In brief, tissue samples were prepared for immunohistochemistry as described above. processes contacted aberrant cone axons in LKB1 mutants. These defects coincided with altered synapse protein organization, and horizontal cell neurites were misdirected to ectopic synapse protein regions. Together, these data suggest that LKB1 instructs the timing and location of connectivity in the outer retina via coordinate regulation of pre and postsynaptic neuron structure and the localization of synapse-associated proteins. test) or as the mean??the s.e.m. (E, **p 0.01, non-parametric Mann-Whitney Rank Sum U-test). Figure 1figure supplement 1. Open in a separate window is highly expressed throughout the retina in early development.In situ XL019 hybridization pattern of over retina development. (ACB) Representative fluorescent in situ hybridization images (A) and quantification (B) of expression patterns across development at P2, P5, P8, and P14 in control mice. Data in (B) are presented as a heatmap indicating the corrected total cell fluorescence of each retinal layer occupied by the signal using a gradient scale where white to blue depicts low to high levels of fluorescent intensity (0C2500, respectively), and black indicates enrichment levels higher than 2500. Scale bars?=?25 m. Figure 1figure supplement 2. Open in a separate window AMPK does not regulate outer retina development.Outer retina emergence and cellular morphology were visualized in Ampk-Ret mice and littermate controls at P5.?(ACC) Representative images (A) and quantification of OPL emergence (B, DAPI, grey) and distance (C) of OPL patches from the apical surface at P5 in Ampk-Ret and littermate controls. The OPL emerges at the proper time and location in Ampk-Ret animals (B) and is located the same distance from the apical surface as controls (C, n?=?187 control cells and n?=?182 Ampk-Ret cells). N?=?3 control and Ampk-Ret animals. (DCE) Representative images (D) and quantification (E) of cone (OPN1SW, green) morphology at P5. Ampk-Ret cones extend their axons to same length as XL019 control mice. N?=?3 control and Ampk-Ret animals. (FCG) Representative images (F) and quantification (G) of horizontal cell (calbindin, cyan) morphology at P5. Ampk-Ret horizontal cells restrict their arbors, spanning the same area as control mice. N?=?3 control and Ampk-Ret animals. Scale bars?=?25 m. Data are represented as the mean??the s.e.m. (B, E, p 0.05, non-parametric Mann-Whitney Rank Sum U-test), as a distribution of the distance of patches from the apical surface (C, p 0.05, unpaired two-tailed Students test), or as the mean fluorescence relative to the distance XL019 from the apical surface (G,?p 0.05, unpaired two-tailed Students test). To begin to resolve these questions, we focused on the serine/threonine kinase LKB1 (Liver Kinase B1, also called STK11 or Par4; encoded by mRNA are highest in early development at P5 when synapses begin to emerge (Number 1figure product 1), with manifestation present in both inner and outer retina. To determine the part of LKB1 in the emergence of synaptic connectivity we generated full retina LKB1 knockout mice using the conditional allele (previously called line (previously Gja7 called in embryonic retinal progenitors to generate animals. This collection is definitely hereafter referred to as Lkb1-Ret. Problems in LKB1 mutant retinas became apparent as the synapse coating started to emerge. While control animals displayed nuclei-free patches at P3 that are localized 39.1 0.3 m away from the apical part of the outer retina, in Lkb1-Ret mice OPL patches were small and hard to visualize (Number 1B), displaced closer to the apical retinal surface relative to control mice (29.6 0.4 m away, (test. Number 3figure product 1. Open in a separate windowpane Horizontal cells fail to restrict their neurites at the appropriate developmental time.Horizontal cells and their neurites were reconstructed in Lkb1-Ret and littermate controls during postnatal development using an antibody to calbindin (cyan).?(ACB) Reconstructed images (A) and quantification (B) of the?quantity of apical neurites per horizontal cell at P3. No significant structural variations were observed. N?=?3 control and Lkb1-Ret animals. (CCD) Reconstructed images (C) and quantification (D) of the?quantity of apical neurites per horizontal cell at P5. There is an increase in the number XL019 of apical neurites in Lkb1-Ret horizontal cells relative to settings, signifying their failure to restrict their arbors at P5. N?=?4 control and N?=?4 Lkb1-Ret animals. Level bars?=?25 m. Data are displayed as the mean??the s.e.m.?*p 0.05, non-parametric Mann-Whitney Rank Sum U test. We next investigated whether the problems in horizontal cell refinement displayed a cell-intrinsic part for LKB1 in shaping horizontal cell architecture..