Michael A. Robichaux

A chemical genetic approach reveals distinct EphB signaling mechanisms during brain development.

EphB receptor tyrosine kinases control multiple steps in nervous system development. However, it remains unclear whether EphBs regulate these different developmental processes directly or indirectly. In addition, given that EphBs signal through multiple mechanisms, it has been challenging to define which signaling functions of EphBs regulate particular developmental events. To address these issues, we engineered triple knock-in mice in which the kinase activity of three neuronally expressed EphBs can be rapidly, reversibly and specifically blocked. We found that the tyrosine kinase activity of EphBs was required for axon guidance in vivo. In contrast, EphB-mediated synaptogenesis occurred normally when the kinase activity of EphBs was inhibited, suggesting that EphBs mediate synapse development by an EphB tyrosine kinase–independent mechanism. Taken together, our data indicate that EphBs control axon guidance and synaptogenesis by distinct mechanisms and provide a new mouse model for dissecting EphB function in development and disease.

EphB2 receptor forward signaling controls cortical growth cone collapse via Nck and Pak.

EphB receptors and their ephrinB ligands transduce bidirectional signals that mediate contact-dependent axon guidance primarily by promoting growth cone repulsion. However, how EphB receptor-mediated forward signaling induces axonal repulsion remains poorly understood. Here, we identify Nck and Pak proteins as essential forward signaling components of EphB2-dependent growth cone collapse in cortical neurons. We show that kinase-active EphB2 binds to Pak and promotes growth cone repulsion via Pak kinase activity, Pak-Nck binding, RhoA signaling and endocytosis. However, Paks function in this context appears to be independent of Rac/Cdc42-GTP, consistent with the absence of Rac-GTP production after ephrinB treatment of cortical neurons. Taken together, our findings suggest that ephrinB-activated EphB2 receptors recruit a novel Nck/Pak signaling complex to mediate repulsive cortical growth cone guidance, which may be relevant for EphB forward signaling-dependent axon guidance in vivo.

EphB receptor forward signaling regulates area-specific reciprocal thalamic and cortical axon pathfinding

Significance In this study, we report a critical role of EphB forward signaling in proper thalamocortical axon guidance. Moreover, our findings provide support for the controversial “handshake hypothesis,” which proposed that cofasciculation of specific thalamic and cortical axons controls proper cortical and thalamic interconnectivity. Finally, growing evidence suggests cortical synaptic connectivity and white matter tract development are abnormal in autism. The recent identification of a de novo EPHB2 kinase domain mutation in an individual with autism suggests that a deficit in EphB forward signaling might result in abnormal brain wiring and enhanced risk for autism. In early brain development, ascending thalamocortical axons (TCAs) navigate through the ventral telencephalon (VTel) to reach their target regions in the young cerebral cortex. Descending, deep-layer cortical axons subsequently target appropriate thalamic and subcortical target regions. However, precisely how and when corticothalamic axons (CTAs) identify their appropriate, reciprocal thalamic targets remains unclear. We show here that EphB1 and EphB2 receptors control proper navigation of a subset of TCA and CTA projections through the VTel. We show in vivo that EphB receptor forward signaling and the ephrinB1 ligand are required during the early navigation of L1-CAM+ thalamic fibers in the VTel, and that the misguided thalamic fibers in EphB1/2 KO mice appear to interact with cortical subregion-specific axon populations during reciprocal cortical axon guidance. As such, our findings suggest that descending cortical axons identify specific TCA subpopulations in the dorsal VTel to coordinate reciprocal cortical–thalamic connectivity in the early developing brain.

Signaling Mechanisms of Axon Guidance and Early Synaptogenesis

The development of the vertebrate nervous system, including the brain and spinal cord, progresses in a step-wise fashion that involves the function of thousands of genes. The birth of new neurons (also known as neurogenesis) and their subsequent migration to appropriate locations within the developing brain mark the earliest stages of CNS development. Subsequently, these newborn neurons extend axons and dendrites to make stereotyped synaptic connections within the developing brain, which is a complex process involving cell intrinsic mechanisms that respond to specific extracellular signals. The extension and navigation of the axon to its appropriate target region in the brain and body is dependent upon many cell surface proteins that detect extracellular cues and transduce signals to the inside of the cell. In turn, intracellular signaling mechanisms orchestrate axon structural reorganization and appropriate turning toward or away from a guidance cue. Once the target region is reached, chemical synapses are formed between the axon and target cell, and again, this appears to involve cell surface proteins signaling to the inside of the neuron to stabilize and mature a synapse. Here, we describe some of the key convergent and, in some cases, divergent molecular pathways that regulate axon guidance and synaptogenesis in early brain development. Mutations in genes involved in early brain wiring and synapse formation and pruning increase the risk for developing autism, further highlighting the relevance of brain development factors in the pathophysiology of neurodevelopmental disorders.

Structural and molecular bases of rod photoreceptor morphogenesis and disease

The rod cell has an extraordinarily specialized structure that allows it to carry out its unique function of detecting individual photons of light. Both the structural features of the rod and the metabolic processes required for highly amplified light detection seem to have rendered the rod especially sensitive to structural and metabolic defects, so that a large number of gene defects are primarily associated with rod cell death and give rise to blinding retinal dystrophies. The structures of the rod, especially those of the sensory cilium known as the outer segment, have been the subject of structural, biochemical, and genetic analysis for many years, but the molecular bases for rod morphogenesis and for cell death in rod dystrophies are still poorly understood. Recent developments in imaging technology, such as cryo-electron tomography and super-resolution fluorescence microscopy, in gene sequencing technology, and in gene editing technology are rapidly leading to new breakthroughs in our understanding of these questions. A summary is presented of our current understanding of selected aspects of these questions, highlighting areas of uncertainty and contention as well as recent discoveries that provide new insights. Examples of structural data from emerging imaging technologies are presented.

EphB1 and EphB2 intracellular domains regulate the formation of the corpus callosum and anterior commissure

The two cortical hemispheres of the mammalian forebrain are interconnected by major white matter tracts, including the corpus callosum (CC) and the posterior branch of the anterior commissure (ACp), that bridge the telencephalic midline. We show here that the intracellular signaling domains of the EphB1 and EphB2 receptors are critical for formation of both the ACp and CC. We observe partial and complete agenesis of the corpus callosum, as well as highly penetrant ACp misprojection phenotypes in truncated EphB1/2 mice that lack intracellular signaling domains. Consistent with the roles for these receptors in formation of the CC and ACp, we detect expression of these receptors in multiple brain regions associated with the formation of these forebrain structures. Taken together, our findings suggest that a combination of forward and reverse EphB1/2 receptor‐mediated signaling contribute to ACp and CC axon guidance.

Defining the Layers of a Sensory Cilium with STORM and Cryo-Electron Nanoscopy

Primary cilia are cylindrical organelles extending from the surface of most animal cells that have been implicated in a host of signaling and sensory functions. Genetic defects in their component molecules, known as 9ciliopathies9 give rise to devastating symptoms, ranging from defective development, to kidney disease, to progressive blindness. The detailed structures of these organelles and the true functions of proteins encoded by ciliopathy genes are poorly understood because of the small size of cilia and the limitations of conventional microscopic techniques. We describe the combination of cryo-electron tomography, enhanced by sub-tomogram averaging, with super-resolution stochastic reconstruction microscopy (STORM) to define substructures and subdomains within the light-sensing rod sensory cilium of the mammalian retina. Longitudinal and radial domains are demarcated by structural features such as the axoneme and its connections to the ciliary membrane, and are correlated with molecular markers of these compartments, including Ca 2+ -binding protein centrin-2 in the lumen of the axoneme, acetylated tubulin forming the axoneme, the glycocalyx extending outward from the surface of the plasma membrane, and molecular residents of the space between axoneme and ciliary membrane, including intraflagellar transport proteins, BBS5, and Arl13B. Within this framework we document that deficiencies in the ciliopathy proteins BBS2, BBS4 and BBS7 lead to inappropriate accumulation of proteins in rod outer segments while largely preserving their sub-domain localization within the connecting cilium region.

A Large Endoplasmic Reticulum-Resident Pool of TRPM1 in Retinal ON-Bipolar Cells

Abstract The chemical signal of light onset, a decrease in glutamate release from rod and cone photoreceptors, is processed by a postsynaptic G protein signaling cascade in ON-bipolar cells (BPCs). The metabotropic glutamate receptor mGluR6, along with other cascade elements, is localized synaptically at the BPC dendritic tips. The effector ion channel protein transient receptor potential melastatin-1 (TRPM1), in contrast, is located not only at the dendritic tips but also in BPC bodies and axons. Little is known about the intracellular localization of TRPM1, or its trafficking route to the dendritic tip plasma membrane. Recombinant TRPM1 expressed in mammalian cells colocalized with endoplasmic reticulum (ER) markers, with little or none detected at the plasma membrane. In mouse retina, somatic TRPM1 was similarly intracellular, and not at the plasma membrane. Labeling of ER membranes by expression of a fluorescent marker showed that in BPCs the ER extends into axons and dendrites, but not dendritic tips. In cell bodies, TRPM1 colocalized with the ER, and not with the Golgi apparatus. Fluorescence protease protection (FPP) assays with TRPM1-GFP fusions in heterologous cells revealed that the N and C termini are both accessible to the cytoplasm, consistent with the transmembrane domain topology of related TRP channels. These results indicate that the majority of TRPM1 is present in the ER, from which it can potentially be transported to the dendritic tips as needed for ON light responses. The excess of ER-resident TRPM1 relative to the amount needed at the dendritic tips suggests a potential new function for TRPM1 in the ER.

SPATA7 maintains a novel photoreceptor-specific zone in the distal connecting cilium

Photoreceptor-specific ciliopathies often affect a structure that is considered functionally homologous to the ciliary transition zone (TZ) called the connecting cilium (CC). However, it is unclear how mutations in certain ciliary genes disrupt the photoreceptor CC without impacting the primary cilia systemically. By applying stochastic optical reconstruction microscopy technology in different genetic models, we show that the CC can be partitioned into two regions: the proximal CC (PCC), which is homologous to the TZ of primary cilia, and the distal CC (DCC), a photoreceptor-specific extension of the ciliary TZ. This specialized distal zone of the CC in photoreceptors is maintained by SPATA7, which interacts with other photoreceptor-specific ciliary proteins such as RPGR and RPGRIP1. The absence of Spata7 results in the mislocalization of DCC proteins without affecting the PCC protein complexes. This collapse results in destabilization of the axonemal microtubules, which consequently results in photoreceptor degeneration. These data provide a novel mechanism to explain how genetic disruption of ubiquitously present ciliary proteins exerts tissue-specific ciliopathy phenotypes.