Crystal structure of an ephrin ectodomain.

Eph receptor tyrosine kinases and their membrane-associated ligands, the ephrins, are essential regulators of axon guidance, cell migration, segmentation, and angiogenesis. There are two classes of vertebrate ephrin ligands which have distinct binding specificities for their cognate receptors. Multimerization of the ligands is required for receptor activation, and ephrin ligands themselves signal intracellularly upon binding Eph receptors. We have determined the structure of the extracellular domain of mouse ephrin-B2. The ephrin ectodomain is an eight-stranded beta barrel with topological similarity to plant nodulins and phytocyanins. Based on the structure, we have identified potential surface determinants of Eph/ephrin binding specificity and a ligand dimerization region. The high sequence similarity among ephrin ectodomains indicates that all ephrins may be modeled upon the ephrin-B2 structure presented here.

An olfactory sensory map develops in the absence of normal projection neurons or GABAergic interneurons.

Olfactory sensory neurons expressing a given odorant receptor project to two topographically fixed glomeruli in the olfactory bulb. We have examined the contribution of different cell types in the olfactory bulb to the establishment of this topographic map. Mice with a homozygous deficiency in Tbr-1 lack most projection neurons, whereas mice with a homozygous deficiency in Dlx-1 and Dlx-2 lack most GABAergic interneurons. Mice bearing a P2-IRES-tau-lacZ allele and deficient in either Tbr-1 or Dlx-1/Dlx-2 reveal the convergence of axons to one medial and one lateral site at positions analogous to those observed in wild-type mice. These observations suggest that the establishment of a topographic map is not dependent upon cues provided by, or synapse formation with, the major neuronal cell types in the olfactory bulb.

Architecture of a mammalian glomerular domain revealed by novel volume electroporation using nanoengineered microelectrodes.

Dense microcircuit reconstruction techniques have begun to provide ultrafine insight into the architecture of small-scale networks. However, identifying the totality of cells belonging to such neuronal modules, the "inputs" and "outputs," remains a major challenge. Here, we present the development of nanoengineered electroporation microelectrodes (NEMs) for comprehensive manipulation of a substantial volume of neuronal tissue. Combining finite element modeling and focused ion beam milling, NEMs permit substantially higher stimulation intensities compared to conventional glass capillaries, allowing for larger volumes configurable to the geometry of the target circuit. We apply NEMs to achieve near-complete labeling of the neuronal network associated with a genetically identified olfactory glomerulus. This allows us to detect sparse higher-order features of the wiring architecture that are inaccessible to statistical labeling approaches. Thus, NEM labeling provides crucial complementary information to dense circuit reconstruction techniques. Relying solely on targeting an electrode to the region of interest and passive biophysical properties largely common across cell types, this can easily be employed anywhere in the CNS.

The genetics of visual system development in Drosophila: specification, connectivity and asymmetry.

Encoding visual information requires a complex neuronal network. Recently, genes regulating early tissue specification, the growth of retinal target structures, the connectivity of photoreceptor axons, and mirror-image retinal symmetry in Drosophila have been identified. The insights gained from studying visual system development in flies promise to inform our understanding of similar processes in vertebrates.

A methionine aminopeptidase and putative regulator of translation initiation is required for cell growth and patterning in Drosophila.

We have isolated mutations in the gene Drosophila methionine aminopeptidase 2 (DMAP2), which encodes a homolog of the type 2 methionine aminopeptidase from yeast, also known as the eukaryotic initiation factor 2alpha (eIF2alpha) associated protein p67. Weak DMAP2 mutations cause ommatidial rotation defects and loss of ventral tissue in the compound eye as well as extra wing veins, whereas stronger alleles impair tissue growth. These limited phenotypes, in conjunction with the differential accumulation of DMAP2 transcripts throughout embryonic and larval development, suggest that a subset of proteins is spatially and temporally regulated at the level of post-translational processing or translation initiation during development. These results provide genetic evidence for post-transcriptional control in the development of multicellular organisms.

In vivo functional analysis of Drosophila Gap1: involvement of Ca2+ and IP4 regulation.

Control of Ras activity is crucial for normal cellular behavior such as fate determination during development. Although several GTPase activating proteins (GAPs) have been shown to act as negative regulators of Ras, the mechanisms involved in regulating their activity in vivo are poorly understood. Here we report the structural requirements for Gap1 activity in cone cell fate decisions during Drosophila eye development. The Gap1 catalytic domain alone is not sufficient for in vivo activity, indicating a requirement for the additional domains. An inositol-1,3,4, 5-tetrakisphosphate (IP4)-sensitive extended PH domain is essential for Gap1 activity, while Ca2+-sensitive C2 domains and a glutamine-rich region contribute equally to full activity in vivo. Furthermore, we find a strong positive genetic interaction between Gap1 and phospholipase Cgamma (PLCgamma), an enzyme which generates inositol-1,4,5-trisphosphate, a precursor for IP4 and a second messenger for intracellular Ca2+ release. These results suggest that Gap1 activity in vivo is stimulated under conditions of elevated intracellular Ca2+ and IP4. Since receptor tyrosine kinases (RTKs) trigger an increase in intracellular Ca2+ and IP4 concentration through stimulation of PLCgamma, RTKs may stimulate not only activation of Ras but also its deactivation by Gap1, thereby moderating the strength and duration of the Ras signal.

Mutations in Hsp83 and cdc37 impair signaling by the sevenless receptor tyrosine kinase in Drosophila.

A highly conserved signal cascade functions subsequent to receptor tyrosine kinase activation. Signaling by the sevenless receptor, required for differentiation of the R7 photoreceptor neuron in Drosophila, is reduced by mutations in E(sev)3A and E(sev)3B. We show here that E(sev)3A is a member of the Hsp90 family of stress proteins and that E(sev)3B encodes a homolog of the cell cycle control protein Cdc37 from S. cerevisiae. Mutations in E(sev)3B also dominantly enhance mutations in Dmcdc2, the gene encoding the p34 protein kinase that regulates the G2/M transition. Together, these data support a role for Hsp90 proteins in tyrosine kinase regulation and suggest that signals promoting neuronal differentiation may involve cell cycle control.

Determination of chirality of alcohol or latent alcohol semiochemicals in individual insects.

A method is described for determining the enantiomeric composition of chiral alcohols, lactones, and hydroxy acids in quantities ranging from 25 ng to 10 µg. Derivatization of the substance with chirally pure acetyl lactate, followed by splitless capillary gas chromatography, enables enantiomeric determinations to be made within 1-3% of the actual value. This technique was applied in the determination of semiochemical inIps pini (Say),Apis mellifera (L.), andCryptolestes ferrugineus (Stephens). The results indicate that considerable variability exists within populations of some insects in the composition of their chiral semiochemicals, whereas others produce substances of constant composition.

Genomic analysis of orthologous mouse and human olfactory receptor loci.

Olfactory receptor (OR) genes represent approximately 1% of genomic coding sequence in mammals, and these genes are clustered on multiple chromosomes in both the mouse and human genomes. We have taken a comparative genomics approach to identify features that may be involved in the dynamic evolution of this gene family and in the transcriptional control that results in a single OR gene expressed per olfactory neuron. We sequenced approximately 350 kb of the murine P2 OR cluster and used synteny, gene linkage, and phylogenetic analysis to identify and sequence approximately 111 kb of an orthologous cluster in the human genome. In total, 18 mouse and 8 human OR genes were identified, including 7 orthologs that appear to be functional in both species. Noncoding homology is evident between orthologs and generally is confined within the transcriptional unit. We find no evidence for common regulatory features shared among paralogs, and promoter regions generally do not contain strong promoter motifs. We discuss these observations, as well as OR clustering, in the context of evolutionary expansion and transcriptional regulation of OR repertoires.

Complex phenotype of mice homozygous for a null mutation in the Sp4 transcription factor gene.

BACKGROUND: Sp4 is a zinc finger transcription factor which is closely related to Sp1 and Sp3. All three proteins recognize the same DNA elements and can act as transcriptional activators through glutamine-rich activation domains. Unlike Sp1 and Sp3, which are ubiquitous proteins, Sp4 is highly abundant in the central nervous system, but also detectable in many other tissues. RESULTS: We have disrupted the mouse Sp4 gene by a targeted deletion of the exons encoding the N-terminal activation domains. Sp4 knockout mice show a complete absence of Sp4 expression. They develop until birth without obvious abnormalities. After birth, two-thirds die within 4 weeks. Surviving mice are growth retarded. Male Sp4null mice do not breed. The cause for the breeding defect remains obscure since they show complete spermatogenesis. In addition, pheromone receptor genes in the vomeronasal organ appear unaffected. Female Sp4null mice have a smaller thymus, spleen and uterus. In addition, they exhibit a pronounced delay in sexual maturation. CONCLUSIONS: The phenotype of the Sp4null mice differs significantly from those described for Sp1-/- and Sp3-/- mice. Thus, the structural similarities, the common recognition motif and the overlapping expression pattern of these three transcription factors do not reflect similar physiological functions.