Inflammatory Mediator TAK1 Regulates Hair Follicle Morphogenesis and Anagen Induction Shown by Using Keratinocyte-Specific TAK1-Deficient Mice

Transforming growth factor-β-activated kinase 1 (TAK1) is a member of the NF-κB pathway and regulates inflammatory responses. We previously showed that TAK1 also regulates keratinocyte growth, differentiation, and apoptosis. However, it is unknown whether TAK1 has any role in epithelial–mesenchymal interactions. To examine this possibility, we studied the role of TAK1 in mouse hair follicle development and cycling as an instructive model system. By comparing keratinocyte-specific TAK1-deficient mice (Map3k7 ^fl/flK5-Cre) with control mice, we found that the number of hair germs (hair follicles precursors) in Map3k7 ^fl/flK5-Cre mice was significantly reduced at E15.5, and that subsequent hair follicle morphogenesis was retarded. Next, we analyzed the role of TAK1 in the cyclic remodeling in follicles by analyzing hair cycle progression in mice with a tamoxifen-inducible keratinocyte-specific TAK1 deficiency (Map3k7 ^fl/flK14-Cre-ER^T2). After active hair growth (anagen) was induced by depilation, TAK1 was deleted by topical tamoxifen application. This resulted in significantly retarded anagen development in TAK1-deficient mice. Deletion of TAK1 in hair follicles that were already in anagen induced premature, apoptosis-driven hair follicle regression, along with hair follicle damage. These studies provide the first evidence that the inflammatory mediator TAK1 regulates hair follicle induction and morphogenesis, and is required for anagen induction and anagen maintenance.     

PROX1: A Lineage Tracer for Cortical Interneurons Originating in the Lateral/Caudal Ganglionic Eminence and Preoptic Area

The homeobox-encoding gene Prox1 and its Drosophila homologue prospero are key regulators of cell fate-specification. In the developing rodent cortex a sparse population of cells thought to correspond to late-generated cortical pyramidal neuron precursors expresses PROX1. Using a series of transgenic mice that mark cell lineages in the subcortical telencephalon and, more specifically, different populations of cortical interneurons, we demonstrate that neurons expressing PROX1 do not represent pyramidal neurons or their precursors but are instead subsets of cortical interneurons. These correspond to interneurons originating in the lateral/caudal ganglionic eminence (LGE/CGE) and a small number of preoptic area (POA)-derived neurons. Expression within the cortex can be detected from late embryonic stages onwards when cortical interneurons are still migrating. There is persistent expression in postmitotic cells in the mature brain mainly in the outer cortical layers. PROX1^+ve interneurons express neurochemical markers such as calretinin, neuropeptide Y, reelin and vasoactive intestinal peptide, all of which are enriched in LGE/CGE- and some POA-derived cells. Unlike in the cortex, in the striatum PROX1 marks nearly all interneurons regardless of their origin. Weak expression of PROX1 can also be detected in oligodendrocyte lineage cells throughout the forebrain. Our data show that PROX1 can be used as a genetic lineage tracer of nearly all LGE/CGE- and subsets POA-derived cortical interneurons at all developmental and postnatal stages in vivo.     

Regulation of iNOS function and cellular redox state by macrophage Gch1 reveals specific requirements for tetrahydrobiopterin in NRF2 activation

Inducible nitric oxide synthase (iNOS) is a key enzyme in the macrophage inflammatory response, which is the source of nitric oxide (NO) that is potently induced in response to proinflammatory stimuli. However, the specific role of NO production, as distinct from iNOS induction, in macrophage inflammatory responses remains unproven. We have generated a novel mouse model with conditional deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme in the biosynthesis of tetrahydrobiopterin (BH4) that is a required cofactor for iNOS NO production. Mice with a floxed Gch1 allele (Gch1^fl/fl) were crossed with Tie2cre transgenic mice, causing Gch1 deletion in leukocytes (Gch1^fl/flTie2cre). Macrophages from Gch1^fl/flTie2cre mice lacked GTPCH protein and de novo biopterin biosynthesis. When activated with LPS and IFNγ, macrophages from Gch1^fl/flTie2cre mice induced iNOS protein in a manner indistinguishable from wild-type controls, but produced no detectable NO, as judged by L-citrulline production, EPR spin trapping of NO, and by nitrite accumulation. Incubation of Gch1^fl/flTie2cre macrophages with dihydroethidium revealed significantly increased production of superoxide in the presence of iNOS expression, and an iNOS-independent, BH4-dependent increase in other ROS species. Normal BH4 levels, nitric oxide production, and cellular redox state were restored by sepiapterin, a precursor of BH4 production by the salvage pathway, demonstrating that the effects of BH4 deficiency were reversible. Gch1^fl/flTie2cre macrophages showed only minor alterations in cytokine production and normal cell migration, and minimal changes in basal gene expression. However, gene expression analysis after iNOS induction identified 78 genes that were altered between wild-type and Gch1^fl/flTie2cre macrophages. Pathway analysis identified decreased NRF2 activation, with reduced induction of archetypal NRF2 genes (gclm, prdx1, gsta3, nqo1, and catalase) in BH4-deficient Gch1^fl/flTie2cre macrophages. These findings identify BH4-dependent iNOS regulation and NO generation as specific requirements for NRF2-dependent responses in macrophage inflammatory activation.     

Noncanonical Transforming Growth Factor β (TGFβ) Signaling in Cranial Neural Crest Cells Causes Tongue Muscle Developmental Defects

Microglossia is a congenital birth defect in humans and adversely impacts quality of life. In vertebrates, tongue muscle derives from the cranial mesoderm, whereas tendons and connective tissues in the craniofacial region originate from cranial neural crest (CNC) cells. Loss of transforming growth factor β (TGFβ) type II receptor in CNC cells in mice (Tgfbr2^fl/fl;Wnt1-Cre) causes microglossia due to a failure of cell-cell communication between cranial mesoderm and CNC cells during tongue development. However, it is still unclear how TGFβ signaling in CNC cells regulates the fate of mesoderm-derived myoblasts during tongue development. Here we show that activation of the cytoplasmic and nuclear tyrosine kinase 1 (ABL1) cascade in Tgfbr2^fl/fl;Wnt1-Cre mice results in a failure of CNC-derived cell differentiation followed by a disruption of TGFβ-mediated induction of growth factors and reduction of myogenic cell proliferation and differentiation activities. Among the affected growth factors, the addition of fibroblast growth factor 4 (FGF4) and neutralizing antibody for follistatin (FST; an antagonist of bone morphogenetic protein (BMP)) could most efficiently restore cell proliferation, differentiation, and organization of muscle cells in the tongue of Tgfbr2^fl/fl;Wnt1-Cre mice. Thus, our data indicate that CNC-derived fibroblasts regulate the fate of mesoderm-derived myoblasts through TGFβ-mediated regulation of FGF and BMP signaling during tongue development.     

The role of TGF-beta signaling in regulating chondrogenesis and osteogenesis during mandibular development

During craniofacial development, Meckel's cartilage and the mandible bone derive from the first branchial arch, and their development depends upon the contribution of cranial neural crest (CNC) cells. We previously demonstrated that conditional inactivation of Tgfbr2 in the neural crest of mice (Tgfbr2^fl/fl;Wnt1-Cre) results in severe defects in mandibular development, although the specific cellular and molecular mechanisms by which TGF-β signaling regulates the fate of CNC cells during mandibular development remain unknown. We show here that loss of Tgfbr2 does not affect the migration of CNC cells during mandibular development. TGF-β signaling is specifically required for cell proliferation in Meckel's cartilage and the mandibular anlagen and for the formation of the coronoid, condyle and angular processes. TGF-β-mediated connective tissue growth factor (CTGF) signaling is critical for CNC cell proliferation. Exogenous CTGF rescues the cell proliferation defect in Meckel's cartilage of Tgfbr2^fl/fl;Wnt1-Cre mutants, demonstrating the biological significance of this signaling cascade in chondrogenesis during mandibular development. Furthermore, TGF-β signaling controls Msx1 expression to regulate mandibular osteogenesis as Msx1 expression is significantly reduced in Tgfbr2^fl/fl;Wnt1-Cre mutants. Collectively, our data suggest that there are differential signal cascades in response to TGF-β to control chondrogenesis and osteogenesis during mandibular development.     

Deletion of Fgfr1 in Osteoblasts Enhances Mobilization of EPCs into Peripheral Blood in a Mouse Endotoxemia Model

Endothelial progenitor cells (EPCs) contribute to neovascularization and vascular repair, and may exert a beneficial effect on the clinical outcome of sepsis. Osteoblasts act as a component of “niche” in bone marrow, which provides a nest for stem/progenitor cells and are involved in the formation and maintenance of stem/progenitor cells. Fibroblast growth factor receptor 1 (FGFR1) can regulate osteoblast activity and influence bone mass. So we explored the role of FGFR1 in EPC mobilization. Male mice with osteoblast-specific knockout of Fgfr1 (Fgfr1^fl/fl;OC-Cre) and its wild-type littermates (Fgfr1^fl/fl) were used in this study. Mice intraperitoneally injected with lipopolysaccharide (LPS) were used to measure the number of circulating EPCs in peripheral blood and serum stromal cell-derived factor 1α (SDF-1α). The circulating EPC number and the serum level of SDF-1α were significantly higher in Fgfr1^fl/fl;OC-Cre mice than those in Fgfr1^fl/fl mice after LPS injection. In cell culture system, SDF-1α level was also significantly higher in Fgfr1^fl/fl;OC-Cre osteoblasts compared with that in Fgfr1^fl/fl osteoblasts after LPS treatment. TRAP staining showed that there was no significant difference between the osteoclast activity of septic Fgfr1^fl/fland Fgfr1^fl/fl;OC-Cre mice. This study suggests that targeted deletion of Fgfr1 in osteoblasts enhances mobilization of EPCs into peripheral blood through up-regulating SDF-1α secretion from osteoblasts.     

Abnormal podocyte TRPML1 channel activity and exosome release in mice with podocyte-specific Asah1 gene deletion

Podocytopathy and associated nephrotic syndrome (NS) have been reported in a knockout mouse strain (Asah1^fl/fl/Podo^Cre) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Ac). However, the pathogenesis of podocytopathy of these mice remains unknown. The present study tested whether exosome release from podocytes is enhanced due to Asah1 gene knockout, which may serve as a pathogenic mechanism switching on podocytopathy and associated NS in Asah1^fl/fl/Podo^Cre mice. We first demonstrated the remarkable elevation of urinary exosome excretion in Asah1^fl/fl/Podo^Cre mice compared with WT/WT mice, which was accompanied by significant Annexin-II (an exosome marker) accumulation in glomeruli of Asah1^fl/fl/Podo^Cre mice, as detected by immunohistochemistry. In cell studies, we also confirmed that Asah1 gene knockout enhanced exosome release in the primary cultures of podocyte isolated from Asah1^fl/fl/Podo^Cre mice compared to WT/WT mice. In the podocytes from Asah1^fl/fl/Podo^Cre mice, the interactions of lysosome and multivesicular body (MVB) were demonstrated to be decreased in comparison with those from their control littermates, suggesting reduced MVB degradation that may lead to increase in exosome release. Given the critical role of transient receptor potential mucolipin 1 (TRPML1) channel in Ca²⁺-dependent lysosome trafficking and consequent lysosome-MVB interaction, we tested whether lysosomal Ca²⁺ release through TRPML1 channels is inhibited in the podocytes of Asah1^fl/fl/Podo^Cre mice. By GCaMP3 Ca²⁺ imaging, it was found that lysosomal Ca²⁺ release through TRPML1 channels was substantially suppressed in podocytes with Asah1 gene deletion. As an Ac product, sphingosine was found to rescue TRPML1 channel activity and thereby recover lysosome-MVB interaction and reduce exosome release of podocytes from Asah1^fl/fl/Podo^Cre mice. Combination of N, N-dimethylsphingosine (DMS), a potent sphingosine kinase inhibitor, and sphingosine significantly inhibited urinary exosome excretion of Asah1^fl/fl/Podo^Cre mice. Moreover, rescue of Aash1 gene expression in podocytes of Asah1^fl/fl/Podo^Cre mice showed normal ceramide metabolism and exosome secretion. Based on these results, we conclude that the normal expression of Ac importantly contributes to the control of TRPML1 channel activity, lysosome-MVB interaction, and consequent exosome release from podocytes. Asah1 gene defect inhibits TRPML1 channel activity and thereby enhances exosome release, which may contribute to the development of podocytopathy and associated NS.     

PKMζ is essential for spinal plasticity underlying the maintenance of persistent pain

Oncostatin M (OSM) is a member of the interleukin-6 cytokine family and regulates eg. gene activation, cell survival, proliferation and differentiation. OSM binds to a receptor complex consisting of the ubiquitously expressed signal transducer gp130 and the ligand binding OSM receptor subunit, which is expressed on a specific subset of primary afferent neurons. In the present study, the effect of OSM on heat nociception was investigated in nociceptor-specific gp130 knock-out (SNS-gp130 ^-/- ) and gp130 floxed (gp130 ^fl/fl ) mice. Subcutaneous injection of pathophysiologically relevant concentrations of OSM into the hind-paw of C57BL6J wild type mice significantly reduced paw withdrawal latencies to heat stimulation. In contrast to gp130 ^fl/fl mice, OSM did not induce heat hypersensitivity in vivo in SNS-gp130 ^-/- mice. OSM applied at the receptive fields of sensory neurons in in vitro skin-nerve preparations showed that OSM significantly increased the discharge rate during a standard ramp-shaped heat stimulus. The capsaicin- and heat-sensitive ion channel TRPV1, expressed on a subpopulation of nociceptive neurons, has been shown to play an important role in inflammation-induced heat hypersensitivity. Stimulation of cultured dorsal root ganglion neurons with OSM resulted in potentiation of capsaicin induced ionic currents. In line with these recordings, mice with a null mutation of the TRPV1 gene did not show any signs of OSM-induced heat hypersensitivity in vivo. The present data suggest that OSM induces thermal hypersensitivity by directly sensitizing nociceptors via OSMR-gp130 receptor mediated potentiation of TRPV1.     

Shp2 in Forebrain Neurons Regulates Synaptic Plasticity,Locomotion, and Memory Formation in Mice

Shp2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) regulates neural cell differentiation. It is also expressed in postmitotic neurons, however, and mutations of Shp2 are associated with clinical syndromes characterized by mental retardation. Here we show that conditional-knockout (cKO) mice lacking Shp2 specifically in postmitotic forebrain neurons manifest abnormal behavior, including hyperactivity. Novelty-induced expression of immediate-early genes and activation of extracellular-signal-regulated kinase (Erk) were attenuated in the cerebral cortex and hippocampus of Shp2 cKO mice, suggestive of reduced neuronal activity. In contrast, ablation of Shp2 enhanced high-K⁺-induced Erk activation in both cultured cortical neurons and synaptosomes, whereas it inhibited that induced by brain-derived growth factor in cultured neurons. Posttetanic potentiation and paired-pulse facilitation were attenuated and enhanced, respectively, in hippocampal slices from Shp2 cKO mice. The mutant mice also manifested transient impairment of memory formation in the Morris water maze. Our data suggest that Shp2 contributes to regulation of Erk activation and synaptic plasticity in postmitotic forebrain neurons and thereby controls locomotor activity and memory formation.     

Characteristics of Multi-Organ Lymphangiectasia Resulting from Temporal Deletion of Calcitonin Receptor-Like Receptor in Adult Mice

Adrenomedullin (AM) and its receptor complexes, calcitonin receptor-like receptor (Calcrl) and receptor activity modifying protein 2/3, are highly expressed in lymphatic endothelial cells and are required for embryonic lymphatic development. To determine the role of Calcrl in adulthood, we used an inducible Cre-loxP system to temporally and ubiquitously delete Calcrl in adult mice. Following tamoxifen injection, Calcrl^fl/fl/CAGGCre-ER™ mice rapidly developed corneal edema and inflammation that was preceded by and persistently associated with dilated corneoscleral lymphatics. Lacteals and submucosal lymphatic capillaries of the intestine were also dilated, while mesenteric collecting lymphatics failed to properly transport chyle after an acute Western Diet, culminating in chronic failure of Calcrl^fl/fl/CAGGCre-ER™ mice to gain weight. Dermal lymphatic capillaries were also dilated and chronic edema challenge confirmed significant and prolonged dermal lymphatic insufficiency. In vivo and in vitro imaging of lymphatics with either genetic or pharmacologic inhibition of AM signaling revealed markedly disorganized lymphatic junctional proteins ZO-1 and VE-cadherin. The maintenance of AM signaling during adulthood is required for preserving normal lymphatic permeability and function. Collectively, these studies reveal a spectrum of lymphatic defects in adult Calcrl^fl/fl/CAGGCre-ER™ mice that closely recapitulate the clinical symptoms of patients with corneal, intestinal and peripheral lymphangiectasia.     

Impaired Clearance of Influenza A Virus in Obese,Leptin Receptor Deficient Mice Is Independent of Leptin Signaling in the Lung Epithelium and Macrophages

Rationale

During the recent H1N1 outbreak, obese patients had worsened lung injury and increased mortality. We used a murine model of influenza A pneumonia to test the hypothesis that leptin receptor deficiency might explain the enhanced mortality in obese patients.

Methods

We infected wild-type, obese mice globally deficient in the leptin receptor (db/db) and non-obese mice with tissue specific deletion of the leptin receptor in the lung epithelium (SPC-Cre/LepR^fl/fl) or macrophages and alveolar type II cells (LysM-Cre/Lepr^fl/fl) with influenza A virus (A/WSN/33 [H1N1]) (500 and 1500 pfu/mouse) and measured mortality, viral clearance and several markers of lung injury severity.

Results

The clearance of influenza A virus from the lungs of mice was impaired in obese mice globally deficient in the leptin receptor (db/db) compared to normal weight wild-type mice. In contrast, non-obese, SP-C-Cre^+/+/LepR^fl/fl and LysM-Cre^+/+/LepR^fl/fl had improved viral clearance after influenza A infection. In obese mice, mortality was increased compared with wild-type mice, while the SP-C-Cre^+/+/LepR^fl/fl and LysM-Cre^+/+/LepR^{fl /fl} mice exhibited improved survival.

Conclusions

Global loss of the leptin receptor results in reduced viral clearance and worse outcomes following influenza A infection. These findings are not the result of the loss of leptin signaling in lung epithelial cells or macrophages. Our results suggest that factors associated with obesity or with leptin signaling in non-myeloid populations such as natural killer and T cells may be associated with worsened outcomes following influenza A infection.     

Efficient neuronal in vitro and in vivo differentiation after immunomagnetic purification of mESC derived neuronal precursors

The cellular heterogeneity that is generated during the differentiation of pluripotent stem cells into specific neural subpopulations represents a major obstacle for experimental and clinical progress. To address this problem we developed an optimized strategy for magnetic isolation of PSA-NCAM positive neuronal precursors from embryonic stem cells (ESCs) derived neuronal cultures. PSA-NCAM enrichment at an early step of the in vitro differentiation process increased the number of ES cell derived neurons and reduced cellular diversity. Gene expression analysis revealed that mainly genes involved in neuronal activity were over-represented after purification. In vitro derived PSA-NCAM⁺ enriched precursors were characterized in vivo through grafting into the forebrain of adult mice. While unsorted control cells 40 days post graft gave rise to a mixed population composed of immature precursors, early postmitotic neurons and glial cells, PSA-NCAM⁺ enriched cells differentiated predominantly into NeuN positive cells. Furthermore, PSA-NCAM enriched population showed efficient migration towards the olfactory bulb after transplantation into the rostral migratory stream of the forebrain neurogenic system. Thus, enrichment of neuronal precursors based on PSA-NCAM expression represents a general and straightforward approach to narrow cellular heterogeneity during neuronal differentiation of pluripotent cells.     

ALK5-Mediated Transforming Growth Factor β Signaling in Neural Crest Cells Controls Craniofacial Muscle Development via Tissue-Tissue Interactions

The development of the craniofacial muscles requires reciprocal interactions with surrounding craniofacial tissues that originate from cranial neural crest cells (CNCCs). However, the molecular mechanism involved in the tissue-tissue interactions between CNCCs and muscle progenitors during craniofacial muscle development is largely unknown. In the current study, we address how CNCCs regulate the development of the tongue and other craniofacial muscles using Wnt1-Cre; Alk5^fl/fl mice, in which loss of Alk5 in CNCCs results in severely disrupted muscle formation. We found that Bmp4 is responsible for reduced proliferation of the myogenic progenitor cells in Wnt1-Cre; Alk5^fl/fl mice during early myogenesis. In addition, Fgf4 and Fgf6 ligands were reduced in Wnt1-Cre; Alk5^fl/fl mice and are critical for differentiation of the myogenic cells. Addition of Bmp4 or Fgf ligands rescues the proliferation and differentiation defects in the craniofacial muscles of Alk5 mutant mice in vitro. Taken together, our results indicate that CNCCs play critical roles in controlling craniofacial myogenic proliferation and differentiation through tissue-tissue interactions.