergence of CFRD. We evaluated the structure and function of the neonatal CF endocrine pancreas using a new CFTR-knockout ferret model. Although CF kits are born with only mild exocrine pancreas disease, progressive exocrine and endocrine pancreatic loss during the first months of life was associated with pancreatic inflammation, spontaneous hyperglycemia, and glucose intolerance. Interestingly, prior to major exocrine pancreas disease, CF kits demonstrated significant abnormalities in blood glucose and insulin regulation, including diminished first-phase and accentuated peak insulin secretion in response to glucose, elevated peak glucose levels following glucose challenge, and variably elevated insulin and C-peptide levels in the nonfasted state. Although there was no difference in lobular insulin and glucagon expression between genotypes at birth, significant alterations in the frequencies of small and large islets were observed. Newborn cultured CF islets demonstrated dysregulated glucose-dependent insulin secretion in comparison to controls, suggesting intrinsic abnormalities in CF islets. These findings demonstrate that early abnormalities exist in the regulation of insulin secretion by the CF endocrine pancreas.

Lung Phenotype of Juvenile and Adult Cystic Fibrosis Transmembrane Conductance Regulator–Knockout Ferrets

Chronic bacterial lung infections in cystic fibrosis (CF) are caused by defects in the CF transmembrane conductance regulator chloride channel. Previously, we described that newborn CF transmembrane conductance regulator-knockout ferrets rapidly develop lung infections within the first week of life. Here, we report a more slowly progressing lung bacterial colonization phenotype observed in juvenile to adult CF ferrets reared on a layered antibiotic regimen. Even on antibiotics, CF ferrets were still very susceptible to bacterial lung infection. The severity of lung histopathology ranged from mild to severe, and variably included mucus obstruction of the airways and submucosal glands, air trapping, atelectasis, bronchopneumonia, and interstitial pneumonia. In all CF lungs, significant numbers of bacteria were detected and impaired tracheal mucociliary clearance was observed. Although Streptococcus, Staphylococcus, and Enterococcus were observed most frequently in the lungs of CF animals, each animal displayed a predominant bacterial species that accounted for over 50% of the culturable bacteria, with no one bacterial taxon predominating in all animals. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry fingerprinting was used to quantify lung bacteria in 10 CF animals and demonstrated Streptococcus, Staphylococcus, Enterococcus, or Escherichia as the most abundant genera. Interestingly, there was significant overlap in the types of bacteria observed in the lung and intestine of a given CF animal, including bacterial taxa unique to the lung and gut of each CF animal analyzed. These findings demonstrate that CF ferrets develop lung disease during the juvenile and adult stages that is similar to patients with CF, and suggest that enteric bacterial flora may seed the lung of CF ferrets.

A mutation in the Srrm4 gene causes alternative splicing defects and deafness in the Bronx waltzer mouse.

Sensory hair cells are essential for hearing and balance. Their development from epithelial precursors has been extensively characterized with respect to transcriptional regulation, but not in terms of posttranscriptional influences. Here we report on the identification and functional characterization of an alternative-splicing regulator whose inactivation is responsible for defective hair-cell development, deafness, and impaired balance in the spontaneous mutant Bronx waltzer (bv) mouse. We used positional cloning and transgenic rescue to locate the bv mutation to the splicing factor-encoding gene Ser/Arg repetitive matrix 4 (Srrm4). Transcriptome-wide analysis of pre–mRNA splicing in the sensory patches of embryonic inner ears revealed that specific alternative exons were skipped at abnormally high rates in the bv mice. Minigene experiments in a heterologous expression system confirmed that these skipped exons require Srrm4 for inclusion into the mature mRNA. Sequence analysis and mutagenesis experiments showed that the affected transcripts share a novel motif that is necessary for the Srrm4-dependent alternative splicing. Functional annotations and protein–protein interaction data indicated that the encoded proteins cluster in the secretion and neurotransmission pathways. In addition, the splicing of a few transcriptional regulators was found to be Srrm4 dependent, and several of the genes known to be targeted by these regulators were expressed at reduced levels in the bv mice. Although Srrm4 expression was detected in neural tissues as well as hair cells, analyses of the bv mouse cerebellum and neocortex failed to detect splicing defects. Our data suggest that Srrm4 function is critical in the hearing and balance organs, but not in all neural tissues. Srrm4 is the first alternative-splicing regulator to be associated with hearing, and the analysis of bv mice provides exon-level insights into hair-cell development.

Gastrointestinal pathology in juvenile and adult CFTR-knockout ferrets.

Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included steatosis, necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (<2 μg elastase-1 per gram of feces). These findings suggest that genetic factors likely influence the extent of exocrine pancreas disease in CF ferrets and have implications for the etiology of pancreatic sufficiency in CF patients. In summary, these studies demonstrate that the CF ferret model develops gastrointestinal pathology similar to CF patients.

Defective innate immunity and hyperinflammation in newborn cystic fibrosis transmembrane conductance regulator-knockout ferret lungs.

Mucociliary clearance (MCC) and submucosal glands are major components of airway innate immunity that have impaired function in cystic fibrosis (CF). Although both of these defense systems develop postnatally in the ferret, the lungs of newborn ferrets remain sterile in the presence of a functioning cystic fibrosis transmembrane conductance regulator gene. We evaluated several components of airway innate immunity and inflammation in the early CF ferret lung. At birth, the rates of MCC did not differ between CF and non-CF animals, but the height of the airway surface liquid was significantly reduced in CF newborn ferrets. CF ferrets had impaired MCC after 7 days of age, despite normal rates of ciliogenesis. Only non-CF ferrets eradicated Pseudomonas directly introduced into the lung after birth, whereas both genotypes could eradicate Staphylococcus. CF bronchoalveolar lavage fluid (BALF) had significantly lower antimicrobial activity selectively against Pseudomonas than non-CF BALF, which was insensitive to changes in pH and bicarbonate. Liquid chromatography-tandem mass spectrometry and cytokine analysis of BALF from sterile Caesarean-sectioned and nonsterile naturally born animals demonstrated CF-associated disturbances in IL-8, TNF-α, and IL-β, and pathways that control immunity and inflammation, including the complement system, macrophage functions, mammalian target of rapamycin signaling, and eukaryotic initiation factor 2 signaling. Interestingly, during the birth transition, IL-8 was selectively induced in CF BALF, despite no genotypic difference in bacterial load shortly after birth. These results suggest that newborn CF ferrets have defects in both innate immunity and inflammatory signaling that may be important in the early onset and progression of lung disease in these animals.

Efficient Term Development of Vitrified Ferret Embryos Using a Novel Pipette Chamber Technique

Abstract Development of an efficient cryopreservation technique for the domestic ferret is key for the long-term maintenance of valuable genetic specimens of this species and for the conservation of related endangered species. Unfortunately, current cryopreservation procedures, such as slow-rate freezing and vitrification with open pulled straws, are inefficient. In this report, we describe a pipette tip-based vitrification method that significantly improves the development of thawed ferret embryos following embryo transfer (ET). Ferret embryos at the morula (MR), compact morula (CM), and early blastocyst (EB) stages were vitrified using an Eppendorf microloader pipette tip as the chamber vessel. The rate of in vitro development was significantly (P < 0.05) higher among embryos vitrified at the CM (93.6%) and EB (100%) stages relative to those vitrified at the MR stages (58.7%). No significant developmental differences were observed when comparing CM and EB vitrified embryos with nonvitrified control CM (100%) and EB (100%) embryos. In addition, few differences in the ultrastructure of intracellular lipid droplets or in microfilament structure were observed between control embryos and embryos vitrified at any developmental stage. Vitrified-thawed CM/EB embryos cultured for 2 or 16 h before ET resulted in live birth rates of 71.3% and 77.4%, respectively. These rates were not significantly different from the control live birth rate (79.2%). However, culture for 32 h (25%) or 48 h (7.8%) after vitrification significantly reduced the rate of live births. These data indicate that the pipette chamber vitrification technique significantly improves the live birth rate of transferred ferret embryos relative to current state-of-the-art methods..

Sox2 modulates Lef-1 expression during airway submucosal gland development

Tracheobronchial submucosal glands (SMGs) are derived from one or more multipotent glandular stem cells that coalesce to form a placode in surface airway epithelium (SAE). Wnt/β-catenin-dependent induction of lymphoid enhancer factor (Lef-1) gene expression during placode formation is an early event required for SMG morphogenesis. We discovered that Sox2 expression is repressed as Lef-1 is induced within airway SMG placodes. Deletion of Lef-1 did not activate Sox2 expression in SMG placodes, demonstrating that Lef-1 activation does not directly inhibit Sox2 expression. Repression of Sox2 protein in SMG placodes occurred posttranscriptionally, since the activity of its endogenous promoter remained unchanged in SMG placodes. Thus we hypothesized that Sox2 transcriptionally represses Lef-1 expression in the SAE and that suppression of Sox2 in SMG placodes activates Wnt/β-catenin-dependent induction of Lef-1 during SMG morphogenesis. Consistent with this hypothesis, transcriptional reporter assays, ChIP analyses, and DNA-protein binding studies revealed a functional Sox2 DNA binding site in the Lef-1 promoter that is required for suppressing β-catenin-dependent transcription. In polarized primary airway epithelium, Wnt induction enhanced Lef-1 expression while also inhibiting Sox2 expression. Conditional deletion of Sox2 also enhanced Lef-1 expression in polarized primary airway epithelium, but this induction was significantly augmented by Wnt stimulation. Our findings provide the first evidence that Sox2 acts as a repressor to directly modulate Wnt-responsive transcription of the Lef-1 gene promoter. These studies support a model whereby Wnt signals and Sox2 dynamically regulate the expression of Lef-1 in airway epithelia and potentially also during SMG development.

A Transient Metabolic Recovery from Early Life Glucose Intolerance in Cystic Fibrosis Ferrets Occurs During Pancreatic Remodeling

Cystic fibrosis (CF)-related diabetes in humans is intimately related to exocrine pancreatic insufficiency, yet little is known about how these 2 disease processes simultaneously evolve in CF. In this context, we examined CF ferrets during the evolution of exocrine pancreatic disease. At 1 month of age, CF ferrets experienced a glycemic crisis with spontaneous diabetic-level hyperglycemia. This occurred during a spike in pancreatic inflammation that was preceded by pancreatic fibrosis and loss of β-cell mass. Surprisingly, there was spontaneous normalization of glucose levels at 2-3 months, with intermediate hyperglycemia thereafter. Mixed meal tolerance was impaired at all ages, but glucose intolerance was not detected until 4 months. Insulin secretion in response to hyperglycemic clamp and to arginine was impaired. Insulin sensitivity, measured by euglycemic hyperinsulinemic clamp, was normal. Pancreatic inflammation rapidly diminished after 2 months of age during a period where β-cell mass rose and gene expression of islet hormones, peroxisome proliferator-activated receptor-γ, and adiponectin increased. We conclude that active CF exocrine pancreatic inflammation adversely affects β-cells but is followed by islet resurgence. We predict that very young humans with CF may experience a transient glycemic crisis and postulate that pancreatic inflammatory to adipogenic remodeling may facilitate islet adaptation in CF.