SWV Strain Research Articles

Genetic and pathologic aspects of retinoic acid‐induced limb malformations in the mouse

Because all-trans retinoic acid (atRA) is teratogenic in all species tested and many of the specific defects induced are common across the phylogenetic spectrum, it would be logical to predict that murine strain differences in teratology to this agent are minimal. However, for specific defects, strain susceptibilities are vastly different. Studies with atRA have shown stark differences between C57BL/6 and SWV mouse strains in susceptibility to postaxial forelimb ectrodactyly and ectopic hindlimb formation, with the C57 strain being more susceptible for both defects. Various approaches were used to determine why these strains differ in susceptibility, but the mechanisms remain unknown. Hindlimb duplications were hypothesized to be caused by the formation of ectopic posterior body axes. For forelimb ectrodactyly, a locus on chromosome 11, Rafar, has linkage to the strain difference, and mRNA localization has shown that specific genes (Fgf8, Dlx3, Bmp4, and Sp8) in the postaxial preAER (prior to formation of the apical ectodermal ridge) of the developing limb bud (the site of the defect) were downregulated hours after atRA administration more in the susceptible C57 than in the SWV strain. Because both atRA and divalent cadmium induce postaxial forelimb ectrodactyly (right-sided predominance) at a high rate in C57BL/6 and low in the SWV strain, there is debate as to whether they share a common mechanism. These teratogens cause a greater-than-additive level of forelimb ectrodactyly when coadministered at low doses, but cadmium does not induce ectopic hindlimb formation. The hypothesis is that these agents have separate molecular pathologic pathways that converge to perturb a common anatomic structure.

Differential perturbation of the Fgf/Erk1/2 and Shh pathways in the C57BL/6N and SWV embryonic limb buds after mid-gestational cadmium chloride administration

The differential susceptibility of inbred mouse strains to teratogen-induced malformations can serve as a model to assess the molecular pathogenesis of dysmorphology. Using such a model, the teratogenic effect of cadmium chloride (CdCl 2), which results in limb reduction deformities in the C57BL/6N mouse strain, but not in the SWV strain, was found to correlate with reduction of the expression domains of Fgf8/4 (fibroblast growth factor-8 and -4) in the apical ectodermal ridge (AER) and Shh (sonic hedgehog) in the posterior mesenchyme, as well as reduction of MAPK/Erk1/2 (the mitogen-activated protein kinase/extracellular regulated kinase 1/2) phosphorylation (pErk1/2) in the mesenchyme throughout the limb bud. The pattern of pErk1/2 reduction did not consistently reflect the pattern of Fgf8/4 reduction suggesting that CdCl 2 might affect pErk1/2 through an Fgf-independent pathway. Other potential downstream mediators of the Fgf pathway including Mkp3 and Fgf10 as well as pMek (phosphorylated MAPK/Erk1/2 kinase) were not different in limb buds between the two strains at the studied time points. The effect of CdCl 2 on skeletogenesis was traced in time to the early stages of pre-chondrogenic condensation as determined by the Sox9 expression domain. The data of the present study indicate that a differential strain response to CdCl 2-induced forelimb digital loss may be due to a polymorphic interference with the Fgf/Shh positive feedback loop and Erk1/2 phosphorylation.

Priming the search for HOX mutations

Background Neural tube defects (NTDs) are among the most common human congenital malformations. Although clinical investigations have reported that periconceptional folic acid supplementation can reduce the occurrence of these defects, its mechanism remains unknown. Therefore, the murine mutant Splotch, which has a high incidence of spontaneous NTDs, along with the inbred strains SWV and LM/Bc, were used to investigate the relationship between folate and NTDs. Methods To investigate whether folates could reduce spontaneous NTDs, heterozygous Splotch dams (+/Sp) were treated with either folate or folinic acid throughout neurulation, gestational day (GD) 6.5 to 10.5. On GD 18.5 the dams were sacrificed and the fetuses examined for any neural tube defects. Subsequently, Sp/+ dams were treated with arsenic while receiving either a folate or folinic acid supplementation. Similar experiments were performed in the LM/Bc and SWV strains. Results Neither folate nor folinic acid supplements reduced the frequency of spontaneous NTDs in the embryos from Splotch heterozygote crosses. Arsenic increased the frequency of NTDs and embryonic death in the Splotch, LM/Bc and SWV litters and folinic acid failed to ameliorate the teratogenic effect of this metal. A folate supplement given to arsenic-treated dams proved to be maternally lethal in all three strains. Conclusions Splotch embryos were not protected from either spontaneous or arsenic-induced NTDs by folinic or folic acid supplementation. Furthermore, folinic acid supplements did not reduce the incidence of arsenic-induced NTDs in either the LM/Bc or SWV litters. Teratology 65:63–69, 2002. © 2002 Wiley-Liss, Inc.

A two-locus model for experience-conditioned direction of paw usage in the mouse is suggested by dominant and recessive constitutive paw usage behaviours.

Left-right direction of paw usage in the mouse depends on the genotype and the directional nature of the test. There are two phenotypic classes; in some strains, direction of paw usage is learned or conditioned by the direction of the initial test chamber and the experience of reaching and, in other strains, paw usage is a constitutive behaviour not affected by previous experience. We report the evidence for locus heterogeneity in the cause of constitutive versus experience-conditioned paw usage from a phenotypic analysis of F1 hybrid generations from the experience-conditioned C57BL/6J, C3H/HeHa, and SWV strains and the constitutive CDS/Lay and DBA/2J strains. The F1 hybrids between strains of different phenotypic classes provide evidence of locus heterogeneity. Constitutive paw usage in CDS/Lay is phenotypically dominant to experience-conditioned behaviour in both C57BL/6J and SWV. However, constitutive paw usage in DBA/2J is phenotypically recessive to experience-conditioned behaviour in C57BL/6J and dominant to experience-conditioned behaviour in SWV. Among the experience-conditioned strains, C57BL/6J is highly lateralized but SWV is only weakly lateralized. Our data suggest a model in which C57BL/6J may have a "strong" allele that identifies a functional difference between the constitutive paw usage of CDS/Lay and DBA/2J. DBA/2J may have a loss-of-function mutation at the same locus that is recessive to the strong C57BL/6J allele. SWV may have a "weak" allele and the (SWV x D2)F1 compound heterozygote may be below a threshold for detectability of experience-conditioned behaviour, making the constitutive behaviour of DBA/2J appear to be dominant to the experience-conditioned behaviour of SWV. CDS/Lay may have a dominant allele at a second locus that suppresses experience-conditioned behaviour in all F1 hybrids.

Does genomic imprinting contribute to valproic acid teratogenicity?☆

Reciprocal outcrosses and backcrosses were made between strains of mice with different susceptibilities to valproic acid (VPA) teratogenicity. Relatively resistant C57BL/6J (C) and more susceptible SWV (S) strains of mice produced F1 hybrids in which the female parent was C and the male parent was S (CS-F1) as well as the reciprocal with S dams and C sires (SC-F1). Each was backcrossed to each strain, producing 8 types of backcross matings: CS × C, SC × C, CS × S, SC × S; C × CS, C × SC, S × CS, S × SC (for all matings dams are listed first). At 8d:12 ± 5h of gestation, a teratogenic dose, 600 mg/kg, of aqueous VPA was injected ip into the dams. Fetuses were examined on gestation day (gd) 18 for abnormality, mortality, litter size, and weight. Genomic imprinting (imprinting) is a phenomenon at least in part involving hyper- or hypomethylation of bases in DNA, which is believed to determine whether or not the imprinted gene will be expressed. Imprinting has been reported to occur differentially in the male and female for a number of gene loci. Thus, in crosses between strains with differing susceptibility to VPA, if imprinting is occurring, the susceptibility of a fetus might be predicted to be disproportionately influenced by susceptibility of its grandparents. Significant differences in frequency (%) of occurrence of exencephaly in progeny of all backcrosses with F1 dams consistent with those expected for imprinting were found in the present study (CS-F1× C = 21.8 ± 3.9%, SC-F1× C = 10.8 ± 3.2%, P < 0.03; CS-F1× S = 14.8 ± 3.1%, SC-F1× S = 6.3 ± 2.3%, P < 0.03). SWV dams revealed the same pattern (S × SC-F1 = 50.0 ± 8.3%, S × CS-F1 = 37.1 ± 4.7%, P < 0.04). Differences in prenatal mortality also consistent with genomic imprinting occurred in backcrosses with pure-line SWV dams (S × SC = 64.4 ± 8.0%, S × CS = 30.5 ± 4.5%, P < 0.001). Fetal weight was reduced in a manner consistent with imprinting in backcrosses involving SWV (S × SC = 0.50 ± 0.18 g, S × CS = 0.96 ± 0.05, P < 0.01). Three of four of the parameters investigated showed differences in some of the backcrosses of reciprocal F1’s consistent with those expected if genomic imprinting were occurring.

Identification of a Murine Locus Conveying Susceptibility to Cadmium-Induced Forelimb Malformations

The heavy metal cadmium (Cd), an environmentally ubiquitous contaminant, is a potent teratogen in mice. When administered parenterally, it induces an array of malformations that vary in scope and severity with the route, dose, time of administration, and the strain of the animal. When administered intraperitoneally on day 9.0 of gestation, 4 mg/kg cadmium chloride produces forelimb defects (predominantly ectrodactyly) in over 80% of fetuses of the C57BL/6 mouse strain, while no limb defects are observed in the identically treated SWV strain. Like other examples of strain-specific teratogenic activity, the underlying nature of the differential susceptibility remains unknown. The present study investigates the segregation of sensitivity to Cd-induced forelimb defects in crosses between C57BL/6 and SWV mice and provides evidence for the involvement of both maternal and fetal factors in the determination of defect expression. In addition, quantitative trait loci (QTL) analysis of the fetal genetic component was performed among 198 backcross progeny, utilizing a genomic linkage map of 149 informative microsatellite markers. One QTL demonstrating significant linkage to expression of the defect, designated Cadfar (cadmium-induced forelimb autopod reduction), was mapped to the distal end of chromosome 6 with a lod score of 3.1.

Differential sensitivity of the SWV and C57BL/6 mouse strains to the teratogenic action of single administrations of cadmium given throughout the period of anterior neuropore closure.

When administered to mice during gestation, the heavy metal, cadmium, is known to induce malformations of the neural tube, craniofacial region, limbs, trunk, viscera, and axial skeleton that vary in scope and severity among inbred strains of mice. Two strains, C57BL/6 and SWV, were previously shown to differ in their susceptibility to exencephaly induced by many teratogenic treatments, including sodium 2-ethylhexanoate, hyperthermia, valproic acid, and carbon dioxide, with the SWV strain being consistently more sensitive than the C57BL/6 strain. These findings support the observation of Finnell et al. ([1988] Teratology 38:313-320) of shared hierarchies of relative susceptibility to exencephaly induced by biochemically distinct teratogens, and suggest that the SWV strain would also be more sensitive to exencephaly induced by cadmium. In the present study, pregnant mice from the two strains were exposed to single i.p. injections of cadmium chloride at 4 mg/kg-BW on day 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0 of gestation. Fetuses were removed by cesarean section on day 18 of gestation and scored for malformations. The C57BL/6 strain was observed to be more sensitive than the SWV strain to the induction of exencephaly by cadmium on days 6.5, 7.0, and 8.0, with mean litter percentages of 3.6%, 88.3%, and 62.2%, respectively, compared to 0.0%, 4.1%, and 27.7% for the SWV strain. This finding provides evidence in contrast to the hypothesis of shared hierarchies of sensitivity to teratogen-induced exencephaly. Data on a number of other cadmium-induced malformations are also presented.

Strain-dependent alterations in the expression of folate pathway genes following teratogenic exposure to valproic acid in a mouse model

The molecular basis for the well-established hierarchy of susceptibility to valproic acid-induced neural tube defects in inbred mouse strains was examined using in situ transcription and anti-sense RNA amplification methodologies with both univariate and multivariate analyses of the resulting gene expression data. The highly sensitive SWV strain demonstrated a significant reduction in the expression of the folate binding protein (FBP-1) following the teratogenic insult at gestational day 8:18, while the more resistant LM/Bc embryos were up-regulating this gene in response to valproic acid treatment. More importantly, at all 3 gestational timepoints spanning the period of murine neural tube closure examined in this study, the LM/Bc embryos had significantly higher MTHFR (5,10-methylenetetrahydrofolate reductase) gene expression levels compared to the SWV embryos. As this folate pathway enzyme is important in homocysteine and methionine metabolism, it suggests that the SWV embryos may be hypomethylated, and essential gene expression during critical periods of neural tube closure is compromised by the teratogenic exposure to valproic acid. This study represents the first evidence of a strain difference in transcriptional activity in response to a teratogenic exposure that might be causally related to the development of the teratogen-induced congenital malformations.

Strain‐dependent alterations in the expression of folate pathway genes following teratogenic exposure to valproic acid in a mouse model

The molecular basis for the well-established hierarchy of susceptibility to valproic acid-induced neural tube defects in inbred mouse strains was examined using in situ transcription and anti-sense RNA amplification methodologies with both univariate and multivariate analyses of the resulting gene expression data. The highly sensitive SWV strain demonstrated a significant reduction in the expression of the folate binding protein (FBP-1) following the teratogenic insult at gestational day 8:18, while the more resistant LM/Bc embryos were up-regulating this gene in response to valproic acid treatment. More importantly, at all 3 gestational timepoints spanning the period of murine neural tube closure examined in this study, the LM/Bc embryos had significantly higher MTHFR (5,10-methylenetetra-hydrofolate reductase) gene expression levels compared to the SWV embryos. As this folate pathway enzyme is important in homocysteine and methionine metabolism, it suggests that the SWV embryos may be hypomethylated, and essential gene expression during critical periods of neural tube closure is compromised by the teratogenic exposure to valproic acid. This study represents the first evidence of a strain difference in transcriptional activity in response to a teratogenic exposure that might be causally related to the development of the teratogen-induced congenital malformations. Am. J. Med. Genet. 70:303–311, 1997. © 1997 Wiley-Liss, Inc.

Expression of the IV (reversed and/or heterotaxic) phenotype in SWV mice.

Approximately 50% of iv/iv mice have situs inversus (mirror image reversal of viscera) and 40% have heterotaxia (anomalous arrangement of viscera). The occurrence of heterotaxia is independent of situs. Using the cross-intercross breeding system to put the iv gene on the SWV background, an occasional presumed iv/+ mouse was found that had an IV (situs inversus and/or heterotaxic) phenotype. Testcrosses of these reversed animals indicated an iv/+ genotype. Since iv is linked tightly to Igh-C on chromosome 12, we inferred the genotype with a polymorphism of Igh-C demonstrated using the polymerase chain reaction (PCR). This confirmed them to be iv/+. The expression of the IV phenotype in animals heterozygous for the iv gene may be due to an interaction of iv with an autosomal recessive gene found in SWV. We have not found the IV phenotype in heterozygous iv/+ mice following placement of the iv gene on six other inbred strains. Rarely, we also found that presumed SWV +/+ mice had the IV phenotype. Test matings of these phenodeviants, corroborated by PCR, have confirmed them to be +/+. Although the phenotypes of the affected SWV +/+ and iv/+ mice resembled those found in iv/iv mice, the occurrence of situs inversus and heterotaxia were not independent of each other, and most of the SWV mice with the IV phenotype had heterotaxia with situs solitus. This infrequent dominant expression of the iv gene has so far only been seen when iv is on the SWV background. These findings are consistent with the idea that this phenomenon is due to the interaction of the iv gene with another gene found so far only in the SWV strain.

Acetazolamide with caffeine causes exencephaly in “resistant” SWV mice

Pregnant SWV mice were treated on day 9 of gestation (PC) with 50 mg/kg of caffeine (CAFF), 200 mg/kg (LD) or 1000 mg/kg (HD) of acetazolamide (ACZM), or a combination of both agents, or on day 8 PC with both agents (ACZM + CAFF). Untreated (UNTD) and vehicle-treated (VEH) groups served as control. The SWV strain is widely reported to be resistant to ACZM; it was resistant to ACZM or CAFF + ACZM when treated on day 9 of gestation, but a significant frequency of malformations, primarily exencephaly, was produced by ACZM + CAFF on day 8 PC. This study provides evidence that ACZM, coupled with a subteratogenic dose of caffeine can produce abnormalities in the “resistant” SWV mice, using the endpoint of exencephaly on day 8 of gestation. The mean number of ossified caudal vertebrae in day-9 treatments and ossified cervical vertebral centra in day-8 treatments were reduced. The frequency of ossification of the first cervical vertebra (C1) was reduced from 93% in UNTD to 39% in HD-ACZM day 9 PC and 69% in HD-ACZM + CAFF day 9 PC groups, and was also significantly reduced in the HD-ACZM + CAFF day-8 treated group.

Intermittent pattern of neural tube closure in two strains of mice

Development of the neural tube is often described as a continuous process that begins in the cervical region of the embryo and proceeds both rostrally and caudally. Examination of neural tube closure in the cranial region of LM/Bc and SWV/Bc mice revealed an intermittent pattern with four distinct areas of closure. Closure I begins at the level of somites 1-3 and proceeds bidirectionally. Closure II is initiated at the prosencephalic-mesencephalic border and also proceeds bidirectionally. Closure III is unidirectional, beginning adjacent to the stomodeum and proceeding caudally to meet closure II. Finally, closure IV takes place over the rhombencephalon where it meets closure II to complete rostral neural tube closure. In these two strains of mice anterior neural tube closure progressed as somite number increased. However, the SWV strain required a longer gestational time to develop equal numbers of somites and therefore to complete closure. In light of the intermittent pattern of closure observed in mice, the development of the rostral nervous system in other mammals, including humans, should be reconsidered.

Genetic differences in heat-induced tolerance to cadmium in cultured mouse embryos are not correlated with changes in a 68-kD heat shock protein.

Heat-induced cross-tolerance to cadmium was investigated in two inbred strains of mice, BALB/c and SWV, using a whole embryo culture system. Embryos were exposed to a pretreatment of 5 min at 43 degrees C and subsequently to an embryotoxic concentration of cadmium, 1.75 microM. The two types of embryos responded differently to the heat pretreatment, as cross-tolerance was induced in SWV but not in BALB/c mice. In SWV embryos, prior exposure to 43 degrees C for 5 min essentially eliminated the negative effects of cadmium on embryonic development and growth. However, in BALB/c embryos, no protection was observed. The variation in development of cross-tolerance in embryos from the two strains of mice was not correlated with differences in the induction of a 68-kD heat-shock protein (hsp68). There was a rapid increase in this protein in both strains after the initial heat exposure but not excess induction in the SWV strain that developed tolerance. The induction of hsp68 is therefore not sufficient to elicit cross-tolerance, and other mechanisms are likely to be important in the protective response of the embryo.

Murine teratology and pharmacokinetics of the enantiomers of sodium 2-ethylhexanoate

A mouse model for the induction of exencephaly with sodium (±)-2-ethylhexanoate has been developed using multiple administration regimes. With three consecutive administrations at one-half-day intervals, the most sensitive time to induce exencephaly was Gestational Days 8–9. Using the racemic substance it was determined that the SWV strain was more sensitive to the induction of exencephaly than the C57BL 6NCrlBR strain. The enantiomers of 2-ethylhexanoic acid were separated via preparative HPLC to greater than 99.8% optical purity, and greater than 99% purity according to a gas chromatographic analysis. It was demonstrated that the ( R)-enantiomer is a more potent teratogen than the ( S)-enantiomer for the induction of exencephaly as well as malformations of other organ systems. Pharmacokinetic analyses for each of the enantiomers were performed in maternal plasma, maternal muscle, and embryo. The pharmacokinetics showed that the peak concentration ( C max) for both enantiomers in the three compartments was approximately equivalent and was attained within 15 min following the third administration. The area under the concentration versus time curve values for the two enantiomers were approximately 10% higher for the ( R)-antipode because of a slightly slower elimination of this compound. There was negligible (or no) racemization of the two enantiomers in the biological samples. The results suggest that teratologic differences in the enantiomers of sodium 2-ethylhexanoate are not due to differences in the concentrations of these antipodes in the embryo, but more likely result from the specific interaction of the enantiomers with chiral molecules in the embryo.

Common hierarchies of susceptibility to the induction of neural tube defects in mouse embryos by valproic acid and its 4-propyl-4-pentenoic acid metabolite.

The teratogenic effects of valproic acid and its 4-propyl-4-pentenoic acid (4-en) metabolite were investigated in three inbred mouse strains that were known to possess differing sensitivity to heat-induced neural tube defects. In the heat-resistant DBA/2J strain, administration of either valproic acid or the metabolite during the critical period of neural tube development failed to produce any abnormal offspring. Similar treatment in the moderately heat-sensitive LM/Bc strain resulted in up to 19.8% exencephalic fetuses. The highly heat-sensitive SWV strain was also very susceptible to the induction of neural tube defects by either valproic acid or its 4-en metabolite. When administered on gestational day 8 plus 12 hours, the parent compound produced 35% exencephalic fetuses, while the metabolite had a response frequency of 32.4%. Thus, the hierarchy of susceptibility for the induction of neural tube defects in these inbred mouse strains was exactly the same whether the teratogen was a physical agent such as hyperthermia or a chemical compound such as valproic acid. If such diverse agents as these should interact to produce malformations, then it is possible that a wide variety of other agents might interact in a similar manner to produce neural tube defects.

Gene-teratogen interaction and its morphological basis in retinoic acid-induced mouse spina bifida.

Homozygotes for the splotch (Sp) mutation in the mouse have spina bifida, whereas the heterozygotes have a white belly spot but otherwise appear normal. Spina bifida can be induced by maternal treatment with retinoic acid. Female SWV strain mice were treated intraperitoneally with retinoic acid suspended in peanut oil 8 days/12 hours after they had been mated to either Sp/+ or +/+ males. Probit analysis of the dose-response data suggests that the presence of the Sp gene causes an increased susceptibility of the embryo to the spina bifida-causing effects of retinoic acid. To study the nature of this increase litters were obtained on gestation day 9 from untreated SWV females mated as above. The mean length of the posterior neuropore (the length of the posterior neural tube that has not yet closed) was determined for each somite number between 14 and 26 and was found to be significantly greater in embryos from the Sp/+ cross. This delay of closure of the neural tube in Sp/+ cross embryos could explain the observed increase in their susceptibility to retinoic acid.

Acetazolamide teratology and its association with carbonic anhydrase inhibition in the mouse.

Acetazolamide, an inhibitor of the enzyme carbonic anhydrase (E.C. 4.2.1.1.), causes a unique congenital anomaly characterized by postaxial reduction of the distal portion of the right forelimb. To gain an understanding of the mechanism of teratogenesis, the activity of carbonic anhydrase in sensitive and resistant mouse strains, and its inhibition by acetazolamide, were examined. Differences in teratologic sensitivity were found not to be attributable to differences in maternal or embryonic drug levels. Enzyme inhibition at acetazolamide concentrations ranging between 10(-11) and 10(-5) M did not differ between the mouse strains when adult erythrocytes or day 12 embryos were assayed. However, in day 10 embryos, the period of maximum teratologic susceptibility, a small strain difference was found which suggested that the form of carbonic anhydrase in the susceptible CBA/J strain at this time is somewhat more sensitive to inhibition by acetazolamide than the form found in the resistant SWV strain. The results suggest further that more than one isozyme of carbonic anhydrase may be present in all three samples.

The chemical status of cadmium ion in the placenta

The chemical status of cadmium ion in the chorioallantoic and yolk sac placentas of SWV strain mice during the critical phase of embryonic organogenesis was investigated. Twenty-four hours after a single teratogenic dose of cadmium ion, both placentas contained significant amounts of a macromolecular chelator. Gel filtration data and disc electrophoresis data suggest that the cadmium-binding macromolecule of the chorioallantois (which binds 61% of the cadmium ion content of the tissue) is a metallothionein dimer.

Teratogenesis of acetazolamide in the CBA/J and SWV strains of mice. II. Genetic control of the teratogenic response.

Three independently segregating loci appear to control the difference in response of CBA and SWV mouse embryos to the ectrodactyly-inducing effect of acetazolamide. Response of the CBA embryo appears to depend on recessive genes for sensitivity being present in the homozygous condition at all 3 loci, and resistance to be conferred by the presence of a dominant allele at any one of the 3 loci. The genetic variability for the ectrodactyly response of the mouse is discussed in the light of the reported physiological factors necessary for acetazolamide-induced ectrodactyly in the rat.

Hereditary Vasopressin-Resistant Diabetes Insipidus in SWV Mice

Old female mice of the SWV strain have an hereditary polydipsia–polyuria defect with a severe increase in water turnover and with hypotonic urine which contains no glucose, blood, or protein; that is, they have diabetes insipidus. Their responses to exogenous vasopressin (negative) and water deprivation (positive) and the normal amount of neurosecretory material in their posterior pituitaries indicate that they have nephrogenic diabetes insipidus. The females also have a progressive kidney defect which, although it resembles nephronophthisis in some aspects and hypokalemia in others, is unique. There is a progressive anemia. Amyloid kidneys, polycystic kidneys, diabetes mellitus, and nephronophthisis have been ruled out as the cause of the defect but hypokalemia and hypercalcemia are still possibilities. The SWV males have a milder form of the defect which develops at an older age and shows no signs of histopathology.