Functional Diversity of Robo Receptor Immunoglobulin Domains Promotes Distinct Axon Guidance Decisions

SummaryRecognition molecules of the immunoglobulin (Ig) superfamily control axon guidance in the developing nervous system. Ig-like domains are among the most widely represented protein domains in the human genome, and the number of Ig superfamily proteins is strongly correlated with cellular complexity [1]. In Drosophila, three Roundabout (Robo) Ig superfamily receptors respond to their common Slit ligand to regulate axon guidance at the midline: Robo and Robo2 mediate midline repulsion, Robo2 and Robo3 control longitudinal pathway selection, and Robo2 can promote midline crossing [2–5]. How these closely related receptors mediate distinct guidance functions is not understood. We report that the differential functions of Robo2 and Robo3 are specified by their ectodomains and do not reflect differences in cytoplasmic signaling. Functional modularity of Robo2's ectodomain facilitates multiple guidance decisions: Ig1 and Ig3 of Robo2 confer lateral positioning activity, whereas Ig2 confers promidline crossing activity. Robo2's distinct functions are not dependent on greater Slit affinity but are instead due in part to differences in multimerization and receptor-ligand stoichiometry conferred by Robo2's Ig domains. Together, our findings suggest that diverse responses to the Slit guidance cue are imparted by intrinsic structural differences encoded in the extracellular Ig domains of the Robo receptors

Repulsive Axon Guidance Abelson and Enabled Play Opposing Roles Downstream of the Roundabout Receptor

AbstractDrosophila Roundabout (Robo) is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Little is known about the signaling mechanisms which function downstream of Robo to mediate repulsion. Here, we present genetic and biochemical evidence that the Abelson (Abl) tyrosine kinase and its substrate Enabled (Ena) play direct and opposing roles in Robo signal transduction. Genetic interactions support a model in which Abl functions to antagonize Robo signaling, while Ena is required in part for Robo's repulsive output. Both Abl and Ena can directly bind to Robo's cytoplasmic domain. A mutant form of Robo that interferes with Ena binding is partially impaired in Robo function, while a mutation in a conserved cytoplasmic tyrosine that can be phosphorylated by Abl generates a hyperactive Robo receptor

Non-invasive assessment of adrenocortical activity as a measure of stress in giraffe (Giraffa camelopardalis)

BACKGROUND : Numbers of giraffes are declining rapidly in their native habitat. As giraffe research and conservation efforts increase, the demand for more complete measures of the impact of conservation interventions and the effects of captive environments on animal health and welfare have risen. We compared the ability of six different enzyme immunoassays to quantify changes in fecal glucocorticoid metabolites (FGM) resulting from three sources: adrenocorticotropic hormone stimulation test, transport, and time of day that samples were collected. RESULTS : Two male giraffes underwent ACTH injections; all six assays detected FGM increases following injection for Giraffe 1, while only three assays detected FGM increases following injection for Giraffe 2. Consistent with other ruminant species, the two 11-oxoetiocholanolone assays (one for 11,17-dioxoandrostanes and the other for 3α,11-oxo metabolites) measured the most pronounced and prolonged elevation of FGM, while an assay for 3β,11β-diol detected peaks of smaller magnitude and duration. Both of the 11-oxoetiocholanolone assays detected significant FGM increases after transport in Giraffes 3–7, and preliminary data suggest FGM detected by the assay for 11,17-dioxoandrostanes may differ across time of day. CONCLUSIONS : We conclude the assay for 11,17-dioxoandrostanes is the most sensitive assay tested for FGM in giraffes and the assay for FGM with a 5β-3α-ol-11-one structure is also effective. 11-oxoetiocholanolone enzyme immunoassays have now been demonstrated to be successful in a wide variety of ruminant species, providing indirect evidence that 5β-reduction may be a common metabolic pathway for glucocorticoids in ruminants. As FGM peaks were detected in at least some giraffes using all assays tested, giraffes appear to excrete a wide variety of different FGM. The assays validated here will provide a valuable tool for research on the health, welfare, and conservation of giraffes.Additional file 1: Full dataset in Microsoft Excel workbook format.The Association of Friends and Supporters of Goethe University Frankfurt provided financial support for F. Sicks to travel to Vienna to analyze fecal samples and von Opel Hessische Zoostiftung supported a studentship for F. Sicks. One commercial funder [Tierpark Berlin] provided support in the form of salary for F. Sicks during data analysis and preparation of this manuscript. The specific role of this author is articulated in the ‘Author Contributions’ section.http://www.biomedcentral.com/bmcvetresam2016Anatomy and PhysiologyParaclinical Science

Evidence for a Role of srGAP3 in the Positioning of Commissural Axons within the Ventrolateral Funiculus of the Mouse Spinal Cord

Slit-Robo signaling guides commissural axons away from the floor-plate of the spinal cord and into the longitudinal axis after crossing the midline. In this study we have evaluated the role of the Slit-Robo GTPase activating protein 3 (srGAP3) in commissural axon guidance using a knockout (KO) mouse model. Co-immunoprecipitation experiments confirmed that srGAP3 interacts with the Slit receptors Robo1 and Robo2 and immunohistochemistry studies showed that srGAP3 co-localises with Robo1 in the ventral and lateral funiculus and with Robo2 in the lateral funiculus. Stalling axons have been reported in the floor-plate of Slit and Robo mutant spinal cords but our axon tracing experiments revealed no dorsal commissural axon stalling in the floor plate of the srGAP3 KO mouse. Interestingly we observed a significant thickening of the ventral funiculus and a thinning of the lateral funiculus in the srGAP3 KO spinal cord, which has also recently been reported in the Robo2 KO. However, axons in the enlarged ventral funiculus of the srGAP3 KO are Robo1 positive but do not express Robo2, indicating that the thickening of the ventral funiculus in the srGAP3 KO is not a Robo2 mediated effect. We suggest a role for srGAP3 in the lateral positioning of post crossing axons within the ventrolateral funiculus

A model of impaired Langerhans cell maturation associated with HPV induced epithelial hyperplasia.

Funder: National Health and Medical Research CouncilLangerhans cells (LC) are skin-resident antigen-presenting cells that regulate immune responses to epithelial microorganisms. Human papillomavirus (HPV) infection can promote malignant epithelial transformation. As LCs are considered important for controlling HPV infection, we compared the transcriptome of murine LCs from skin transformed by K14E7 oncoprotein and from healthy skin. We identified transcriptome heterogeneity at the single cell level amongst LCs in normal skin, associated with ontogeny, cell cycle, and maturation. We identified a balanced co-existence of immune-stimulatory and immune-inhibitory LC cell states in normal skin that was significantly disturbed in HPV16 E7-transformed skin. Hyperplastic skin was depleted of immune-stimulatory LCs and enriched for LCs with an immune-inhibitory gene signature, and LC-keratinocyte crosstalk was dysregulated. We identified reduced expression of interleukin (IL)-34, a critical molecule for LC homeostasis. Enrichment of an immune-inhibitory LC gene signature and reduced levels of epithelial IL-34 were also found in human HPV-associated cervical epithelial cancers

Requirement of Male-Specific Dosage Compensation in Drosophila Females—Implications of Early X Chromosome Gene Expression

Dosage compensation equates between the sexes the gene dose of sex chromosomes that carry substantially different gene content. In Drosophila, the single male X chromosome is hypertranscribed by approximately two-fold to effect this correction. The key genes are male lethal and appear not to be required in females, or affect their viability. Here, we show these male lethals do in fact have a role in females, and they participate in the very process which will eventually shut down their function—female determination. We find the male dosage compensation complex is required for upregulating transcription of the sex determination master switch, Sex-lethal, an X-linked gene which is specifically activated in females in response to their two X chromosomes. The levels of some X-linked genes are also affected, and some of these genes are used in the process of counting the number of X chromosomes early in development. Our data suggest that before the female state is set, the ground state is male and female X chromosome expression is elevated. Females thus utilize the male dosage compensation process to amplify the signal which determines their fate

Quantification of SLIT-ROBO transcripts in hepatocellular carcinoma reveals two groups of genes with coordinate expression

<p>Abstract</p> <p>Background</p> <p>SLIT-ROBO families of proteins mediate axon pathfinding and their expression is not solely confined to nervous system. Aberrant expression of <it>SLIT-ROBO </it>genes was repeatedly shown in a wide variety of cancers, yet data about their collective behavior in hepatocellular carcinoma (HCC) is missing. Hence, we quantified <it>SLIT-ROBO </it>transcripts in HCC cell lines, and in normal and tumor tissues from liver.</p> <p>Methods</p> <p>Expression of <it>SLIT-ROBO </it>family members was quantified by real-time qRT-PCR in 14 HCC cell lines, 8 normal and 35 tumor tissues from the liver. ANOVA and Pearson's correlation analyses were performed in R environment, and different clinicopathological subgroups were pairwise compared in Minitab. Gene expression matrices of cell lines and tissues were analyzed by Mantel's association test.</p> <p>Results</p> <p>Genewise hierarchical clustering revealed two subgroups with coordinate expression pattern in both the HCC cell lines and tissues: <it>ROBO1</it>, <it>ROBO2</it>, <it>SLIT1 </it>in one cluster, and <it>ROBO4</it>, <it>SLIT2</it>, <it>SLIT3 </it>in the other, respectively. Moreover, <it>SLIT-ROBO </it>expression predicted <it>AFP</it>-dependent subgrouping of HCC cell lines, but not that of liver tissues. <it>ROBO1 </it>and <it>ROBO2 </it>were significantly up-regulated, whereas <it>SLIT3 </it>was significantly down-regulated in cell lines with high-<it>AFP </it>background. When compared to normal liver tissue, <it>ROBO1 </it>was found to be significantly overexpressed, while <it>ROBO4 </it>was down-regulated in HCC. We also observed that <it>ROBO1 </it>and <it>SLIT2 </it>differentiated histopathological subgroups of liver tissues depending on both tumor staging and differentiation status. However, <it>ROBO4 </it>could discriminate poorly differentiated HCC from other subgroups.</p> <p>Conclusion</p> <p>The present study is the first in comprehensive and quantitative evaluation of <it>SLIT-ROBO </it>family gene expression in HCC, and suggests that the expression of <it>SLIT-ROBO </it>genes is regulated in hepatocarcinogenesis. Our results implicate that <it>SLIT-ROBO </it>transcription profile is bi-modular in nature, and that each module shows intrinsic variability. We also provide quantitative evidence for potential use of <it>ROBO1</it>, <it>ROBO4 </it>and <it>SLIT2 </it>for prediction of tumor stage and differentiation status.</p

A requirement for filopodia extension toward Slit during Robo-mediated axon repulsion

Axons navigate long distances through complex 3D environments to interconnect the nervous system during development. Although the precise spatiotemporal effects of most axon guidance cues remain poorly characterized, a prevailing model posits that attractive guidance cues stimulate actin polymerization in neuronal growth cones whereas repulsive cues induce actin disassembly. Contrary to this model, we find that the repulsive guidance cue Slit stimulates the formation and elongation of actin-based filopodia from mouse dorsal root ganglion growth cones. Surprisingly, filopodia form and elongate toward sources of Slit, a response that we find is required for subsequent axonal repulsion away from Slit. Mechanistically, Slit evokes changes in filopodium dynamics by increasing direct binding of its receptor, Robo, to members of the actin-regulatory Ena/VASP family. Perturbing filopodium dynamics pharmacologically or genetically disrupts Slit-mediated repulsion and produces severe axon guidance defects in vivo. Thus, Slit locally stimulates directional filopodial extension, a process that is required for subsequent axonal repulsion downstream of the Robo receptor.National Institutes of Health (U.S.) (Grant F32-CA165700)National Institutes of Health (U.S.) (Grant R01-GM068678)National Institutes of Health (U.S.) (Grant P30-CA014051

Sex and the Single Cell. II. There Is a Time and Place for Sex

In both male and female Drosophila, only a subset of cells have the potential to sexually differentiate, making both males and females mosaics of sexually differentiated and sexually undifferentiated cells