Specificity and structure of a high affinity activin receptor-like kinase 1 (ALK1) signaling complex.

Activin receptor-like kinase 1 (ALK1), an endothelial cell-specific type I receptor of the TGF-ß superfamily, is an important regulator of normal blood vessel development as well as pathological tumor angiogenesis. As such, ALK1 is an important therapeutic target. Thus, several ALK1-directed agents are currently in clinical trials as anti-angiogenic cancer therapeutics. Given the biological and clinical importance of the ALK1 signaling pathway, we sought to elucidate the biophysical and structural basis underlying ALK1 signaling. The TGF-ß family ligands BMP9 and BMP10 as well as the three type II TGF-ß family receptors ActRIIA, ActRIIB, and BMPRII have been implicated in ALK1 signaling. Here, we provide a kinetic and thermodynamic analysis of BMP9 and BMP10 interactions with ALK1 and type II receptors. Our data show that BMP9 displays a significant discrimination in type II receptor binding, whereas BMP10 does not. We also report the crystal structure of a fully assembled ternary complex of BMP9 with the extracellular domains of ALK1 and ActRIIB. The structure reveals that the high specificity of ALK1 for BMP9/10 is determined by a novel orientation of ALK1 with respect to BMP9, which leads to a unique set of receptor-ligand interactions. In addition, the structure explains how BMP9 discriminates between low and high affinity type II receptors. Taken together, our findings provide structural and mechanistic insights into ALK1 signaling that could serve as a basis for novel anti-angiogenic therapies.

Cryosectioning and Immunohistochemistry of Peripheral Tissues of Mosquitoes.

Immunohistochemistry analysis of mosquitoes is complicated by the outer cuticle that prevents reagents from penetrating peripheral tissues. This protocol incorporates a cryosectioning method that provides a higher resolution of the internal architecture of mosquito peripheral sensory tissues and enables the visualization of protein expression. This eliminates the need for enzymatic steps to digest the outer cuticle that encases these tissues. This protocol can also be adapted for other tissues, such as the brain and the legs, as chitin exoskeleton thickness does not affect antibody penetration once the sample is sectioned.

RNA In Situ Hybridization and Immunohistochemistry to Visualize Gene Expression in Peripheral Chemosensory Tissues of Mosquitoes.

Mosquito-borne diseases such as malaria, Zika virus, and dengue virus are a menace to the human population. Although many mosquito species are not attracted to humans and do not feed on blood, human-biting female mosquitoes are strongly attracted to people and use chemosensory cues to identify a suitable host for a blood meal. Mosquitoes need blood components to reproduce, rendering them excellent vectors for blood-borne diseases. The three genera (Culex, Anopheles, and Aedes) responsible for most of these diseases find hosts by using their peripheral sensory organs. These organs include the antennae, maxillary palps, and proboscis. All three contain diverse populations of highly sensitive neurons that express sensory receptors that can detect odorants, temperature, chemicals, and tastants. Although these organs are essential to the host-seeking behavior that results in biting, their small size and thick outer cuticle can hinder typical histochemical analyses. Here, we briefly review the role the peripheral sensory organs play in mosquito behavior. Then, we introduce how to investigate their gene expression profiles using immunohistochemical and RNA in situ approaches for both whole-mount and frozen-section preparations.

Humidity sensors that alert mosquitoes to nearby hosts and egg-laying sites.

To reproduce and to transmit disease, female mosquitoes must obtain blood meals and locate appropriate sites for egg laying (oviposition). While distinct sensory cues drive each behavior, humidity contributes to both. Here, we identify the mosquito's humidity sensors (hygrosensors). Using generalizable approaches designed to simplify genetic analysis in non-traditional model organisms, we demonstrate that the ionotropic receptor Ir93a mediates mosquito hygrosensation as well as thermosensation. We further show that Ir93a-dependent sensors drive human host proximity detection and blood-feeding behavior, consistent with the overlapping short-range heat and humidity gradients these targets generate. After blood feeding, gravid females require Ir93a to seek high humidity associated with preferred egg-laying sites. Reliance on Ir93a-dependent sensors to promote blood feeding and locate potential oviposition sites is shared between the malaria vector Anopheles gambiae and arbovirus vector Aedes aegypti. These Ir93a-dependent systems represent potential targets for efforts to control these human disease vectors.

Mosquito heat seeking is driven by an ancestral cooling receptor.

Mosquitoes transmit pathogens that kill >700,000 people annually. These insects use body heat to locate and feed on warm-blooded hosts, but the molecular basis of such behavior is unknown. Here, we identify ionotropic receptor IR21a, a receptor conserved throughout insects, as a key mediator of heat seeking in the malaria vector Anopheles gambiae Although Ir21a mediates heat avoidance in Drosophila, we find it drives heat seeking and heat-stimulated blood feeding in Anopheles At a cellular level, Ir21a is essential for the detection of cooling, suggesting that during evolution mosquito heat seeking relied on cooling-mediated repulsion. Our data indicate that the evolution of blood feeding in Anopheles involves repurposing an ancestral thermoreceptor from non-blood-feeding Diptera.

Ctip1 Controls Acquisition of Sensory Area Identity and Establishment of Sensory Input Fields in the Developing Neocortex.

While transcriptional controls over the size and relative position of cortical areas have been identified, less is known about regulators that direct acquisition of area-specific characteristics. Here, we report that the transcription factor Ctip1 functions in primary sensory areas to repress motor and activate sensory programs of gene expression, enabling establishment of sharp molecular boundaries defining functional areas. In Ctip1 mutants, abnormal gene expression leads to aberrantly motorized corticocortical and corticofugal output connectivity. Ctip1 critically regulates differentiation of layer IV neurons, and selective loss of Ctip1 in cortex deprives thalamocortical axons of their receptive "sensory field" in layer IV, which normally provides a tangentially and radially defined compartment of dedicated synaptic territory. Therefore, although thalamocortical axons invade appropriate cortical regions, they are unable to organize into properly configured sensory maps. Together, these data identify Ctip1 as a critical control over sensory area development.

Some like it hot, but not too hot.

A temperature-sensitive receptor prevents mosquitoes from being attracted to targets that are hotter than a potential host.