Hwai Jong Cheng

Complementary gradients in expression and binding of ELF-1 and Mek4 in development of the topographic retinotectal projection map

Topographic maps with a defined spatial ordering of neuronal connections are a key feature of brain organization. Such maps are believed to develop in response to complementary position-specific labels in presynaptic and postsynaptic fields. However, the complementary labeling molecules are not known. In the well-studied visual map of retinal axons projecting to the tectum, the labels are hypothesized to be in gradients, without needing large numbers of cell-specific molecules. We recently cloned ELF-1 as a ligand for Eph family receptors. Here, RNA hybridization shows matching expression gradients for ELF-1 in the tectum and its receptor Mek4 in the retina. Binding activity detected with alkaline phosphatase fusions of ELF-1 and Mek4 also reveals gradients and provides direct evidence for molecular complementarity of gradients in reciprocal fields. ELF-1 and Mek4 may therefore play roles in retinotectal development and have properties predicted of topographic mapping labels.

Disrupted-In-Schizophrenia 1 Regulates Integration of Newly Generated Neurons in the Adult Brain

Adult neurogenesis occurs throughout life in discrete regions of the adult mammalian brain. Little is known about the mechanism governing the sequential developmental process that leads to integration of new neurons from adult neural stem cells into the existing circuitry. Here, we investigated roles of Disrupted-In-Schizophrenia 1 (DISC1), a schizophrenia susceptibility gene, in adult hippocampal neurogenesis. Unexpectedly, downregulation of DISC1 leads to accelerated neuronal integration, resulting in aberrant morphological development and mispositioning of new dentate granule cells in a cell-autonomous fashion. Functionally, newborn neurons with DISC1 knockdown exhibit enhanced excitability and accelerated dendritic development and synapse formation. Furthermore, DISC1 cooperates with its binding partner NDEL1 in regulating adult neurogenesis. Taken together, our study identifies DISC1 as a key regulator that orchestrates the tempo of functional neuronal integration in the adult brain and demonstrates essential roles of a susceptibility gene for major mental illness in neuronal development, including adult neurogenesis.

Topographically Specific Effects of ELF-1 on Retinal Axon Guidance In Vitro and Retinal Axon Mapping In Vivo

Topographic maps, which maintain the spatial order of neurons in the order of their axonal connections, are found throughout the nervous system. In the visual retinotectal projection, ELF-1, a ligand in the tectum, and its receptors in the retina show complementary gradients in expression and binding, indicating they may be positional labels for map development. Here we show that ELF-1 acts as a repellent axon guidance factor in vitro. In vivo, when the tectal ELF-1 pattern is modified by retroviral overexpression, retinal axons avoid ectopic ELF-1 patches and map to abnormally anterior positions. All these effects were seen on axons from temporal but not nasal retina, indicating that ELF-1 could determine nasal versus temporal retinotectal specificity, and providing a direct demonstration of a cell recognition molecule with topographically specific effects on neural map development.

Identification and cloning of ELF-1, a developmentally expressed ligand for the Mek4 and Sek receptor tyrosine kinases

Mek4 and Sek are tyrosine kinases with expression patterns in the mouse embryo that suggest important functions in early development. However, like all Eph family kinases, both were identified as orphan receptors without known ligands. We show that Mek4 and Sek soluble receptor-alkaline phosphatase fusion proteins can be used in a procedure termed RAP in situ to identify regions of ligand expression in the mouse embryo. Based on this spatial information, a cDNA expression library was prepared, and was screened with the fusion proteins to identify Eph ligand family-1 (ELF-1). In cell lines and embryos, ELF-1 is membrane bound by a phosphatidylinositol tail, a feature that may account for unique biological functions. Its sequence is homologous with B61, a ligand for the Eck kinase, defining a family of related ligands. The expression domains of ELF-1, Mek4 and Sek indicate potential roles in embryonic patterning.

Stereotyped Pruning of Long Hippocampal Axon Branches Triggered by Retraction Inducers of the Semaphorin Family

Like naturally occurring neuronal cell death, stereotyped pruning of long axon branches to temporary targets is a widespread regressive phenomenon in the developing mammalian brain that helps sculpt the pattern of neuronal connections. The mechanisms controlling stereotyped pruning are, however, poorly understood. Here, we provide evidence that semaphorins, activating the Plexin-A3 receptor, function as retraction inducers to trigger-stereotyped pruning of specific hippocampal mossy fiber and pyramidal axon branches. Both pruning events are defective in Plexin-A3 mutants, reflecting a cell-autonomous requirement for Plexin-A3. The distribution of mRNAs for Sema3F and Sema3A makes them candidates for triggering the pruning. In vitro, hippocampal neurons respond to semaphorins by retracting axon branches. These results implicate semaphorins as retraction inducers controlling stereotyped pruning in the mammalian brain.

Plexin-A3 Mediates Semaphorin Signaling and Regulates the Development of Hippocampal Axonal Projections

Plexins are receptors implicated in mediating signaling by semaphorins, a family of axonal chemorepellents. The role of specific plexins in mediating semaphorin function in vivo has not, however, yet been examined in vertebrates. Here, we show that plexin-A3 is the most ubiquitously expressed plexin family member within regions of the developing mammalian nervous system known to contain semaphorin-responsive neurons. Using a chimeric receptor construct, we provide evidence that plexin-A3 can transduce a repulsive signal in growth cones in vitro. Analysis of plexin-A3 knockout mice shows that plexin-A3 contributes to Sema3F and Sema3A signaling and that plexin-A3 regulates the development of hippocampal axonal projections in vivo.

Differential Requirement for Plexin-A3 and -A4 in Mediating Responses of Sensory and Sympathetic Neurons to Distinct Class 3 Semaphorins

The class 3 Semaphorins Sema3A and Sema3F are potent axonal repellents that cause repulsion by binding Neuropilin-1 and Neuropilin-2, respectively. Plexins are implicated as signaling coreceptors for the Neuropilins, but the identity of the Plexins that transduce Sema3A and Sema3F responses in vivo is uncertain. Here, we show that Plexin-A3 and -A4 are key determinants of these responses, through analysis of a Plexin-A3/Plexin-A4 double mutant mouse. Sensory and sympathetic neurons from the double mutant are insensitive to Sema3A and Sema3F in vitro, and defects in axonal projections in vivo correspond to those seen in Neuropilin-1 and -2 mutants. Interestingly, we found a differential requirement for these two Plexins: signaling via Neuropilin-1 is mediated principally by Plexin-A4, whereas signaling via Neuropilin-2 is mediated principally by Plexin-A3. Thus, Plexin-A3 and -A4 contribute to the specificity of axonal responses to class 3 Semaphorins.

Development of hippocampal mossy fiber synaptic outputs by new neurons in the adult brain

New neurons are continuously generated in restricted regions of the adult mammalian brain. Although these adult-born neurons have been shown to receive synaptic inputs, little is known about their synaptic outputs. Using retrovirus-mediated birth-dating and labeling in combination with serial section electron microscopic reconstruction, we report that mossy fiber en passant boutons of adult-born dentate granule cells form initial synaptic contacts with CA3 pyramidal cells within 2 weeks after their birth and reach morphologic maturity within 8 weeks in the adult hippocampus. Knockdown of Disrupted-in-Schizophrenia-1 (DISC1) in newborn granule cells leads to defects in axonal targeting and development of synaptic outputs in the adult brain. Together with previous reports of synaptic inputs, these results demonstrate that adult-born neurons are fully integrated into the existing neuronal circuitry. Our results also indicate a role for DISC1 in presynaptic development and may have implications for the etiology of schizophrenia and related mental disorders.

ELF-2, a new member of the Eph ligand family, is segmentally expressed in mouse embryos in the region of the hindbrain and newly forming somites.

The Eph receptors are the largest known family of receptor tyrosine kinases and are notable for distinctive expression patterns in the nervous system and in early vertebrate development. However, all were identified as orphan receptors, and only recently have there been descriptions of a corresponding family of ligands. We describe here a new member of the Eph ligand family, designated ELF-2 (Eph ligand family 2). The cDNA sequence for mouse ELF-2 indicates that it is a transmembrane ligand. It shows closest homology to the other known transmembrane ligand in the family, ELK-L/LERK-2/Cek5-L, with 57% identity in the extracellular domain. There is also striking homology in the cytoplasmic domain, including complete identity of the last 33 amino acids, suggesting intracellular interactions. On cell surfaces, and in a cell-free system, ELF-2 binds to three closely related Eph family receptors, Elk, Cek10 (apparent ortholog of Sek-4 and HEK2), and Cek5 (apparent ortholog of Nuk/Sek-3), all with dissociation constants of approximately 1 nM. In situ hybridization of mouse embryos shows ELF-2 RNA expression in a segmental pattern in the hindbrain region and the segmenting mesoderm. Comparable patterns have been described for Eph family receptors, including Sek-4 and Nuk/Sek-3, suggesting roles for ELF-2 in patterning these regions of the embryo.

Alkaline phosphatase fusions of ligands or receptors as in situ probes for staining of cells, tissues, and embryos.

Publisher Summary Polypeptide ligands and their cell surface receptors bind to one another with high affinity and specificity. These biological properties can be exploited to make affinity probes to detect their cognate ligands or receptors. This approach has been applied for decades, using radiolabeled ligands as probes to detect their receptors. More recently, it has also been found that receptor ectodomains can be used as soluble probes to detect their ligands. When producing soluble receptor or ligand affinity probes, it has been common to produce the probe as a fusion protein with a tag. This can make detection and purification procedures much easier. Two tags that are widely used for this purpose are alkaline phosphatase (AP) or the immunoglobulin Fc region. Both of these tags are dimeric, and are expected to produce a fusion protein with a pair of ligand or receptor moieties facing away from the tag in the same direction. This chapter describes the production of alkaline phosphatase (AP) fusion proteins, and also describes in situ procedures in which these affinity probes are used to detect the distribution of cognate ligands or receptors in tissues or cells.