Radial glia regulate vascular patterning around the developing spinal cord

Vascular networks surrounding individual organs are important for their development, maintenance, and function; however, how these networks are assembled remains poorly understood. Here we show that CNS progenitors, referred to as radial glia, modulate vascular patterning around the spinal cord by acting as negative regulators. We found that radial glia ablation in zebrafish embryos leads to excessive sprouting of the trunk vessels around the spinal cord, and exclusively those of venous identity. Mechanistically, we determined that radial glia control this process via the Vegf decoy receptor sFlt1: sflt1 mutants exhibit the venous over-sprouting observed in radial glia-ablated larvae, and sFlt1 overexpression rescues it. Genetic mosaic analyses show that sFlt1 function in trunk endothelial cells can limit their over-sprouting. Together, our findings identify CNS-resident progenitors as critical angiogenic regulators that determine the precise patterning of the vasculature around the spinal cord, providing novel insights into vascular network formation around developing organs

Sema6B, Sema6C, and Sema6D Expression and Function during Mammalian Retinal Development

In the vertebrate retina, the formation of neural circuits within discrete laminae is critical for the establishment of retinal visual function. Precise formation of retinal circuits requires the coordinated actions of adhesive and repulsive molecules, including repulsive transmembrane semaphorins (Sema6A, Sema5A, and Sema5B). These semaphorins signal through different Plexin A (PlexA) receptors, thereby regulating distinct aspects of retinal circuit assembly. Here, we investigate the physiological roles of three Class 6 transmembrane semaphorins (Sema6B, Sema6C, and Sema6D), previously identified as PlexA receptor ligands in non-retinal tissues, inmammalian retinal development.Weperformedexpressionanalysis andalsophenotypic analysesofmice thatcarrynullmutations ineachofgenesencoding theseproteinsusingabroad rangeof innerandouter retinalmarkers.Wefind that these Class 6 semaphorins are uniquely expressed throughout postnatal retinal development in specific domains and cell typesofthedevelopingretina.However,wedonotobservedefects instereotypical lamina-specificneuritestratificationof retinal neuron subtypes in Sema6B2/2 or Sema6C2/2; Sema6D2/2 retinas. These findings indicate these Class 6 transmembrane semaphorins areunlikely to serve asmajor PlexA receptor ligands for the assemblyofmurine retinal circuit laminar organization

Sema6B, Sema6C, and Sema6D expression and function during mammalian retinal development.

In the vertebrate retina, the formation of neural circuits within discrete laminae is critical for the establishment of retinal visual function. Precise formation of retinal circuits requires the coordinated actions of adhesive and repulsive molecules, including repulsive transmembrane semaphorins (Sema6A, Sema5A, and Sema5B). These semaphorins signal through different Plexin A (PlexA) receptors, thereby regulating distinct aspects of retinal circuit assembly. Here, we investigate the physiological roles of three Class 6 transmembrane semaphorins (Sema6B, Sema6C, and Sema6D), previously identified as PlexA receptor ligands in non-retinal tissues, in mammalian retinal development. We performed expression analysis and also phenotypic analyses of mice that carry null mutations in each of genes encoding these proteins using a broad range of inner and outer retinal markers. We find that these Class 6 semaphorins are uniquely expressed throughout postnatal retinal development in specific domains and cell types of the developing retina. However, we do not observe defects in stereotypical lamina-specific neurite stratification of retinal neuron subtypes in Sema6B-/- or Sema6C-/-; Sema6D-/- retinas. These findings indicate these Class 6 transmembrane semaphorins are unlikely to serve as major PlexA receptor ligands for the assembly of murine retinal circuit laminar organization

Neurite stratification of amacrine cell and RGC subtypes in the IPL of <i>Sema6B^−/−</i> and <i>Sema6C^−/−; Sema6D^−/−</i> retinas.

<p>Wild-type (<b>A, D, G, J, M, P</b>), <i>Sema6B^−/−</i> (<b>B, E, H, K, N, Q</b>), and <i>Sema6C^−/−</i>; <i>Sema6D^−/−</i> (<b>C, F, I, L, O, R</b>) adult retina sections were immunostained with antibodies against tyrosine hydroxylase (TH) (<b>A–C</b>), a marker for dopaminergic amacrine cells, choline acetyltransferase (ChAT) (<b>D–F</b>), a marker for cholinergic amacrine cells, calretinin (<b>G–I</b>), a marker for subsets of amacrine cells and RGCs, vGlut3 (<b>J–L</b>), a marker for subsets of amacrine cells, Dab-1 (<b>M–O</b>), a marker for AII amacrine cells, or calbindin (<b>P–R</b>), a marker for subsets of amacrine cells and RGCs. TO-PRO-3 dye (blue) was used to visualize the organization of nuclear layers in the retina. The amacrine cell and RGC subtypes labelled by these markers do not show obvious defects in their sublaminar neurite targeting and stratification in <i>Sema6B^−/−</i> or <i>Sema6C^−/−</i>; <i>Sema6D^−/−</i> retinas, as compared to those in wild-type retinas. Scale bar: 50 µm in R for <b>A–R</b>.</p

Neurite stratification of outer retinal neuron cell types in the OPL of <i>Sema6B^−/−</i> and <i>Sema6C^−/−; Sema6D^−/−</i> retinas.

<p>Wild-type (<b>A, D, G, J</b>), <i>Sema6B^−/−</i> (<b>B, E, H, K</b>), and <i>Sema6C^−/−</i>; <i>Sema6D^−/−</i> (<b>C, F, I, L</b>) adult retina sections were immunostained with antibodies against cone arrestin (<b>A–C</b>, a marker for cone photoreceptors), calbindin (<b>D–F</b>, a marker for horizontal cells), PKCα (<b>G–I</b>, a marker for rod bipolar cells), and vGlut1 (<b>J–L</b>, a marker for photoreceptor axonal terminals). Neurite stratification of photoreceptors, horizontal cells, and bipolar cells visualized by each of these markers does not show any obvious defects in <i>Sema6B^−/−</i> and <i>Sema6C^−/−; Sema6D^−/−</i> retinas as compared to wild-type retinas. Scale bar: 50 µm in L for <b>A–L</b>.</p

<i>Sema6B</i>, <i>Sema6C</i>, and <i>Sema6D</i> mRNA expression during postnatal retinal development.

<p><i>In situ</i> hybridization was performed using antisense probes specific for <i>Sema6B</i> (<b>A</b>, <b>G</b>), <i>Sema6C</i> (<b>C</b>, <b>I</b>), or <i>Sema6D</i> (<b>E</b>, <b>K</b>) on retina sections from P7 (<b>A</b>–<b>F</b>) and P14 (<b>G</b>–<b>L</b>) postnatal ages of mice. Adjacent P7 (<b>B</b>, <b>D</b>, <b>F</b>) and P14 (<b>H</b>, <b>J</b>, <b>L</b>) retina sections incubated with sense probes for <i>Sema6B</i> (<b>B</b>, <b>H</b>), <i>Sema6C</i> (<b>D</b>, <b>J</b>), or <i>Sema6D</i> (<b>F</b>, <b>L</b>) were shown as references for background staining. <i>Sema6B</i>, <i>Sema6C</i>, and <i>Sema6D</i> are all expressed at these developmental time points. Strong <i>Sema6B</i> expression is found in the GCL and INL at P7 and P14 (<b>A</b>, <b>G</b>). <i>Sema6C</i> is expressed broadly in the INL and sparsely in the GCL (<b>C</b>, <b>I</b>) at P7 and P14. <i>Sema6D</i> is strongly expressed in specific cell types that reside in the GCL and INL close to the OPL at P7 and P14. <i>Sema6D</i> expression is also observed sparsely in the inner INL (<b>E</b>, <b>K</b>). GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer. Scale bar: 50 µm in <b>L</b> for <b>A</b>–<b>L</b>.</p

Neurite stratification of ipRGCs, PlexA2-positive, PlexA4-positive cells in the IPL of <i>Sema6B^−/−</i> and <i>Sema6C^−/−; Sema6D^−/−</i> retinas.

<p>(<b>A–I</b>) Wild-type (<b>A, D, G</b>), <i>Sema6B^−/−</i> (<b>B, E, H</b>), and <i>Sema6C^−/−</i>; <i>Sema6D^−/−</i> (<b>C, F, I</b>) P17 retina sections were immunostained with antibodies against melanopsin (<b>A–C</b>), PlexA2 (<b>D–F</b>), or PlexA4 (<b>G–I</b>). Retinal neuron subtypes labelled by each of these markers exhibit neurite stratification within specific sublaminae in wild-type retinas, and these sublaminar targeting and stratification patterns are not apparently affected in <i>Sema6B^−/−</i> or <i>Sema6C^−/−</i>; <i>Sema6D^−/−</i> retinas. Scale bar: 50 µm in I for <b>A–I</b>.</p

Targeting by distinct subtypes of bipolar cell axons in the IPL of <i>Sema6B^−/−</i> and <i>Sema6C^−/−; Sema6D^−/−</i> retinas.

<p>Wild-type (<b>A, D, G</b>), <i>Sema6B^−/−</i> (<b>B, E, H</b>), and <i>Sema6C^−/−</i>; <i>Sema6D^−/−</i> (<b>C, F, I</b>) adult retina sections were immunostained with antibodies against protein kinase C alpha (PKCα, <b>A–C</b>, a marker for rod bipolar cells), synaptotagmin2 (Syt2, D–F, a marker for type-2 cone OFF and type-6 cone ON bipolar cells), and neurokinin-3 receptor (NK3R, <b>G–I</b>, a marker for type-5 cone ON and cone OFF bipolar cells). Distinct subtypes of bipolar cells labelled by these three markers elaborate axon terminals within specific sublaminae of wild-type retinas, and these axon termination patterns do not apparently differ in <i>Sema6B^−/−</i> and <i>Sema6C^−/−</i>; <i>Sema6D^−/−</i> retinas. Scale bar: 50 µm in I for <b>A–I</b>.</p

On and Off Retinal Circuit Assembly by Divergent Molecular Mechanisms

Direction-selective responses to motion can be to the onset (On) or cessation (Off) of illumination. Here, we show that the transmembrane protein semaphorin 6A and its receptor plexin A2 are critical for achieving radially symmetric arborization of On starburst amacrine cell (SAC) dendrites and normal SAC stratification in the mouse retina. Plexin A2 is expressed in both On and Off SACs; however, semaphorin 6A is expressed in On SACs. Specific On-Off bistratified direction-selective ganglion cells in semaphorin 6A(-/-) mutants exhibit decreased tuning of On directional motion responses. These results correlate the elaboration of symmetric SAC dendritic morphology and asymmetric responses to motion, shedding light on the development of visual pathways that use the same cell types for divergent outputs