tissue-specific-mirna-expression-profiling-mouse-heart-sections-using

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using <em>In Situ</em> Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

University College London

Gonadotropin-releasing hormone (GnRH) neurons are neuroendocrine cells that are born in the nasal placode during embryonic development and migrate through the nose and forebrain to the hypothalamus, where they regulate reproduction. Many molecular pathways that guide their migration have been identified, but little is known about the factors that control the survival of the migrating GnRH neurons as they negotiate different environments. We previously reported that the class 3 semaphorin SEMA3A signals through its neuropilin receptors, NRP1 and NRP2, to organise the axons that guide migrating GnRH neurons from their birthplace into the brain. By combining analysis of genetically altered mice with in vitro models, we show here that the alternative neuropilin ligand VEGF164 promotes the survival of migrating GnRH neurons by co-activating the ERK and AKT signalling pathways through NRP1. We also demonstrate that survival signalling relies on neuronal, but not endothelial, NRP1 expression and that it occurs independently of KDR, the main VEGF receptor in blood vessels. Therefore, VEGF164 provides survival signals directly to developing GnRH neurons, independently of its role in blood vessels. Finally, we show that the VEGF164-mediated neuronal survival and SEMA3A-mediated axon guidance cooperate to ensure that migrating GnRH neurons reach the brain. Thus, the loss of both neuropilin ligands leads to an almost complete failure to establish the GnRH neuron system.

VEGF signalling controls GnRH neuron survival via NRP1 independently of KDR and blood vessels.

Gonadotropin-releasing hormone (GnRH) neurons are born in the nasal placode and migrate along olfactory and vomeronasal axons to reach the forebrain and settle in the hypothalamus, where they control reproduction. The molecular cues that guide their migration have not been fully identified, but are thought to control either cell movement directly or the patterning of their axonal substrates. Using genetically altered mouse models we show that the migration of GnRH neurons is directly modulated by Slit2 and Robo3, members of the axon guidance Slit ligand and Robo receptor families. Mice lacking Slit2 or Robo3 have a reduced number of GnRH neurons in the forebrain, but a normal complement of their supporting axons, pointing to a direct role for these molecules in GnRH neuron migration.

Slit2 and Robo3 modulate the migration of GnRH-secreting neurons.

En route to the neocortex, interneurons migrate around and avoid the developing striatum. This is due to the chemorepulsive cues of class 3 semaphorins (Sema3A and Sema3F) acting through neuropilin and plexin co-receptors expressed in interneurons. In a recent genetic screen aimed at identifying novel components that may play a role in interneuron migration, we identified LIM-kinase 2 (Limk2), a kinase previously shown to be involved in cell movement and in Sema7A-PlexinC1 signalling. Here we show that Limk2 is differentially expressed in interneurons, with a higher expression in the subpallium compared to cortex, suggesting it may play a role in their migration through the subpallium. Chemotactic assays, carried out with small interfering RNAs (siRNAs), revealed that Limk2-siRNA transfected interneurons are less responsive to Sema3A, but respond to Sema3F. Lack of responsiveness to Sema3A resulted in their aberrant invasion of the developing striatum, as demonstrated in brain slice preparations and in in utero electroporated mouse embryos with the same siRNAs. Our results reveal a previously unknown role for Limk2 in interneuron migration and Sema3A signalling.

Limk2 mediates semaphorin signalling in cortical interneurons migrating through the subpallium.

Gonadotropin-releasing hormone (GnRH) neurons are neuroendocrine cells, located in the hypothalamus, that play an essential role in mammalian reproduction. These neurons originate in the nasal placode and migrate during embryonic development, in association with olfactory/vomeronasal nerves, first in the nose, then through the cribriform plate to enter the forebrain, before settling in the hypothalamus. One of the molecules required for their early migration in the nose is the chemokine CXCL12, which is expressed in the embryonic nasal mesenchyme in an increasing ventral to dorsal gradient, presumably guiding GnRH neurons toward the forebrain. Mice lacking CXCR4, the receptor for CXCL12, exhibit defective GnRH cell movement and a significant reduction in their number, suggesting that CXCL12/CXCR4 signaling is important in the migration and survival of these neurons. Here, we investigated the role of the more recently identified second CXCL12 receptor, CXCR7, in GnRH neuron development. We demonstrate that CXCR7 is expressed along the migratory path of GnRH neurons in the nasal cavity and, although not expressed by GnRH neurons, it affects their migration as indicated by the ectopic accumulation of these cells in the nasal compartment in CXCR7(-/-) mice. Absence of CXCR7 caused abnormal accumulation of CXCL12-RFP at CXCR4-positive sites in the nasal area of CXCL12-RFP-transgenic mice and excessive CXCL12-dependent intracellular clustering of CXCR4 in GnRH neurons, suggesting internalization. These findings imply that CXCR7 regulates CXCL12 availability by acting as a scavenger along the migratory path of GnRH neurons and, thus, influences the migration of these cells in a noncell-autonomous manner. 

CXC chemokine receptor 7 (CXCR7) affects the migration of GnRH neurons by regulating CXCL12 availability.

Cortical interneurons are generated predominantly in the medial ganglionic eminence of the ventral telencephalon and migrate to the cortex during embryonic development. These cells express neuropilin (Nrp1 and Nrp2) receptors which mediate their response to the chemorepulsive class 3 semaphorin (Sema) ligands. We show here that semaphorins Sema3A and Sema3F are expressed in layers adjacent to cortical interneuron migratory streams as well as in the striatum, suggesting they may have a role in guiding these cells throughout their journey. Analysis of Sema3A  and Sema3F  mice during corticogenesis showed that absence of Sema3A, but not Sema3F, leads to aberrant migration of cortical interneurons through the striatum. Reduced number of cortical interneurons was found in the cortex of Sema3A , Nrp1  and Nrp2  mice, as well as altered distribution in Sema3F , Nrp1 , Nrp2  animals and especially in neuropilin double mutants. The observed decrease in interneurons in Sema3A  and Nrp1  mice was due to altered proliferative activity of their progenitors highlighted by changes in their mitotic spindle positioning and angle of cleavage plane during cell division. These findings point to a novel role for Sema3A-Nrp1 signalling in progenitor cell dynamics and in the generation of interneurons in the ventral telencephalon.

Semaphorin3A-neuropilin1 signalling is involved in the generation of cortical interneurons.

Sonic Hedgehog (Shh) plays an instrumental role in brain development, fine-tuning processes such as cell proliferation, patterning, and fate specification. Although, mutations in the SHH pathway in humans are associated with various neurodevelopmental disorders, ranging from holoprosencephaly to schizophrenia, its expression pattern in the developing human brain is not well established. We now determined the previously not reported wide expression of SHH in the human fetal cerebral cortex during most of the gestation period (10-40 gestational weeks). This spatiotemporal distribution puts Shh in a position to influence the fundamental processes involved in corticogenesis. SHH expression increased during development, shifting from progenitor cells in the proliferative zones to neurons, both glutamatergic and GABAergic, and astrocytes in upper cortical compartments. Importantly, the expression of its downstream effectors and complementary receptors revealed evolutionary differences in SHH-pathway gene expression between humans and rodents.

Multiple roles of Sonic Hedgehog in the developing human cortex are suggested by its widespread distribution.

Vascular endothelial growth factor (Vegfa) is essential for promoting the vascularization of the embryonic murine forebrain. In addition, it directly influences neural development, although its role in the forming forebrain is less well elucidated. It was recently suggested that Vegfa may influence the development of GABAergic interneurons, inhibitory cells with crucial signaling roles in cortical neuronal circuits. However, the mechanism by which it affects interneuron development remains unknown. Here we investigated the developmental processes by which Vegfa may influence cortical interneuron development by analyzing transgenic mice that ubiquitously express the Vegfa120 isoform to perturb its signaling gradient. We found that interneurons reach the dorsal cortex at mid phases of corticogenesis despite an aberrant vascular network. Instead, endothelial ablation of Vegfa alters cortical interneuron numbers, their intracortical distribution and spatial proximity to blood vessels. We show for the first time that vascular-secreted guidance factors promote early-migrating interneurons in the intact forebrain in vivo and identify a novel role for vascular-Vegfa in this process.

Fani Memi

Department of Cell and Developmental Biology

University College London

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization