Src family kinases are required for limb trajectory selection by spinal motor axons

Signal relay by guidance receptors at the axonal growth cone is a process essential for the assembly of a functional nervous system. We investigated the in vivo function of Src family kinases (SFKs) as growth cone guidance signaling intermediates in the context of spinal lateral motor column (LMC) motor axon projection toward the ventral or dorsal limb mesenchyme. Using in situ mRNA detection we determined that Src and Fyn are expressed in LMC motor neurons of chick and mouse embryos at the time of limb trajectory selection. Inhibition of SFK activity by C-terminal Src kinase (Csk) overexpression in chickLMCaxons using in ovo electroporation resulted inLMC axons selecting the inappropriate dorsoventral trajectory within the limb mesenchyme, with medial LMC axon projecting into the dorsal and ventral limb nerve with apparently random incidence. We also detected LMC axon trajectory choice errors in Src mutant mice demonstrating a nonredundant role for Src in motor axon guidance in agreement with gain and loss of Src function in chickLMCneurons which led to the redirection ofLMCaxons. Finally, Csk-mediated SFK inhibition attenuated the retargeting ofLMCaxons caused by EphA or EphB over-expression, implying the participation of SFKs in Eph-mediated LMC motor axon guidance. In summary, our findings demonstrate that SFKs are essential for motor axon guidance and suggest that they play an important role in relaying ephrin:Eph signals that mediate the selection of motor axon trajectory in the limb.This work was supported by a grant from the Canadian Institutes of Health Research (Institute of Genetics and Institute of Neurosciences, Mental Health, and Addiction) to A.K. (MOP-77556 and IG-74068)

Foxp1 and lhx1 coordinate motor neuron migration with axon trajectory choice by gating Reelin signalling.

Topographic neuronal maps arise as a consequence of axon trajectory choice correlated with the localisation of neuronal soma, but the identity of the pathways coordinating these processes is unknown. We addressed this question in the context of the myotopic map formed by limb muscles innervated by spinal lateral motor column (LMC) motor axons where the Eph receptor signals specifying growth cone trajectory are restricted by Foxp1 and Lhx1 transcription factors. We show that the localisation of LMC neuron cell bodies can be dissociated from axon trajectory choice by either the loss or gain of function of the Reelin signalling pathway. The response of LMC motor neurons to Reelin is gated by Foxp1- and Lhx1-mediated regulation of expression of the critical Reelin signalling intermediate Dab1. Together, these observations point to identical transcription factors that control motor axon guidance and soma migration and reveal the molecular hierarchy of myotopic organisation

Cell-Type Specific Roles for PTEN in Establishing a Functional Retinal Architecture

BACKGROUND: The retina has a unique three-dimensional architecture, the precise organization of which allows for complete sampling of the visual field. Along the radial or apicobasal axis, retinal neurons and their dendritic and axonal arbors are segregated into layers, while perpendicular to this axis, in the tangential plane, four of the six neuronal types form patterned cellular arrays, or mosaics. Currently, the molecular cues that control retinal cell positioning are not well-understood, especially those that operate in the tangential plane. Here we investigated the role of the PTEN phosphatase in establishing a functional retinal architecture. METHODOLOGY/PRINCIPAL FINDINGS: In the developing retina, PTEN was localized preferentially to ganglion, amacrine and horizontal cells, whose somata are distributed in mosaic patterns in the tangential plane. Generation of a retina-specific Pten knock-out resulted in retinal ganglion, amacrine and horizontal cell hypertrophy, and expansion of the inner plexiform layer. The spacing of Pten mutant mosaic populations was also aberrant, as were the arborization and fasciculation patterns of their processes, displaying cell type-specific defects in the radial and tangential dimensions. Irregular oscillatory potentials were also observed in Pten mutant electroretinograms, indicative of asynchronous amacrine cell firing. Furthermore, while Pten mutant RGC axons targeted appropriate brain regions, optokinetic spatial acuity was reduced in Pten mutant animals. Finally, while some features of the Pten mutant retina appeared similar to those reported in Dscam-mutant mice, PTEN expression and activity were normal in the absence of Dscam. CONCLUSIONS/SIGNIFICANCE: We conclude that Pten regulates somal positioning and neurite arborization patterns of a subset of retinal cells that form mosaics, likely functioning independently of Dscam, at least during the embryonic period. Our findings thus reveal an unexpected level of cellular specificity for the multi-purpose phosphatase, and identify Pten as an integral component of a novel cell positioning pathway in the retina

Cell-type specific roles for PTEN in establishing a functional retinal architecture.

BackgroundThe retina has a unique three-dimensional architecture, the precise organization of which allows for complete sampling of the visual field. Along the radial or apicobasal axis, retinal neurons and their dendritic and axonal arbors are segregated into layers, while perpendicular to this axis, in the tangential plane, four of the six neuronal types form patterned cellular arrays, or mosaics. Currently, the molecular cues that control retinal cell positioning are not well-understood, especially those that operate in the tangential plane. Here we investigated the role of the PTEN phosphatase in establishing a functional retinal architecture.Methodology/principal findingsIn the developing retina, PTEN was localized preferentially to ganglion, amacrine and horizontal cells, whose somata are distributed in mosaic patterns in the tangential plane. Generation of a retina-specific Pten knock-out resulted in retinal ganglion, amacrine and horizontal cell hypertrophy, and expansion of the inner plexiform layer. The spacing of Pten mutant mosaic populations was also aberrant, as were the arborization and fasciculation patterns of their processes, displaying cell type-specific defects in the radial and tangential dimensions. Irregular oscillatory potentials were also observed in Pten mutant electroretinograms, indicative of asynchronous amacrine cell firing. Furthermore, while Pten mutant RGC axons targeted appropriate brain regions, optokinetic spatial acuity was reduced in Pten mutant animals. Finally, while some features of the Pten mutant retina appeared similar to those reported in Dscam-mutant mice, PTEN expression and activity were normal in the absence of Dscam.Conclusions/significanceWe conclude that Pten regulates somal positioning and neurite arborization patterns of a subset of retinal cells that form mosaics, likely functioning independently of Dscam, at least during the embryonic period. Our findings thus reveal an unexpected level of cellular specificity for the multi-purpose phosphatase, and identify Pten as an integral component of a novel cell positioning pathway in the retina

Aberrant cellular mosaicism in <i>Pten</i> cKOs.

<p>(A–H) Immunolabeling of P21 wild-type (A) and <i>Pten</i> cKO (B) retinal flatmounts with TH. Voronoi diagrams depicting the distribution of TH⁺ amacrine cells in P21 wild-type (C) and <i>Pten</i> cKO (D) retinae. Calculation of TH⁺ Voronoi domain areas and their relative distributions in these two fields for P21 wild-type (C′) and <i>Pten</i> cKO (D′) retinae. Near neighbors of a TH⁺ reference cell in P21 wild-type (E) and <i>Pten</i> cKO (F) retinae, with the nearest neighbour indicated in red. Frequency distribution of nearest neighbor distances between TH⁺ amacrine cells in these two fields for P21 wild-type (E′) and <i>Pten</i> cKO (F′) retinae. Calculation of Voronoi domain (G) and Nearest Neighbor (H) regularity indices for TH⁺ amacrine cells in wild-type and <i>Pten</i> cKO retinae. (I–P) Immunolabeling of P21 wild-type (I) and <i>Pten</i> cKO (J) retinal flatmounts with calbindin. Voronoi diagrams depicting the distribution of calbindin⁺ horizontal cells in P21 wild-type (K) and <i>Pten</i> cKO (L) retinae. Calculation of TH⁺ Voronoi domain areas and their frequency distributions in P21 wild-type (K′) and <i>Pten</i> cKO (L′) retinae in these two fields. Near neighbors of a calbindin⁺ reference cell in P21 wild-type (M) and <i>Pten</i> cKO (N) retinae, with the nearest neighbour indicated in red. Frequency distribution of distances between TH⁺ amacrine cells in P21 wild-type (M′) and <i>Pten</i> cKO (N′) retinae in these two fields. Calculation of Voronoi domain (O) and Nearest Neighbor (P) regularity indices for calbindin⁺ horizontal cells in wild-type and <i>Pten</i> cKO retinae. p values are denoted as follows: <0.05 *, <0.01 **, <0.005 ***. Scale bars = 600 µm (A,B), 100 µm (C,D).</p

Synaptic contacts in the <i>Pten</i> cKO retinal IPL.

<p>(A–F) Electron microscopy (EM) of adult wild-type and <i>Pten</i> cKO retinae. Schematic illustration of retinal architecture (A′). Low magnification EM images of wild-type (A) and <i>Pten</i> cKO (B) retinae, shown to scale, illustrating expansion of mutant retinae. Higher magnification images of <i>Pten</i> cKO IPL (C–F), with boxed areas in C shown in higher magnification in D,E. Asterisks in C mark ectopic cells in the IPL. Color scheme in D–F′ is as follows: Blue denotes rod bipolar cell terminal with ribbons (labeled R) in the <i>Pten</i> cKO IPL (D). Pink denotes amacrine cell synapses on ectopic somata within the IPL (E,F). GCL, ganglion cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; onl, outer nuclear layer; opl, outer plexiform layer. Scale bars = 10 µm (A,B,C), 1 µm (D,E), 2 µm (F), 100 µm (G–J), 50 µm (G′–J′).</p

Aberrant RGC fasciculation and subcortical visual responses in <i>Pten</i> cKOs.

<p>(A–B) Low (A,B) and high (A′,B′) power photomicrographs of SMI-32 labeling of P21 wild-type and <i>Pten</i> cKO retinal wholemounts. (C–E) Photomicrographs of wild-type and <i>Pten</i> cKO P21 optic nerves (C) and corresponding cross sections stained with hematoxylin-eosin (E). Optic nerve diameters are shown in D. (F,G) AP staining of P21 <i>Pax6::Cre</i>⁺;Z/AP⁺ (“wild-type”; F) and <i>Pten^f</i>^l/fl;<i>Pax6::Cre</i>⁺;Z/AP⁺ (<i>Pten</i> cKO, G) whole brains with the overlying cortex removed to reveal the visual pathway. The center of the superior colliculus (SC) is unstained as it is innervated by RGCs in the central retina, where cre activity is low. (H,I) Behavioural measures of the optokinetic reflex in adult wild-type and <i>Pten</i> cKOs that are either pooled (H) or separated into affected and unaffected groups (I). Scale bars = 300 µm (A,B), 100 µm (A′,B′), 750 µm (C), 200 µm (E), 2.5 mm (F,G).</p

Altered ERG oscillatory potential responses in <i>Pten</i> cKO animals.

<p>(A–F) Scotopic ERG with representative trace (A; wild-type is black; <i>Pten</i> cKO is red) and OP scalogram (B,C) at flash intensity of 0.38 cd*s/m². (D–F) Graphical representation of OP amplitude (D), frequency (E) and latency (F) across 19 steps (−5.22 to 2.86 cd*s/m²). (G–L) Double flash ERG with representative trace (G; wild-type is black; <i>Pten</i> cKO is red) and OP scalogram (H,I) at flash intensity of 0.38 cd*s/m². (J–L) Graphical representation of OP amplitude (J), frequency (K) and latency (L) across 10 steps (−5.22 to 2.86 cd*s/m²).</p

Abnormal retinal architecture and increased retinal cell sizes in <i>Pten</i> cKOs.

<p>(A–D) Low (A,B) and high (C,D) magnification images of hematoxylin-eosin (H&E) stained sections of adult wild-type (A,C) and <i>Pten</i> cKO (B,D) retinae. (E–G) Calbindin labelling of P7 wild-type and <i>Pten</i> cKO retinal sections (E,F) and area measurements of calbindin⁺ horizontal cells (G). (H–S) Labeling of retinal flatmounts from P21 wild-type and <i>Pten</i> cKOs with calbindin (H,I), ChAT (K,L), TH (N,O), and SMI32 (Q,R). Calculation of cell areas for P21 calbindin⁺ horizontal cells (J), ChAT⁺ (M) and calbindin⁺ (P) amacrine cells and SMI32⁺ RGCs (S). p values are denoted as follows: <0.05 *, <0.01 **, <0.005 ***. Scale bars = 100 µm (A,B,N,O), 50 µm (C–L,Q,R).</p