Otx1 null mutant mice show partial segregation of sensory epithelia comparable to lamprey ears

We investigated the development of inner ear innervation in Otx1 null mutants, which lack a horizontal canal, between embryonic day 12 (E12) and postnatal day 7 (P7) with DiI and immunostaining for acetylated tubulin. Comparable to control animals, horizontal crista-like fibers were found to cross over the utricle in Otx1 null mice. In mutants these fibers extend toward an area near the endolymphatic duct, not to a horizontal crista. Most Otx1 null mutants had a small patch of sensory hair cells at this position. Measurement of the area of the utricular macula suggested it to be enlarged in Otx1 null mutants. We suggest that parts of the horizontal canal crista remain incorporated in the utricular sensory epithelium in Otx1 null mutants. Other parts of the horizontal crista appear to be variably segregated to form the isolated patch of hair cells identifiable by the unique fiber trajectory as representing the horizontal canal crista. Comparison with lamprey ear innervation reveals similarities in the pattern of innervation with the dorsal macula, a sensory patch of unknown function. SEM data confirm that all foramina are less constricted in Otx1 null mutants. We propose that Otx1 is not directly involved in sensory hair cell formation of the horizontal canal but affects the segregation of the horizontal canal crista from the utricle. It also affects constriction of the two main foramina in the ear, but not their initial formation. Otx1 is thus causally related to horizontal canal morphogenesis as well as morphogenesis of these foramina.     

Cardiac ErbB-1/ErbB-2 mutant expression in young adult mice leads to cardiac dysfunction

Multiple factors lead to the development and maintenance of chronic heart failure. Blockade of ErbB-2 or ErbB-4 tyrosine kinase receptor signaling leads to dilated cardiomyopathy. ErbB-1 may protect the heart against stress-induced injury and its ligand; epidermal growth factor (EGF) increases myocardial contractility, whereas heparin-binding EGF is essential for normal cardiac function. However, the role of ErbB-1 in control of cardiac function is not clear. We hypothesized that ErbB-1 is essential for maintaining adult cardiac function. Using the ecdysone-inducible gene expression system, we expressed humanized cardiomyocyte-specific dominant-negative ErbB-1 mutant receptors (hErbB-1-mut) in young adult mice that block endogenous cardiac ErbB-1 signaling. Molecular, morphological, and physiological tests (under anesthesia) were performed. As a result, hErbB-1-mut was expressed selectively in cardiomyocytes leading to the blockade of endogenous ErbB-1 phosphorylation and ErbB-2 transphosphorylation. An increase in left ventricular mass, atrial natriuretic factor expression, and histological changes were indicative of cardiac hypertrophy. Cardiac dilation, numerous cardiac lesions, and the loss of the clear boundary between cardiac fibrils were noted histologically. Early and long-term hErbB-1-mut induction led to a significant decrease in fractional shortening and to significant increases in left ventricular end-systolic diameter and volume. The treatment of adenylyl cyclase activator (forskolin analog) normalized the depressed cardiac function. Resting cardiac function returned to normal after reversing mutant expression. A 4-day survival rate of transverse-aortic constricted hErbB-1-mut mice was only 20% compared with 100% in controls. In conclusion, these observations indicate that the blockade of cardiac ErbB-1 signaling leads to the blockade of ErbB-2 signaling and that together they result in cardiac dysfunction.     

Epidermal hyperplasia and expansion of the interfollicular stem cell compartment in mutant mice with a C-terminal truncation of Patched1

Hedgehog (Hh) signaling is conserved from flies to humans and is indispensable in embryogenesis and adulthood. Patched (Ptc) encodes a receptor for Hh ligands and functions as a tumor suppressor. PTCH1 mutations in humans are found in basal cell carcinoma (BCC) and irradiated Ptc1(+/-) mice recapitulate this phenotype. However, due to embryonic lethality associated with the Ptc1 null mutation, its normal function in embryonic and adult skin remains unknown. Here we describe the epidermal phenotypes of a spontaneous and viable allele of Ptc1, Ptc1(mes), in which the C-terminal domain (CTD) is truncated. Ptc1(mes/mes) embryos display normal epidermal and hair follicle development. Postnatal Ptc1(mes/mes) skin displays severe basal cell layer hyperplasia and increased proliferation, while stratification of the suprabasal layers is mostly normal. Interestingly, truncation of the Ptc1 CTD did not result in skin tumors. However, long term labeling studies revealed a greater than three-fold increase in label-retaining cells in the interfollicular epidermis of Ptc1(mes/mes) adults, indicating possible expansion of the epidermal stem cell compartment. Increased expression of regulators of epidermal homeostasis, c-Myc and p63, was also observed in Ptc1(mes/mes) adult skin. These results suggest that the CTD of Ptc1 is involved in regulating epidermal homeostasis in mature skin.     

Aberrant myofibril assembly in tropomodulin1 null mice leads to aborted heart development and embryonic lethality

Tropomodulin1 (Tmod1) caps thin filament pointed ends in striated muscle, where it controls filament lengths by regulating actin dynamics. Here, we investigated myofibril assembly and heart development in a Tmod1 knockout mouse. In the absence of Tmod1, embryonic development appeared normal up to embryonic day (E) 8.5. By E9.5, heart defects were evident, including aborted development of the myocardium and inability to pump, leading to embryonic lethality by E10.5. Confocal microscopy of hearts of E8-8.5 Tmod1 null embryos revealed structures resembling nascent myofibrils with continuous F-actin staining and periodic dots of alpha-actinin, indicating that I-Z-I complexes assembled in the absence of Tmod1. Myomesin, a thick filament component, was also assembled normally along these structures, indicating that thick filament assembly is independent of Tmod1. However, myofibrils did not become striated, and gaps in F-actin staining (H zones) were never observed. We conclude that Tmod1 is required for regulation of actin filament lengths and myofibril maturation; this is critical for heart morphogenesis during embryonic development.     

Epithelial cell stretching and luminal acidification lead to a retarded development of stria vascularis and deafness in mice lacking pendrin

Loss-of-function mutations of SLC26A4/pendrin are among the most prevalent causes of deafness. Deafness and vestibular dysfunction in the corresponding mouse model, Slc26a4(-/-), are associated with an enlargement and acidification of the membranous labyrinth. Here we relate the onset of expression of the HCO(3) (-) transporter pendrin to the luminal pH and to enlargement-associated epithelial cell stretching. We determined expression with immunocytochemistry, cell stretching by digital morphometry and pH with double-barreled ion-selective electrodes. Pendrin was first expressed in the endolymphatic sac at embryonic day (E) 11.5, in the cochlear hook-region at E13.5, in the utricle and saccule at E14.5, in ampullae at E16.5, and in the upper turn of the cochlea at E17.5. Epithelial cell stretching in Slc26a4(-/-) mice began at E14.5. pH changes occurred first in the cochlea at E15.5 and in the endolymphatic sac at E17.5. At postnatal day 2, stria vascularis, outer sulcus and Reissner's membrane epithelial cells, and utricular and saccular transitional cells were stretched, whereas sensory cells in the cochlea, utricle and saccule did not differ between Slc26a4(+/-) and Slc26a4(-/-) mice. Structural development of stria vascularis, including vascularization, was retarded in Slc26a4(-/-) mice. In conclusion, the data demonstrate that the enlargement and stretching of non-sensory epithelial cells precedes luminal acidification in the cochlea and the endolymphatic sac. Stretching and luminal acidification may alter cell-to-cell communication and lead to the observed retarded development of stria vascularis, which may be an important step on the path to deafness in Slc26a4(-/-) mice, and possibly in humans, lacking functional pendrin expression.     

Developmental expression of otoconin-22 in the bullfrog endolymphatic sac and inner ear.

In amphibians, calcium carbonate crystals are present in the endolymphatic sac and the inner ear. The formation of these crystals is considered to be facilitated by a protein called otoconin-22. We examined the spatial and temporal expression of otoconin-22 during the development of the bullfrog (Rana catesbeiana) using RT-PCR, in situ hybridization (ISH), and immunofluorescence techniques. By RT-PCR, otoconin-22 mRNA was first detected in embryos at Shumway stage 20, and this expression pattern continues in late stages. The first otoconin-22 mRNA-positive reaction was detected in stage 22 embryos in the placode of the endolymphatic sac. Otoconin-22 protein was observed in the epithelial cells of the endolymphatic sac at stage 24. On the other hand, a whole-mount ISH technique showed the first expression of otoconin-22 mRNA in the inner ear, in addition to the endolymphatic sac, at the mid-phase of Shumway stage 25. We discuss the role of otoconin-22 in the formation of calcium carbonate crystals in the endolymphatic sac and inner ear.     

Renal cortical and medullary blood flow responses to L-NAME and ANG II in wild-type, nNOS null mutant, and eNOS null mutant mice

Experiments in wild-type (WT; C57BL/6J) mice, endothelial nitric oxide synthase null mutant [eNOS(-/-)] mice, and neuronal NOS null mutant [nNOS(-/-)] mice were performed to determine which NOS isoform regulates renal cortical and medullary blood flow under basal conditions and during the infusion of ANG II. Inhibition of NOS with N(omega)-nitro-l-arginine methyl ester (l-NAME; 50 mg/kg iv) in Inactin-anesthetized WT and nNOS(-/-) mice increased arterial blood pressure by 28-31 mmHg and significantly decreased blood flow in the renal cortex (18-24%) and the renal medulla (13-18%). In contrast, blood pressure and renal cortical and medullary blood flow were unaltered after l-NAME administration to eNOS(-/-) mice, indicating that NO derived from eNOS regulates baseline vascular resistance in mice. In subsequent experiments, intravenous ANG II (20 ng x kg(-1) x min(-1)) significantly decreased renal cortical blood flow (by 15-25%) in WT, eNOS(-/-), nNOS(-/-), and WT mice treated with l-NAME. The infusion of ANG II, however, led to a significant increase in medullary blood flow (12-15%) in WT and eNOS(-/-) mice. The increase in medullary blood flow following ANG II infusion was not observed in nNOS(-/-) mice, in WT or eNOS(-/-) mice pretreated with l-NAME, or in WT mice administered the nNOS inhibitor 5-(1-imino-3-butenyl)-l-ornithine (1 mg x kg(-1) x h(-1)). These data demonstrate that NO from eNOS regulates baseline blood flow in the mouse renal cortex and medulla, while NO produced by nNOS mediates an increase in medullary blood flow in response to ANG II.     

Anxiety and increased 5-HT1A receptor response in NCAM null mutant mice.

Mice deficient in the neural cell adhesion molecule (NCAM) show behavioral abnormalities as adults, including altered exploratory behavior, deficits in spatial learning, and increased intermale aggression. Here, we report increased anxiety-like behavior of homozygous (NCAM-/-) and heterozygous (NCAM/-) mutant mice in a light/dark avoidance test, independent of genetic background and gender. Anxiety-like behavior was reduced in both NCAM+/+ and NCAM-/- mice by systemic administration of the benzodiazepine agonist diazepam and the 5-HT1A receptor agonists buspirone and 8-OH-DPAT. However, NCAM-/- mice showed anxiolytic-like effects at lower doses of buspirone and 8-OH-DPAT than NCAM+/+ mice. Such increased response to 5-HT1A receptor stimulation suggests a functional change in the serotonergic system of NCAM-/- mice, likely involved in the control of anxiety and aggression. However, 5-HT1A receptor binding and tissue content of serotonin and its metabolite 5-hydroxyindolacetic acid were found unaltered in every brain area of NCAM-/- mice investigated, indicating that expression of 5-HT1A receptors as well as synthesis and release of serotonin are largely unchanged in NCAM-/- mice. We hypothesize a critical involvement of endogenous NCAM in serotonergic transmission via 5-HT1A receptors and inwardly rectifying K+ channels as the respective effector systems.     

Proliferation failure and gamma radiation sensitivity of Fen1 null mutant mice at the blastocyst stage

Flap endonuclease 1 (FEN1) has been shown to remove 5' overhanging flap intermediates during base excision repair and to process the 5' ends of Okazaki fragments during lagging-strand DNA replication in vitro. To assess the in vivo role of the mammalian enzyme in repair and replication, we used a gene-targeting approach to generate mice lacking a functional Fen1 gene. Heterozygote animals appear normal, whereas complete depletion of FEN1 causes early embryonic lethality. Fen1(-/-) blastocysts fail to form inner cell mass during cellular outgrowth, and a complete inactivation of DNA synthesis in giant cells of blastocyst outgrowth was observed. Exposure of Fen1(-/-) blastocysts to gamma radiation caused extensive apoptosis, implying an essential role for FEN1 in the repair of radiation-induced DNA damage in vivo. Our data thus provide in vivo evidence for an essential function of FEN1 in DNA repair, as well as in DNA replication.     

Interferon expression in the testes of transgenic mice leads to sterility

A plasmid containing the mouse interferon-alpha 1 gene under control of the mouse metallothionein-I promoter was used for the construction of transgenic mice. Four transgenic mice (two males and two females) were obtained containing 1 to over 10 copies of the introduced DNA. Both males appeared to be sterile. One of the female mice founded a transgenic strain in which the foreign DNA was transmitted to her offspring in a Mendelian fashion. In this strain most male animals are sterile or turn sterile with time. Northern blot analysis of several tissues of these animals shows that expression of the introduced interferon gene occurs only in the testis. In some of the animals biologically active interferon could also be detected in testes homogenates. Histological examination of testis tissue shows an ongoing degeneration of spermatogenic cells leading to calcium deposits and complete atrophy of the seminiferous tubules.     

Endocrine expression of the active form of TGF-beta1 in the TGF-beta1 null mice fails to ameliorate lethal phenotype

TGF-beta1 null mice die by 3 to 4 weeks of age due to a severe autoimmune-mediated multifocal inflammation resulting in multi-organ failure. To assess the therapeutic potential of circulating levels of active TGF-beta1, we generated mice with endocrine expression of active TGF-beta1 on a TGF-beta1 null background (TGF-beta1 (-/-/TG)) by crossing TGF-beta1(+/-) mice with transgenic mice (TG) that express recombinant TGF-beta1 specifically in the liver and secrete it in the blood. The TGF-beta1 (-/-/TG) mice exhibit a survival profile similar to the TGF-beta1 (-/-) mice indicating a failure to rescue the lethal phenotype. However, serum TGF-beta1 levels in the TGF-beta1 (-/-/TG) mice were restored to near normal levels with expression in all the tissues, notably in the kidney and spleen. Histopathology showed reduced inflammation in the target tissues, especially in the heart. Interestingly, unlike TGF-beta1 (-/-) mice, the TGF-beta1 (-/-/TG) mice have glomerulonephritis in their kidneys similar to the TG mice. Thus, the phenotype of TGF-beta1 (-/-/TG) animal model indicates the potential role of circulating active-TGF-beta1 in reducing inflammation, but its failure to rescue lethality in TGF-beta1 null mice indicates a critical autocrine role of TGF-beta1.     

Disruption of segmental neural crest migration and ephrin expression in delta-1 null mice

Neural crest cells migrate segmentally through the rostral half of each trunk somite due to inhibitory influences of ephrins and other molecules present in the caudal-half of somites. To examine the potential role of Notch/Delta signaling in establishing the segmental distribution of ephrins, we examined neural crest migration and ephrin expression in Delta-1 mutant mice. Using Sox-10 as a marker, we noted that neural crest cells moved through both rostral and caudal halves of the somites in mutants, consistent with the finding that ephrinB2 levels are significantly reduced in the caudal-half somites. Later, mutant embryos had aberrantly fused and/or reduced dorsal root and sympathetic ganglia, with a marked diminution in peripheral glia. These results show that Delta-1 is essential for proper migration and differentiation of neural crest cells. Interestingly, absence of Delta-1 leads to diminution of both neurons and glia in peripheral ganglia, suggesting a general depletion of the ganglion precursor pool in mutant mice.     

Leptin deficiency leads to the regulation of kinin receptors expression in mice

Kinins are vasoactive and pro-inflammatory peptides generated by the cleavage of the kininogen by kallikreins. Two kinin receptors have been described and denominated B1 and B2. Obesity frequently accompanies other pathologies, such as diabetes and hypertention. The clustering of these pathologies is usually known as "metabolic syndrome". Mice lacking leptin gene (ob/ob) are severely obese and hyperphagic. Using quantitative RT-PCR analysis of B1 and B2 mRNAs expression, we described for the first time a correlation between the kallikrein-kinin system (KKS) and severe obesity in mice. The ob/ob mice presented lower expression of B2 mRNA in the white adipose tissue (WAT) and hypothalamus, both primary sites for neuroendocrine regulation of the energetic metabolism. B1 mRNA, however, is overexpressed in these tissues of ob/ob mice. An upregulation of the B1 mRNA has also been seen in liver, abdominal aorta and stomach fundus. However, different from the lean mice, the expression of the B1 mRNA in brown adipose tissue (BAT) and heart is completely abolished. Our data show that kinin receptors are differently modulated in distinct tissues in obesity. These findings suggest a connection between the KKS and obesity, and suggest that kinin receptors could be involved in the ethiopathogenesis of the metabolic syndrome.     

Anxiety and increased 5‐HT1A receptor response in NCAM null mutant mice

Mice deficient in the neural cell adhesion molecule (NCAM) show behavioral abnormalities as adults, including altered exploratory behavior, deficits in spatial learning, and increased intermale aggression. Here, we report increased anxiety‐like behavior of homozygous (NCAM^−/−) and heterozygous (NCAM^+/−) mutant mice in a light/dark avoidance test, independent of genetic background and gender. Anxiety‐like behavior was reduced in both NCAM^+/+ and NCAM^−/− mice by systemic administration of the benzodiazepine agonist diazepam and the 5‐HT_1A receptor agonists buspirone and 8‐OH‐DPAT. However, NCAM^−/− mice showed anxiolytic‐like effects at lower doses of buspirone and 8‐OH‐DPAT than NCAM^+/+ mice. Such increased response to 5‐HT_1A receptor stimulation suggests a functional change in the serotonergic system of NCAM^−/− mice, likely involved in the control of anxiety and aggression. However, 5‐HT_1A receptor binding and tissue content of serotonin and its metabolite 5‐hydroxyindolacetic acid were found unaltered in every brain area of NCAM^−/− mice investigated, indicating that expression of 5‐HT_1A receptors as well as synthesis and release of serotonin are largely unchanged in NCAM^−/− mice. We hypothesize a critical involvement of endogenous NCAM in serotonergic transmission via 5‐HT_1A receptors and inwardly rectifying K⁺ channels as the respective effector systems. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 343–355, 1999     

Mammary gland development in transforming growth factor beta1 null mutant mice: systemic and epithelial effects

The cytokine-transforming growth factor beta1 (TGFB1) is implicated in development of the mammary gland through regulation of epithelial cell proliferation and differentiation during puberty and pregnancy. We compared mammary gland morphogenesis in virgin Tgfb1(+/+), Tgfb1(+/-), and Tgfb1(-/-) mice and transplanted Tgfb1(+/+) and Tgfb1(-/-) epithelium to determine the impact of TGFB1 deficiency on development. When mammary gland tissue was evaluated relative to the timing of puberty, invasion through the mammary fat pad of the ductal epithelium progressed similarly, irrespective of genotype, albeit fewer terminal end buds were observed in mammary glands from Tgfb1(-/-) mice. The terminal end buds appeared to be normal morphologically, and a comparable amount of epithelial proliferation was evident. When transplanted into wild-type recipients, however, Tgfb1(-/-) epithelium showed accelerated invasion compared with Tgfb1(+/+) epithelium. This suggests that the normal rate of ductal extension in Tgfb1(-/-) null mutant mice is the net result of impaired endocrine or paracrine support acting to limit the consequences of unrestrained epithelial growth. By adulthood, mammary glands in cycling virgin Tgfb1(-/-) mice were morphologically similar to those in Tgfb1(+/+) and Tgfb1(+/-) animals, with a normal branching pattern, and the tissue differentiated into early alveolar structures in the diestrous phase of the ovarian cycle. Transplanted mammary gland epithelium showed a similar extent of ductal branching and evidence of secretory differentiation of luminal cells in pregnancy. These results reveal two opposing actions of TGFB1 during pubertal mammary gland morphogenesis: autocrine inhibition of epithelial ductal growth, and endocrine or paracrine stimulation of epithelial ductal growth.     

Hair cells in the inner ear of the pirouette and shaker 2 mutant mice

The shaker 2 (sh2) and pirouette (pi) mouse mutants display severe inner ear dysfunction that involves both auditory and vestibular manifestation. Pathology of the stereocilia of hair cells has been found in both mutants. This study was designed to further our knowledge of the pathological characteristics of the inner ear sensory epithelia in both the sh2 and pi strains. Measurements of auditory brainstem responses indicated that both mutants were profoundly deaf. The morphological assays were specifically designed to characterize a pathological actin bundle that is found in both the inner hair cells and the vestibular hair cells in all five vestibular organs in these two mutants. Using light microscope analysis of phalloidin-stained specimens, these actin bundles could first be detected on postnatal day 3. As the cochleae matured, each inner hair cell and type I vestibular hair cell contained a bundle that spans from the region of the cuticular plate to the basal end of the cell, then extends along with cytoplasm and membrane, towards the basement membrane. Abnormal contact with the basement membrane was found in vestibular hair cells. Based on the shape of the cellular extension and the actin bundle that supports it, we propose to name these extensions “cytocauds.” The data suggest that the cytocauds in type I vestibular hair cells and inner hair cells are associated with a failure to differentiate and detach from the basement membrane.     

Lack of endothelial diaphragms in fenestrae and caveolae of mutant Plvap-deficient mice

Plasmalemmal vesicle-associated protein (PLVAP, PV-1) is specifically expressed in endothelial cells in which it localizes to diaphragms of fenestrae, caveolae, and transendothelial channels. To learn about its function, we generated mutant mice that lack PLVAP. In a C57BL/6N genetic background, homozygous Plvap-deficient embryos die before birth and suffer from subcutaneous edema, hemorrhages, and defects in the vascular wall of subcutaneous capillaries. In addition, hearts of Plvap ^?/? embryos show ventricular septal defects and thinner ventricular walls. In wild-type embryos, PLVAP and caveolae with a stomatal diaphragm are present in endothelial cells of subcutaneous capillaries and endocardium, while a diaphragm is missing in caveolae of Plvap ^?/? littermates. Plvap ^?/? mice in a mixed C57BL/6N/FVB-N genetic background are born and survive at the most for 4?weeks. Capillaries of exocrine and endocrine pancreas and of kidney peritubular interstitium were investigated in more detail as examples of fenestrated capillaries. In these vascular beds, Plvap ^?/? mice show a complete absence of diaphragms in fenestrae, caveolae, and transendothelial channels, findings which are associated with a substantial decrease in the number of endothelial fenestrae. The changes in the capillary phenotype correlate with a considerable retardation of postnatal growth and anemia. Plvap ^?/? mice provide an animal model to clarify the specific functional role of endothelial fenestrae and their contribution to passage of water and solutes in different organs.