Defective vascular development in connexin 45-deficient mice

In order to reveal the biological function(s) of the gap-junction protein connexin 45 (Cx45), we generated Cx45-deficient mice with targeted replacement of the Cx45-coding region with the lacZ reporter gene. Heterozygous Cx45(+/)(-) mice showed strong expression of the reporter gene in vascular and visceral smooth muscle cells. Cx45-deficient embryos exhibited striking abnormalities in vascular development and died between embryonic day (E) 9.5 and 10.5. Differentiation and positioning of endothelial cells appeared to be normal, but subsequent development of blood vessels revealed impaired formation of vascular trees in the yolk sac, impaired allantoic mesenchymal ingrowth and capillary formation in the labyrinthine part of the placenta, and arrest of arterial growth, including a failure to develop a smooth muscle layer surrounding the major arteries of the embryo proper. As a consequence, the hearts of most Cx45-deficient embryos were dilated. The abnormal development of the vasculature in the yolk sac of Cx45(-)(/)(-) embryos could be caused by defective TGFbeta signalling, as the amount of TGF beta1 protein in the epithelial layer of the yolk sac was largely decreased in the E9.5 Cx45(-)(/)(-) embryo, compared with the wild-type embryo. The defective vascular development was accompanied by massive apoptosis, which began in some embryos at E8.5 and was abundant in virtually all tissues of the embryos at E9.5. We conclude that in Cx45(-)(/)(-) embryos, vasculogenesis was normal, but subsequent transformation into mature vessels was interrupted. Development of different types of vessels was impaired to a varying extent, which possibly reflects the complementation by other connexin(s).     

Immunohistochemical detection of the neuronal connexin36 in the mouse central nervous system in comparison to connexin36-deficient tissues

Investigating the spatial and temporal expression of connexin36 (Cx36) protein in neuronal tissue is of prime importance to understand the molecular mechanisms underlying extensive electrical coupling. Although Cx36 mRNA was shown to be expressed in neurons of the central nervous system in different studies, only the determination of Cx36 protein expression allows a correlation between localization and its functional role in gap junction-mediated neuronal coupling. After the initial use of antibodies recognizing the skate connexin35 protein, antibodies directed to the mammalian Cx36 sequence allowed the detailed investigation of Cx36 cellular localization. However, results on Cx36 protein distribution still remained controversial in some areas of the central nervous system. In the present study, we have investigated: (a) the distribution of Cx36 protein in various areas of the central nervous system and (b) determined the specificity in the immunohistochemical staining of two polyclonal antibodies comparing wildtype and Cx36-deficient mice. In some areas of the central nervous system, for example in the retina and the inferior nuclear olivary complex, Cx36 antibodies were highly specific, and in the cerebellar cortex, Cx36 protein expression was partly specific. In other regions, particularly in pyramidal cells of the hippocampal formation, non-specific staining was prevalent, indicating that Cx36 antibodies also recognize proteins other than Cx36 in these tissues. The present results argue for a re-evaluation of many documented immunohistochemical protein distribution patterns and require, not only in connexin research, their assessment using null-mutant animals.     

Characterization of connexin30.3-deficient mice suggests a possible role of connexin30.3 in olfaction

We have generated connexin30.3-deficient mice in which the coding region of the connexin30.3 gene was replaced by the lacZ reporter gene. The expression pattern of this connexin was characterized using beta-galactosidase staining and immunoblot analyses. In skin, beta-galactosidase/connexin30.3 protein was expressed in the spinous and granulous layers of the epidermis. Specific beta-galactosidase/connexin30.3 expression was also detected in the thin ascending limb of Henle's loop in the kidney. In addition, we found beta-galactosidase/connexin30.3 in progenitor cells of the olfactory epithelium and in a subpopulation of cells in the apical layer of the vomeronasal organ. Connexin30.3-deficient mice were fertile and displayed no abnormalities in the skin or in the chemosensory systems. Furthermore, they showed normal auditory thresholds as measured by brain stem evoked potentials. These mice did, however, exhibit reduced behavioural responses to a vanilla scent.     

Impaired striatal Akt signaling disrupts dopamine homeostasis and increases feeding

Background

The prevalence of obesity has increased dramatically worldwide. The obesity epidemic begs for novel concepts and therapeutic targets that cohesively address “food-abuse” disorders. We demonstrate a molecular link between impairment of a central kinase (Akt) involved in insulin signaling induced by exposure to a high-fat (HF) diet and dysregulation of higher order circuitry involved in feeding. Dopamine (DA) rich brain structures, such as striatum, provide motivation stimuli for feeding. In these central circuitries, DA dysfunction is posited to contribute to obesity pathogenesis. We identified a mechanistic link between metabolic dysregulation and the maladaptive behaviors that potentiate weight gain. Insulin, a hormone in the periphery, also acts centrally to regulate both homeostatic and reward-based HF feeding. It regulates DA homeostasis, in part, by controlling a key element in DA clearance, the DA transporter (DAT). Upon HF feeding, nigro-striatal neurons rapidly develop insulin signaling deficiencies, causing increased HF calorie intake.

Methodology/Principal Findings

We show that consumption of fat-rich food impairs striatal activation of the insulin-activated signaling kinase, Akt. HF-induced Akt impairment, in turn, reduces DAT cell surface expression and function, thereby decreasing DA homeostasis and amphetamine (AMPH)-induced DA efflux. In addition, HF-mediated dysregulation of Akt signaling impairs DA-related behaviors such as (AMPH)-induced locomotion and increased caloric intake. We restored nigro-striatal Akt phosphorylation using recombinant viral vector expression technology. We observed a rescue of DAT expression in HF fed rats, which was associated with a return of locomotor responses to AMPH and normalization of HF diet-induced hyperphagia.

Conclusions/Significance

Acquired disruption of brain insulin action may confer risk for and/or underlie “food-abuse” disorders and the recalcitrance of obesity. This molecular model, thus, explains how even short-term exposure to “the fast food lifestyle” creates a cycle of disordered eating that cements pathological changes in DA signaling leading to weight gain and obesity.     

Characterization of connexin31.1-deficient mice reveals impaired placental development

The gap junction gene Connexin31.1 has been reported to be expressed predominantly in the epidermis of murine skin. To study the function of this gene, we generated mice in which the coding DNA of the Connexin31.1 gene was replaced by lacZ reporter coding DNA. Using β-galactosidase staining, we have shown that lacZ/Connexin31.1 was expressed in the spinous and granular layers of the epidermis, in cells of olfactory epithelium and in the vomeronasal organ. During embryogenesis, Connexin31.1 was co-expressed with another isoform, Connexin31, in the post-implantation trophoblast cell lineage and, later in gestation, in placental glycogen cells. Although homozygous Connexin31.1-deficient mice were fertile and showed no morphological or functional defects in adult organs expressing this gene, 30% of the offspring expected according to Mendelian inheritance were lost between embryonic days 11.5 and 14.5 and surviving embryos were significantly reduced in weight near the end of pregnancy. Placentas of Connexin31.1-deficient embryos were reduced in weight and showed altered morphology of the spongiotrophoblast and labyrinth layer. The spongiotrophoblast formed a compact barrier at the decidual border that might restrict the maternal blood supply. We conclude that Connexin31.1 is critical for normal placental development but appears to be functionally compensated by other connexin isoforms in the embryo proper and adult mouse.     

Plasmodium yoelii sporozoites infect CD36-deficient mice

Sinnis, P. and Febbraio, M. 2001. Plasmodium yoelii sporozoites infect CD36-deficient mice. Experimental Parasitology100, 12-16.     

Impaired long-term potentiation in c-Jun N-terminal kinase 2-deficient mice

c-Jun N-terminal kinases (JNKs) are thought to be involved in regulating synaptic plasticity. We therefore investigated the specific role of JNK2 in modulating long-term potentiation (LTP) in hippocampus during development, using JNK2-deficient mice. The morphological structure and the numbers of both NeuN, a specific neuronal marker, and GABA-positive neurons in the hippocampal areas were similar in wild-type and Jnk2(-/-) mice. Western blot analysis revealed that JNK2 expression was higher and stable at 1 and 3 months of age, but JNK1 levels were lower at 1 month of age and almost undetectable in 3-month-old wild-type mice. In contrast to wild-type mice, there was a significant increase in JNK1 expression in JNK2 mutant mice, especially at 1 month of age. Electrophysiological studies demonstrated that LTP was impaired in both the CA1 and CA3 regions in 1-month-old, but not in adult, Jnk2(-/-) mice, probably owing to decreased presynaptic neurotransmitter release. Moreover, late-phase LTP, but not early-phase LTP, was impaired in the Jnk2(-/-) adult mice, suggesting that JNK2 plays a role in transforming early LTP to late LTP. Together, the data highlight the specific role of JNK2 in hippocampal synaptic plasticity during development.     

ICER-1gamma overexpression drives palmitate-mediated connexin36 down-regulation in insulin-secreting cells

Channels formed by the gap junction protein connexin36 (Cx36) contribute to the proper control of insulin secretion. We investigated the impact of chronic hyperlipidemia on Cx36 expression in pancreatic beta-cells. Prolonged exposure to the saturated free fatty acid palmitate reduced the expression of Cx36 in several insulin-secreting cell lines and isolated mouse islets. The effect of palmitate was fully blocked upon protein kinase A (PKA) inhibition by H89 and (Rp)-cAMP, indicating that the cAMP/PKA pathway is involved in the control of Cx36 expression. Palmitate treatment led to overexpression of the inducible cAMP early repressor (ICER-1gamma), which bound to a functional cAMP-response element located in the promoter of the CX36 gene. Inhibition of ICER-1gamma overexpression prevented the Cx36 decrease, as well as the palmitate-induced beta-cell secretory dysfunction. Finally, freshly isolated islets from mice undergoing a long term high fat diet expressed reduced Cx36 levels and increased ICER-1gamma levels. Taken together, these data demonstrate that chronic exposure to palmitate inhibits the Cx36 expression through PKA-mediated ICER-1gamma overexpression. This Cx36 down-regulation may contribute to the reduced glucose sensitivity and altered insulin secretion observed during the pre-diabetic stage and in the metabolic syndrome.     

Impaired endochondral bone development and osteopenia in Gli2-deficient mice

Mice homozygous for targeted disruption of the zinc finger domain of Gli2 (Gli2(zfd/zfd)) die at birth with developmental defects in several organ systems including the skeleton. The current studies were undertaken to define the role of Gli2 in endochondral bone development by characterizing the molecular defects in the limbs and vertebrae of Gli2(zfd/zfd) mice. The bones of mutant mice removed by cesarian section at E16.5 and E18.5 demonstrated delayed endochondral ossification. This was accompanied by an increase in the length of cartilaginous growth plates, reduced bone tissue in the femur and tibia and by failure to develop the primary ossification centre in vertebral bodies. The growth plates of tibiae and vertebrae exhibited increased numbers of proliferating and hypertrophic chondrocytes with no apparent alteration in matrix mineralisation. The changes in growth plate morphology were accompanied by an increase in expression of FGF2 in proliferating chondrocytes and decreased expression of Indian hedgehog (Ihh), patched (Ptc) and parathyroid-hormone-related protein (PTHrP) in prehypertrophic cells. Furthermore, there was a reduction in expression of angiogenic molecules in hypertrophic chondrocytes, which was accompanied by a decrease in chondroclasts at the cartilage bone interface, fewer osteoblasts lining trabecular surfaces and a reduced volume of metaphyseal bone. These results indicate that functional Gli2 is necessary for normal endochondral bone development and that its absence results in increased proliferation of immature chondrocytes and decreased resorption of mineralised cartilage and bone formation.     

Impaired auditory-vestibular functions and behavioral abnormalities of Slitrk6-deficient mice

A recent study revealed that Slitrk6, a transmembrane protein containing a leucine-rich repeat domain, has a critical role in the development of the inner ear neural circuit. However, it is still unknown how the absence of Slitrk6 affects auditory and vestibular functions. In addition, the role of Slitrk6 in regions of the central nervous system, including the dorsal thalamus, has not been addressed. To understand the physiological role of Slitrk6, Slitrk6-knockout (KO) mice were subjected to systematic behavioral analyses including auditory and vestibular function tests. Compared to wild-type mice, the auditory brainstem response (ABR) of Slitrk6-KO mice indicated a mid-frequency range (8-16 kHz) hearing loss and reduction of the first ABR wave. The auditory startle response was also reduced. A vestibulo-ocular reflex (VOR) test showed decreased vertical (head movement-induced) VOR gains and normal horizontal VOR. In an open field test, locomotor activity was reduced; the tendency to be in the center region was increased, but only in the first 5 min of the test, indicating altered adaptive responses to a novel environment. Altered adaptive responses were also found in a hole-board test in which head-dip behavior was increased and advanced. Aside from these abnormalities, no clear abnormalities were noted in the mood, anxiety, learning, spatial memory, or fear memory-related behavioral tests. These results indicate that the Slitrk6-KO mouse can serve as a model of hereditary sensorineural deafness. Furthermore, the altered responses of Slitrk6-KO mice to the novel environment suggest a role of Slitrk6 in some cognitive functions.     

Impaired protection against Mycobacterium bovis bacillus Calmette-Guerin infection in IL-15-deficient mice

To investigate the potential role of endogenous IL-15 in mycobacterial infection, we examined protective immunity in IL-15-deficient (IL-15(-/-)) mice after infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG) or recombinant OVA-expressing BCG (rBCG-OVA). IL-15(-/-) mice exhibited an impaired protection in the lung on day 120 after BCG infection as assessed by bacterial growth. CD4(+) Th1 response capable of producing IFN-gamma was normally detected in spleen and lung of IL-15(-/-) mice on day 120 after infection. Although Ag-specific CD8 responses capable of producing IFN-gamma and exhibiting cytotoxic activity were detected in the lung on day 21 after infection with rBCG-OVA, the responses were severely impaired on days 70 and 120 in IL-15(-/-) mice. The degree of proliferation of Ag-specific CD8(+) T cells in IL-15(-/-) mice was similar to that in wild-type mice during the course of infection with rBCG-OVA, whereas sensitivity to apoptosis of Ag-specific CD8(+) T cells significantly increased in IL-15(-/-) mice. These results suggest that IL-15 plays an important role in the development of long-lasting protective immunity to BCG infection via sustaining CD8 responses in the lung.     

Impaired cerebral cortex development and blood pressure regulation in FGF-2-deficient mice.

Fibroblast growth factor-2 (FGF-2) has been implicated in various signaling processes which control embryonic growth and differentiation, adult physiology and pathology. To analyze the in vivo functions of this signaling molecule, the FGF-2 gene was inactivated by homologous recombination in mouse embryonic stem cells. FGF-2-deficient mice are viable, but display cerebral cortex defects at birth. Bromodeoxyuridine pulse labeling of embryos showed that proliferation of neuronal progenitors is normal, whereas a fraction of them fail to colonize their target layers in the cerebral cortex. A corresponding reduction in parvalbumin-positive neurons is observed in adult cortical layers. Neuronal defects are not limited to the cerebral cortex, as ectopic parvalbumin-positive neurons are present in the hippocampal commissure and neuronal deficiencies are observed in the cervical spinal cord. Physiological studies showed that FGF-2-deficient adult mice are hypotensive. They respond normally to angiotensin II-induced hypertension, whereas neural regulation of blood pressure by the baroreceptor reflex is impaired. The present genetic study establishes that FGF-2 participates in controlling fates, migration and differentiation of neuronal cells, whereas it is not essential for their proliferation. The observed autonomic dysfunction in FGF-2-deficient adult mice uncovers more general roles in neural development and function.     

Impaired cardiac contractility response to hemodynamic stress in S100A1-deficient mice

Ca(2+) signaling plays a central role in cardiac contractility and adaptation to increased hemodynamic demand. We have generated mice with a targeted deletion of the S100A1 gene coding for the major cardiac isoform of the large multigenic S100 family of EF hand Ca(2+)-binding proteins. S100A1(-/-) mice have normal cardiac function under baseline conditions but have significantly reduced contraction rate and relaxation rate responses to beta-adrenergic stimulation that are associated with a reduced Ca(2+) sensitivity. In S100A1(-/-) mice, basal left-ventricular contractility deteriorated following 3-week pressure overload by thoracic aorta constriction despite a normal adaptive hypertrophy. Surprisingly, heterozygotes also had an impaired response to acute beta-adrenergic stimulation but maintained normal contractility in response to chronic pressure overload that coincided with S100A1 upregulation to wild-type levels. In contrast to other genetic models with impaired cardiac contractility, loss of S100A1 did not lead to cardiac hypertrophy or dilation in aged mice. The data demonstrate that high S100A1 protein levels are essential for the cardiac reserve and adaptation to acute and chronic hemodynamic stress in vivo.     

Impaired germ cell development due to compromised cell cycle progression in Skp2-deficient mice

Background

A high proliferative capacity of tumor cells usually is associated with shortened patient survival. Disruption of the RB pathway, which is critically involved in regulating the G1 to S cell cycle transition, is a frequent target of oncogenic events that are thought to contribute to increased proliferation during tumor progression. Previously, we determined that p18INK4c, an essential gene for normal plasma cell differentiation, was bi-allelically deleted in five of sixteen multiple myeloma (MM) cell lines. The present study was undertaken to investigate a possible role of p18INK4c in increased proliferation of myeloma tumors as they progress.

Results

Thirteen of 40 (33%) human myeloma cell lines do not express normal p18INK4c, with bi-allelic deletion of p18 in twelve, and expression of a mutated p18 fragment in one. Bi-allelic deletion of p18, which appears to be a late progression event, has a prevalence of about 2% in 261 multiple myeloma (MM) tumors, but the prevalence is 6 to10% in the 50 tumors with a high expression-based proliferation index. Paradoxically, 24 of 40 (60%) MM cell lines, and 30 of 50 (60%) MM tumors with a high proliferation index express an increased level of p18 RNA compared to normal bone marrow plasma cells, whereas this occurs in only five of the 151 (3%) MM tumors with a low proliferation index. Tumor progression is often accompanied by increased p18 expression and an increased proliferation index. Retroviral-mediated expression of exogenous p18 results in marked growth inhibition in three MM cell lines that express little or no endogenous p18, but has no effect in another MM cell line that already expresses a high level of p18.

Conclusion

Paradoxically, although loss of p18 appears to contribute to increased proliferation of nearly 10% of MM tumors, most MM cell lines and proliferative MM tumors have increased expression of p18. Apart from a small fraction of cell lines and tumors that have inactivated the RB1 protein, it is not yet clear how other MM cell lines and tumors have become insensitive to the anti-proliferative effects of increased p18 expression.     

Impaired mast cell maturation and degranulation and attenuated allergic responses in Ndrg1-deficient mice

We have previously reported that N-myc downstream regulated gene-1 (NDRG1) is an early inducible protein during the maturation of mouse bone marrow-derived mast cells (BMMCs) toward a connective tissue mast cell-like phenotype. To clarify the function of NDRG1 in mast cells and allergic responses, we herein analyzed mast cell-associated phenotypes of mice lacking the Ndrg1 gene. Allergic responses including IgE-mediated passive systemic and cutaneous anaphylactic reactions were markedly attenuated in Ndrg1-deficient mice as compared with those in wild-type mice. In Ndrg1-deficient mice, dermal and peritoneal mast cells were decreased in number and morphologically abnormal with impaired degranulating ability. Ex vivo, Ndrg1-deficient BMMCs cocultured with Swiss 3T3 fibroblasts in the presence of stem cell factor, a condition that facilitates the maturation of BMMCs toward a CTMC-like phenotype, displayed less exocytosis than replicate wild-type cells after the cross-linking of FcepsilonRI or stimulation with compound 48/80, even though the exocytotic response of IL-3-maintained, immature BMMCs from both genotypes was comparable. Unlike degranulation, the production of leukotriene and cytokines by cocultured BMMCs was unaffected by NDRG1 deficiency. Taken together, the altered phenotypes of Ndrg1-deficient mast cells both in vivo and ex vivo suggest that NDRG1 has roles in the terminal maturation and effector function (degranulation) of mast cells.     

Impaired flow-dependent control of vascular tone and remodeling in P2X4-deficient mice

The structure and function of blood vessels adapt to environmental changes such as physical development and exercise. This phenomenon is based on the ability of the endothelial cells to sense and respond to blood flow; however, the underlying mechanisms remain unclear. Here we show that the ATP-gated P2X4 ion channel, expressed on endothelial cells and encoded by P2rx4 in mice, has a key role in the response of endothelial cells to changes in blood flow. P2rx4(-/-) mice do not have normal endothelial cell responses to flow, such as influx of Ca(2+) and subsequent production of the potent vasodilator nitric oxide (NO). Additionally, vessel dilation induced by acute increases in blood flow is markedly suppressed in P2rx4(-/-) mice. Furthermore, P2rx4(-/-) mice have higher blood pressure and excrete smaller amounts of NO products in their urine than do wild-type mice. Moreover, no adaptive vascular remodeling, that is, a decrease in vessel size in response to a chronic decrease in blood flow, was observed in P2rx4(-/-) mice. Thus, endothelial P2X4 channels are crucial to flow-sensitive mechanisms that regulate blood pressure and vascular remodeling.