Between Slit and Repulsion: Cell and Molecular Mechanisms Underlying Robo-Mediated Midline Guidance

Understanding how axon guidance receptors are activated by their extracellular ligands to regulate growth cone motility is critical to learning how proper wiring is established during development. Roundabout (Robo) is one such guidance receptor that mediates repulsion from its ligand Slit in both invertebrates and vertebrates. Here we show that endocytic trafficking of the Robo receptor in response to Slit-binding is necessary for its repulsive signaling output. Dose-dependent genetic interactions and in vitro Robo activation assays support a role for Clathrin-dependent endocytosis, and entry into both the early and late endosomes as positive regulators of Slit-Robo signaling. We identify two conserved motifs in Robo\u27s cytoplasmic domain that are required for its Clathrin-dependent endocytosis and activation in vitro, and gain of function and genetic rescue experiments provide strong evidence that these trafficking events are required for Robo repulsive guidance activity in vivo. Our data support a model in which Robo\u27s ligand-dependent internalization from the cell surface to the late endosome is essential for receptor activation and proper repulsive guidance at the midline by allowing recruitment of the downstream effector Son of Sevenless in a spatially constrained endocytic trafficking compartment. We then go on to provide evidence for the placement of Robo endocytosis after the previously reported kuzbanian-mediated juxtamembrane activating cleavage and before a newly reported inactivating presenilin-mediated transmembrane cleavage that serves to curtail the timecourse of signaling from activated Robo

The cellular basis of distinct thirst modalities

Fluid intake is an essential innate behaviour that is mainly caused by two distinct types of thirst. Increased blood osmolality induces osmotic thirst that drives animals to consume pure water. Conversely, the loss of body fluid induces hypovolaemic thirst, in which animals seek both water and minerals (salts) to recover blood volume. Circumventricular organs in the lamina terminalis are critical sites for sensing both types of thirst-inducing stimulus. However, how different thirst modalities are encoded in the brain remains unknown. Here we employed stimulus-to-cell-type mapping using single-cell RNA sequencing to identify the cellular substrates that underlie distinct types of thirst. These studies revealed diverse types of excitatory and inhibitory neuron in each circumventricular organ structure. We show that unique combinations of these neuron types are activated under osmotic and hypovolaemic stresses. These results elucidate the cellular logic that underlies distinct thirst modalities. Furthermore, optogenetic gain of function in thirst-modality-specific cell types recapitulated water-specific and non-specific fluid appetite caused by the two distinct dipsogenic stimuli. Together, these results show that thirst is a multimodal physiological state, and that different thirst states are mediated by specific neuron types in the mammalian brain

Complementary networks of cortical somatostatin interneurons enforce layer specific control

The neocortex is functionally organized into layers. Layer four receives the densest bottom up sensory inputs, while layers 2/3 and 5 receive top down inputs that may convey predictive information. A subset of cortical somatostatin (SST) neurons, the Martinotti cells, gate top down input by inhibiting the apical dendrites of pyramidal cells in layers 2/3 and 5, but it is unknown whether an analogous inhibitory mechanism controls activity in layer 4. Using high precision circuit mapping, in vivo optogenetic perturbations, and single cell transcriptional profiling, we reveal complementary circuits in the mouse barrel cortex involving genetically distinct SST subtypes that specifically and reciprocally interconnect with excitatory cells in different layers: Martinotti cells connect with layers 2/3 and 5, whereas non-Martinotti cells connect with layer 4. By enforcing layer-specific inhibition, these parallel SST subnetworks could independently regulate the balance between bottom up and top down input

A multimodal cell census and atlas of the mammalian primary motor cortex

ABSTRACT We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties

Slit-Dependent Endocytic Trafficking of the Robo Receptor Is Required for Son of Sevenless Recruitment and Midline Axon Repulsion

<div><p>Understanding how axon guidance receptors are activated by their extracellular ligands to regulate growth cone motility is critical to learning how proper wiring is established during development. Roundabout (Robo) is one such guidance receptor that mediates repulsion from its ligand Slit in both invertebrates and vertebrates. Here we show that endocytic trafficking of the Robo receptor in response to Slit-binding is necessary for its repulsive signaling output. Dose-dependent genetic interactions and <i>in vitro</i> Robo activation assays support a role for Clathrin-dependent endocytosis, and entry into both the early and late endosomes as positive regulators of Slit-Robo signaling. We identify two conserved motifs in Robo’s cytoplasmic domain that are required for its Clathrin-dependent endocytosis and activation <i>in vitro</i>; gain of function and genetic rescue experiments provide strong evidence that these trafficking events are required for Robo repulsive guidance activity <i>in vivo</i>. Our data support a model in which Robo’s ligand-dependent internalization from the cell surface to the late endosome is essential for receptor activation and proper repulsive guidance at the midline by allowing recruitment of the downstream effector Son of Sevenless in a spatially constrained endocytic trafficking compartment.</p></div

Endocytosis motifs are required for ectopic repulsion <i>in vivo</i>.

<p>The projection pattern of all axons of the ventral nerve cord of late stage 14 <i>Drosophila</i> embryos are imaged with HRP (A-D) and the fascicles of the Eg commissural subset are imaged with a Tau-myc-GFP transgene (E-H). (A) In wild-type embryos, all segments (3 shown here) have two horizontal commissures, which are quantified as 0% of segments with error in the histogram (L n = 88). (B) Overexpressing wild-type Robo transgene in all neurons causes gain of repulsion from midline Slit, resulting in a loss of commissures in 76% of embryonic segments (L, n = 99). In contrast, overexpressing similar levels of Robo transgene that is missing its AP-2 binding motifs (C, D) can not signal ectopic repulsion from the midline, with all segments projecting in a commissural pattern indistinguishable from embryos without transgene (L ΔYQAGL n = 152, ΔYLQY n = 136, ΔYLQYΔYQAGL n = 88). (E) The Ew commissural subset of axons, schematized on the right, cross the midline in each embryonic segment, quantified as 0% error in (M, n = 88). (F) Expressing wild-type Robo transgene (I) specifically in the Ew commissural subset of axons is sufficient to cause ectopic repulsion, with loss of projection across the midline (schematized in dotted gray) in 96% of embryonic segments (M, n = 99). In contrast, expressing either Robo∆YQAGL (G, J) or Robo∆YLQY (H, K) does not cause ectopic repulsion of the Ew projection pattern, with a 0% error in (M, ΔYQAGL n = 152, ΔYLQY n = 136, ΔYLQYΔYQAGL n = 88). Error bars indicate standard error of the mean. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005402#pgen.1005402.s005" target="_blank">S5 Fig</a>.</p

Robo Endocytosis is required for Sos recruitment <i>in vitro</i>.

<p>Co-expression of a version of Son of Sevenless dominant-negative for its RacGEF activity (B) inhibits spreading and branching of processes in response to Slit CM as effectively as deleting Robo’s entire C-terminus (A). Feature extraction of the pixel intensity of endogenous Sos in processes reveals recruitment of Sos to processes in Slit (D) versus Control CM (C) treatment. The increase in Sos signal in processes in response to Slit seen in Robo^<i>WT</i>-expressing cells, quantified in the histogram as a statistically significant increase (*) in average signal intensity (I, n’s displayed on histogram), is missing in cells expressing RoboΔIg1 (E, E’). Cells expressing a Robo∆CC2∆CC3 receptor that can’t bind Ena or Dock, required for Sos binding (F, F’) also show impaired recruitment of endogenous Sos to processes, as do conditions inhibiting endocytosis (G-H’), despite comparable number of pixels (process area) analyzed (J). Statistical significance quantified by two-way ANOVA, Sidak’s 95% Confidence Interval. Error bars indicate standard error of the mean.</p

Slit induces Robo colocalization with Rab5 in cell processes.

<p>S2R+ cells expressing Robo and treated with SlitCM were fixed at an earlier timepoint (2’) and stained for endogenous Rab5, a marker of the early endosome, (A, C, E, F, H, J, K, M, O) and either bound Slit ligand (B, G, L), or Robo’s C-terminal tag (D, I, N). Cells with Slit bound to processes show covariance between ligand and early endosome signal (B, P (n = # cells indicated on histogram bar)). This colocalization is reduced either by reducing Slit-binding (ΔIg1 in P), or by inhibiting Clathrin-dependent endocytosis globally with DN-Shibire (F, G), or the Dynamin inhibitor Dynasore (P), or by deleting Robo’s AP-2-binding motifs (K, L). Treatment with Slit CM induces colocalization between Robo and Rab5 in processes as compared to cells treated with Control CM, quantified as a percent increase of thresholded Mander’s Overlap Coefficient between Slit and Control CM (C-E, Q). Inhibiting Clathrin-dependent endocytosis by coexpression with DN-Shibire (H-J), use of Dynasore, or deleting AP-2 adaptor motifs (M-O), or inhibiting Slit-binding by deleting the first Ig domain, causes a loss of Slit-dependent colocalization between Robo C-terminus and the early endosome in processes, quantified as the percent change in colocalization between Slit and Control CM (Q). The percentage change switches from positive to negative (Q (n’s for Ctrl CM on top, Slit CM on bottom). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005402#pgen.1005402.s004" target="_blank">S4 Fig</a>.</p

Clathrin-dependent Endocytosis is required for removal of Robo from the surface.

<p>An N-terminal pH sensitive tag on Robo (A-F) reveals the pool of Robo expressed on the surface of S2R+ cells after 2’ of conditioned media (CM) bath-treatment. S2R+ cells treated with CM from cells expressing Slit (B) as opposed to empty vector (A) show a decrease in surface levels of Robo, quantified in (G) as a percent decrease in average pixel intensity value of processes in (B) as compared to (A). (C, D) Inhibiting Robo signaling by deleting the entire C-terminus shunts the Slit-dependent reduction in average pixel intensity value of surface Robo, leading to a smaller percentage decrease in (G). (E, F) Deleting both of Robo’s putative AP-2 motifs abrogates the Slit-dependent reduction in surface receptor levels, leading to a smaller % decrease in average pixel intensity in (G), significant (*) according to one-way ANOVA Dunnett’s test (n’s for Control and Slit CM displayed below each bar). (H-M) The ectopic crossing events of a normally ipsilateral subset of axons in the ventral nerve cord of Stage 16 <i>Drosophila</i> embryos are induced by either manipulating entry to the early endosome with expression of DN-Rab5 transgene (H), or by overexpression of an attractive guidance receptor, Frazzled (J, L). Robo transgene is mislocalized to the ectopically crossing segments of axons in embryos defective for endocytic trafficking (I) but not in those with excessive attractive guidance (K), despite the similarity in strength of ectopic crossing phenotype (N). In contrast, Robo transgene defective for AP-2 binding is mislocalized to the ectopically crossing segments of axons (M) in the same gain of attraction background (L). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005402#pgen.1005402.s003" target="_blank">S3 Fig</a>.</p

Robo Endocytosis is required for axon guidance <i>in vivo</i>.

<p>Two ipsilateral subsets of axons are imaged in Stage 17 <i>Drosophila</i> embryos- the FasII+ axons with a monoclonal antibody to FasII (A-E) and the Ap axons (F-J) with a GFP antibody detecting Tau-Myc-GFP transgene. In wild-type embryos these ipsilateral subsets project on either side of the midline, with three fascicles on either side for FasII (A) and one fascicle on either side for Ap (F). In <i>robo</i> mutant embryos, the two medial-most of the FasII+ fascicles (B) and both of the Ap fascicles (G) collapse onto the midline, scored as 100% of embryonic segments having ectopic collapse/circling events. Expressing wild-type Robo transgene is sufficient to restore repulsive signaling and therefore rescue the crossing defects in the FasII+ axons (C, +/+ n = 121, <i>robo</i>^<i>GA285</i><i>/robo</i> ^<i>GA285</i> n = 121, <i>robo</i>^<i>GA285</i><i>/robo</i> ^<i>GA285</i>;ElavGAL4/UAS-Robo^<i>WT</i> n = 121) and the Ap axons (I +/+ n = 120, <i>robo</i>^<i>GA285</i><i>/Ap</i>, <i>robo</i>^<i>z1772</i> n = 120, <i>robo</i>^<i>GA285</i><i>/Ap</i>, <i>robo</i>^<i>z1772</i>;UAS-RoboWT n = 80). In contrast, expressing Robo∆YQAGL (D, I), Robo∆YLQY, or Robo∆YQAGL ∆YLQY (E, J) is not sufficient to rescue the ectopic crossing events, with a large portion of embryonic segments carrying severe errors (crossing/circling events represented by dark gray) remaining. Dark gray indicates a qualitatively more severe crossing error, light gray indicates a less severe crossing error, with the stacked histogram bar height indicating total % of embryonic segments with loss-of-repulsion errors for each genotype. Error bars indicate standard error of the mean. (<i>robo</i>^<i>GA285</i><i>/robo</i> ^<i>GA285</i>; ElavGAL4/UAS-Robo^{<i>∆YQAGL</i>} n = 154, <i>robo</i>^<i>GA285</i><i>/robo</i> ^<i>GA285</i>; ElavGAL4/UAS-Robo^{<i>∆YLQY</i>} n = 99, <i>robo</i>^<i>GA285</i><i>/robo</i> ^<i>GA285</i>; ElavGAL4/UAS-Robo^{<i>∆YLQY∆YQAGL</i>} n = 121. <i>robo</i>^<i>GA285</i><i>/ApGAL4</i>, <i>robo</i>^<i>z1772</i>;UAS-Robo^{<i>∆YQAGL</i>} n = 80, <i>robo</i>^<i>GA285</i><i>/ApGAL4</i>, <i>robo</i>^<i>z1772</i>;UAS-Robo^{<i>∆YLQY</i>} n = 120, <i>robo</i>^<i>GA285</i><i>/ApGAL4</i>, <i>robo</i>^<i>z1772</i>;UAS-Robo^{<i>∆YLQY∆YQAGL</i>} n = 136.)</p