Probes for INS

Deficiency of vascular endothelial growth factor A (VEGF) has been associated with severe craniofacial anomalies in both humans and mice. Cranial neural crest cell (NCC)-derived VEGF regulates proliferation, vascularization and ossification of cartilage and membranous bone. However, the function of VEGF derived from specific subpopulations of NCCs in controlling unique aspects of craniofacial morphogenesis is not clear. In this study a conditional knockdown strategy was used to genetically delete Vegfa expression in Osterix (Osx) and collagen II (Col2)-expressing NCC descendants. No major defects in calvaria and mandibular morphogenesis were observed upon knockdown of VEGF in the Col2+ cell population. In contrast, loss of VEGF in Osx+ osteoblast progenitor cells led to reduced ossification of calvarial and mandibular bones without affecting the formation of cartilage templates in newborn mice. The early stages of ossification in the developing jaw revealed decreased initial mineralization levels and a reduced thickness of the collagen I (Col1)-positive bone template upon loss of VEGF in Osx+ precursors. Increased numbers of proliferating cells were detected within the jaw mesenchyme of mutant embryos. Explant culture assays revealed that mandibular osteogenesis occurred independently of paracrine VEGF action and vascular development. Reduced VEGF expression in mandibles coincided with increased phospho-Smad1/5 (P-Smad1/5) levels and bone morphogenetic protein 2 (Bmp2) expression in the jaw mesenchyme. We conclude that VEGF derived from Osx+ osteoblast progenitor cells is required for optimal ossification of developing mandibular bones and modulates mechanisms controlling BMP-dependent specification and expansion of the jaw mesenchyme.

Stressful life events induce abnormalities in emotional and cognitive behaviour. The endogenous opioid system plays an essential role in stress adaptation and coping strategies. In particular, the µ-opioid receptor (μR), one of the major opioid receptors, strongly influences memory processing in that alterations in μR signalling are associated with various neuropsychiatric disorders. However, it remains unclear whether μR signalling contributes to memory impairments induced by acute stress. Here, we utilized pharmacological methods and cell-type-selective/non-cell-type-selective μR depletion approaches combined with behavioural tests, biochemical analyses, and in vitro electrophysiological recordings to investigate the role of hippocampal μR signalling in memory-retrieval impairment induced by acute elevated platform (EP) stress in mice. Biochemical and molecular analyses revealed that hippocampal μRs were significantly activated during acute stress. Blockage of hippocampal μRs, non-selective deletion of μRs or selective deletion of μRs on GABAergic neurons (μRGABA) reversed EP-stress-induced impairment of memory retrieval, with no effect on the elevation of serum corticosterone after stress. Electrophysiological results demonstrated that stress depressed hippocampal GABAergic synaptic transmission to CA1 pyramidal neurons, thereby leading to excitation/inhibition (E/I) imbalance in a μRGABA-dependent manner. Pharmaceutically enhancing hippocampal GABAAreceptor-mediated inhibitory currents in stressed mice restored their memory retrieval, whereas inhibiting those currents in the unstressed mice mimicked the stress-induced impairment of memory retrieval. Our findings reveal a novel pathway in which endogenous opioids recruited by acute stress predominantly activate μRGABA to depress GABAergic inhibitory effects on CA1 pyramidal neurons, which subsequently alters the E/I balance in the hippocampus and results in impairment of memory retrieval.

PPARγ is one of the three isoforms of the Peroxisome Proliferator-Activated Receptors (PPARs). PPARγ is activated by thiazolidinediones such as pioglitazone, and it is targeted to treat insulin resistance. PPARγ is densely expressed in brain areas involved in regulation of motivational and emotional processes.Here, we investigated the role of PPARγ in the brain and explored its role in anxiety and stress responses in mice. The results show that stimulation of PPARγ by pioglitazone did not affect basal anxiety but fully prevented the anxiogenic effect of acute stress. Using mice with genetic ablation of neuronal PPARγ (PPARγNestinCre), we demonstrated that a lack of receptors, specifically in neurons, exacerbated basal anxiety and enhanced stress sensitivity. The administration of GW9662, a selective PPARγ antagonist, elicited a marked anxiogenic response in PPARγ wild-type (Wt) but not in PPARγNestinCre KO mice. Using c-Fos immunohistochemistry we observed that acute stress exposure resulted in a different pattern of neuronal activation in the amygdala and the hippocampus of PPARγNestinCre KO mice compared with Wt mice. No differences were found between Wt and KO mice in hypothalamic regions responsible for hormonal response to stress, nor in blood corticosterone levels. Microinjection of pioglitazone, into the amygdala but not into the hippocampus abolished the anxiogenic response elicited by acute stress. Results also showed that in both regions PPARγ co-localizes with GABAergic cells. These findings demonstrate that neuronal PPARγ is involved the regulation of the stress response, and that the amygdala is a key substrate for the anxiolytic effect of PPARγ.

SIGNIFICANCE STATEMENT:

PPARγ is a classical target for antidiabetic therapies with thiazolidinedione compounds. PPARγ agonists, such as rosiglitazone and pioglitazone, are in clinical use for the treatment of insulin resistance. PPARγ has recently attracted attention for its involvement in the regulation of CNS immune response and functions. Here, we demonstrate that neuronal PPARγ activation prevented the negative emotional effects of stress and exerted anxiolytic actions without influencing HPA axis function. Conversely, pharmacological blockade or genetic deletion of PPARγ enhanced anxiogenic responses and increased vulnerability to stress. These effects appear to be controlled by PPARγ neuronal-mediated mechanisms in the amygdala.

Microvascular endothelial cells (EC) display a high degree of phenotypic and functional heterogeneity among different organs. Organ-specific EC control their tissue microenvironment by angiocrine factors in health and disease. Liver sinusoidal EC (LSEC) are uniquely differentiated to fulfil important organ-specific functions in development, under homeostatic conditions, and in regeneration and liver pathology. Recently, Bmp2 has been identified by us as an organ-specific angiokine derived from LSEC. To study angiocrine Bmp2 signaling in the liver, we conditionally deleted Bmp2 in LSEC using EC subtype-specific Stab2-Cre mice. Genetic inactivation of hepatic angiocrine Bmp2 signaling in Stab2-Cre;Bmp2fl/fl(Bmp2LSECKO) mice caused massive iron overload in the liver, and increased serum iron levels and iron deposition in several organs similar to classic hereditary hemochromatosis. Iron overload was mediated by decreased hepatic expression of hepcidin, a key regulator of iron homeostasis. Thus, angiocrine Bmp2 signaling within the hepatic vascular niche represents a constitutive pathway indispensable for iron homeostasis in vivo that is non-redundant with Bmp6. Notably, we demonstrate that organ-specific angiocrine signaling is essential not only for the homeostasis of the respective organ, but also for the homeostasis of the whole organism.

Microvascular endothelial cells (ECs) display a high degree of phenotypic and functional heterogeneity among different organs. Organ-specific ECs control their tissue microenvironment by angiocrine factors in health and disease. Liver sinusoidal endothelial cells (LSECs) are uniquely differentiated to fulfill important organ-specific functions in development, under homeostatic conditions, and in regeneration and liver pathology. Recently, Bmp2 has been identified by us as an organ-specific angiokine derived from LSECs. To study angiocrine Bmp2 signaling in the liver, we conditionally deleted Bmp2 in LSECs using EC subtype-specific Stab2-Cre mice. Genetic inactivation of hepatic angiocrine Bmp2 signaling in Stab2-Cre;Bmp2fl/fl (Bmp2LSECKO) mice caused massive iron overload in the liver and increased serum iron levels and iron deposition in several organs similar to classic hereditary hemochromatosis. Iron overload was mediated by decreased hepatic expression of hepcidin, a key regulator of iron homeostasis. Thus, angiocrine Bmp2 signaling within the hepatic vascular niche represents a constitutive pathway indispensable for iron homeostasis in vivo that is nonredundant with Bmp6. Notably, we demonstrate that organ-specific angiocrine signaling is essential not only for the homeostasis of the respective organ but also for the homeostasis of the whole organism.

SHP2 is a ubiquitously expressed protein tyrosine phosphatase, which is involved in many signaling pathways to regulate the skeletal development. In endochondral ossification, SHP2 is known to modify the osteogenic fate of osteochondroprogenitors and to impair the osteoblastic transdifferentiation of hypertrophic chondrocytes. However, how SHP2 regulates osteoblast differentiation in intramembranous ossification remains incompletely understood. To address this question, we generated a mouse model to ablate SHP2 in the Prrx1-expressing mesenchymal progenitors by using "Cre-loxP"-mediated gene excision and examined the development of calvarial bone, in which the main process of bone formation is intramembranous ossification. Phenotypic characterization showed that SHP2 mutants have severe defects in calvarial bone formation. Cell lineage tracing and in situ hybridization data showed less osteoblast differentiation of mesenchymal cells and reduced osteogenic genes expression, respectively. Further mechanistic studies revealed enhanced TGFβ and suppressed BMP2 signaling in SHP2 ablated mesenchymal progenitors and their derivatives. Our study uncovered the critical role of SHP2 in osteoblast differentiation through intramembranous ossification and might provide a potential target to treat craniofacial skeleton disorders.