Ami Okada

Boc is a receptor for sonic hedgehog in the guidance of commissural axons

In the spinal cord, sonic hedgehog (Shh) is secreted by the floor plate to control the generation of distinct classes of ventral neurons along the dorsoventral axis. Genetic and in vitro studies have shown that Shh also later acts as a midline-derived chemoattractant for commissural axons. However, the receptor(s) responsible for Shh attraction remain unknown. Here we show that two Robo-related proteins, Boc and Cdon, bind specifically to Shh and are therefore candidate receptors for the action of Shh as an axon guidance ligand. Boc is expressed by commissural neurons, and targeted disruption of Boc in mouse results in the misguidance of commissural axons towards the floor plate. RNA-interference-mediated knockdown of Boc impairs the ability of rat commissural axons to turn towards an ectopic source of Shh in vitro. Taken together, these data suggest that Boc is essential as a receptor for Shh in commissural axon guidance.

Imaging cells in the developing nervous system with retrovirus expressing modified green fluorescent protein.

To visualize the movements of cells and their processes in developing vertebrates, we constructed replication-incompetent retroviral vectors encoding green fluorescent protein (GFP) that can be detected as a single integrated copy per cell. To optimize GFP expression, the CMV enhancer and avian beta-actin promoter were incorporated within a retrovirus construct to drive transcription of redshifted (F64L, S65T) and codon-modified GFP (EGFP), EGFP tagged with GAP-43 sequences targeting the GFP to the cell membrane, or EGFP with additional mutations that increase its ability to fold properly at 37 degrees C (S147P or V163A, S175G). We have used these viruses to efficiently mark and follow the developmental progression of a large population of cells in rat neocortex and whole avian embryos. In the chick embryo, the migration and development of GFP-marked neural crest cells were monitored using time-lapse videomicroscopy. In the neocortex, GFP clearly delineates the morphology of a variety of neuronal and glial phenotypes. Cells expressing GFP display normal dendritic morphologies, and infected cells persist into adulthood. Cortical neurons appear to form normal local axonal and long-distance projections, suggesting that the presence of cytoplasmic or GAP-43-tagged GFP does not significantly interfere with normal development.

Sonic Hedgehog Expression in Corticofugal Projection Neurons Directs Cortical Microcircuit Formation

VIDEO ABSTRACT The precise connectivity of inputs and outputs is critical for cerebral cortex function; however, the cellular mechanisms that establish these connections are poorly understood. Here, we show that the secreted molecule Sonic Hedgehog (Shh) is involved in synapse formation of a specific cortical circuit. Shh is expressed in layer V corticofugal projection neurons and the Shh receptor, Brother of CDO (Boc), is expressed in local and callosal projection neurons of layer II/III that synapse onto the subcortical projection neurons. Layer V neurons of mice lacking functional Shh exhibit decreased synapses. Conversely, the loss of functional Boc leads to a reduction in the strength of synaptic connections onto layer Vb, but not layer II/III, pyramidal neurons. These results demonstrate that Shh is expressed in postsynaptic target cells while Boc is expressed in a complementary population of presynaptic input neurons, and they function to guide the formation of cortical microcircuitry.

Accessibility control of variable region gene assembly during T‐cell development

Summary: T‐cell development is a complex and ordered process that is regulated in part by the progressive assembly and expression of antigen receptor genes. T cells can be divided into two lineages based on expression of either an αβ or γδ T‐cell antigen receptor (TCR), The genes that encode the TCR β and y chains lie in distinct loci, whereas the genes that encode the TCR a and S chains he in a single locos (TCR α/δ locus). Assembly of TCR variable region genes is mediated by a site‐specific recombination process that is common among all lymphocytes. Despite the common nature of this process, recombination of TCR genes is tightly regulated within the context of the developing T cell. TCR β, γ and δ variable region genes are assembled prior to TCR α variable region genes. Furthermore, assembly of TCR β variable region genes is regulated within the context of allelic exclusion. The regulation of rearrangement arid expression of genes within the TCR α/δ locus presents a complicated problem. TCR α and δ variable region genes are assembled at different stages of T‐cell development, and fully assembled TCR α and δ variable region genes must be expressed in distinct hneages of T cells, αβ and γδ. respectively We have developed several experimental approaches lo assess the role of cis‐acting elements in regulating recombination and expression of TCR genes. Here we describe these approaches and discuss our analyses of the regulation of accessibility of the TCR β and TCR α/δ foci during T‐cell development.

Developmental expression pattern of the cdo gene.

CDO is a cell‐surface protein of the immunoglobulin/fibronectin type III repeat family that positively regulates myogenic differentiation in vitro. To gain a better understanding of the role of cdo during vertebrate development, we carried out an extensive in situ hybridization study to characterize its expression pattern from postimplantation to late stages of mouse embryogenesis and in rat brain from E13 to adult. Our results show a broad pattern of cdo expression that is spatially and temporally restricted during embryogenesis. In the central nervous system (CNS), cdo expression is detected as early as E7.5 and maintained in the dorsal ventricular zones of the brain and spinal cord, becoming increasingly restricted in the adult. High levels of cdo are detected in developing sensory organs, such as the eye and ear. Outside the CNS, cdo is expressed mainly in neural crest and mesodermal derivatives, including skeletal muscle precursors. Overall, the highest levels of cdo expression are seen from E9.0 to E15.5. The temporal onset and restricted expression of cdo suggest that cdo plays a role in the determination and/or differentiation of a number of cell types during embryogenesis.

Regulated nuclear trafficking of the homeodomain protein otx1 in cortical neurons.

Otx1 is a homeodomain protein required for axon refinement by layer 5 neurons in developing cerebral cortex. Otx1 localizes to the cytoplasm of progenitor cells in the rat ventricular zone, and remains cytoplasmic as neurons migrate and begin to differentiate. Nuclear translocation occurs during the first week of postnatal life, when layer 5 neurons begin pruning their long-distance axonal projections. Deletion analysis reveals that Otx1 is imported actively into cell nuclei, that the N-terminus of Otx1 is necessary for nuclear import, and that a putative nuclear localization sequence within this domain is sufficient to direct nuclear import in a variety of cell lines. In contrast, GFP-Otx1 fusion proteins that contain the N-terminus are retained in the cytoplasm of cortical progenitor cells, mimicking the distribution of Otx1 in vivo. These results suggest that ventricular cells actively sequester Otx1 in the cytoplasm, either by preventing nuclear import or by promoting a balance of export over import signals.

Shh/Boc Signaling Is Required for Sustained Generation of Ipsilateral Projecting Ganglion Cells in the Mouse Retina

Sonic Hedgehog (Shh) signaling is an important determinant of vertebrate retinal ganglion cell (RGC) development. In mice, there are two major RGC populations: (1) the Islet2-expressing contralateral projecting (c)RGCs, which both produce and respond to Shh; and (2) the Zic2-expressing ipsilateral projecting RGCs (iRGCs), which lack Shh expression. In contrast to cRGCs, iRGCs, which are generated in the ventrotemporal crescent (VTC) of the retina, specifically express Boc, a cell adhesion molecule that acts as a high-affinity receptor for Shh. In Boc−/− mutant mice, the ipsilateral projection is significantly decreased. Here, we demonstrate that this phenotype results, at least in part, from the misspecification of a proportion of iRGCs. In Boc−/− VTC, the number of Zic2-positive RGCs is reduced, whereas more Islet2/Shh-positive RGCs are observed, a phenotype also detected in Zic2 and Foxd1 null embryos. Consistent with this observation, organization of retinal projections at the dorsal lateral geniculate nucleus is altered in Boc−/− mice. Analyses of the molecular and cellular consequences of introducing Shh into the developing VTC and Zic2 and Boc into the central retina indicate that Boc expression alone is insufficient to fully activate the ipsilateral program and that Zic2 regulates Shh expression. Taking these data together, we propose that expression of Boc in cells from the VTC is required to sustain Zic2 expression, likely by regulating the levels of Shh signaling from the nearby cRGCs. Zic2, in turn, directly or indirectly, counteracts Shh and Islet2 expression in the VTC and activates the ipsilateral program.

The variable region gene assembly mechanism

Publisher Summary The mechanism responsible for generating diversity of antigen receptors is the assembly of the variable (V) region of each antigen receptor chain by the somatic recombination of variable (V), joining (J), and diversity (D) gene segments. This chapter reviews the current understanding of the mechanism of V (D) J recombination. The somatic recombination or V (D) J recombination occurs during early stages of lymphocyte differentiation, and is associated with the program of lymphocyte development. Because the V, D, and J segments that encode each antigen receptor V region can have several members, a significant level of diversity can be generated from the combinatorial assembly of these segments. Additional diversity of the antigen receptor can also be generated during the somatic recombination event. The junction of the assembled V-region gene segments encodes the third complementarity determining region (CDR3) of the immunoglobulin (Ig) V region that contacts the antigen and an analogous antigen contact region of the T-cell receptor. Thus, features of the recombination machinery that modify the joining segment ends during assembly can significantly contribute to increasing the diversity of the antigen receptor repertoires.