LKB1 coordinates neurite remodeling to drive synapse layer emergence in the outer retina.

Structural changes in pre and postsynaptic neurons that accompany synapse formation often temporally and spatially overlap. Thus, it has been difficult to resolve which processes drive patterned connectivity. To overcome this, we use the laminated outer murine retina. We identify the serine/threonine kinase LKB1 as a key driver of synapse layer emergence. The absence of LKB1 in the retina caused a marked mislocalization and delay in synapse layer formation. In parallel, LKB1 modulated postsynaptic horizontal cell refinement and presynaptic photoreceptor axon growth. Mislocalized horizontal cell 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.


Sample-size estimation
• You should state whether an appropriate sample size was computed when the study was being designed • You should state the statistical method of sample size computation and any required assumptions • If no explicit power analysis was used, you should describe how you decided what sample (replicate) size (number) to use Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission:

Replicates
• You should report how often each experiment was performed • You should include a definition of biological versus technical replication • The data obtained should be provided and sufficient information should be provided to indicate the number of independent biological and/or technical replicates • If you encountered any outliers, you should describe how these were handled • Criteria for exclusion/inclusion of data should be clearly stated • High-throughput sequence data should be uploaded before submission, with a private link for reviewers provided (these are available from both GEO and ArrayExpress) Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: No sample size was computed when the study was designed. Full retina knockout phenotypes were confirmed using two different Cres (Six3-Cre and Chx10-Cre). At least two litters per genotype and time point were used. Sample size can be found in the figure legends.
For all experiments, three to eight animals per genotype from at least two litters were used in this study at each time point, with wildtype littermates used as controls. Three to four images per animal were obtained for quantification, and all images were acquired at equivalent retinal eccentricities from the optic nerve head. There were no outliers. This information can be found in the Materials and Methods section.

Statistical reporting
• Statistical analysis methods should be described and justified • Raw data should be presented in figures whenever informative to do so (typically when N per group is less than 10) • For each experiment, you should identify the statistical tests used, exact values of N, definitions of center, methods of multiple test correction, and dispersion and precision measures (e.g., mean, median, SD, SEM, confidence intervals; and, for the major substantive results, a measure of effect size (e.g., Pearson's r, Cohen's d) • Report exact p-values wherever possible alongside the summary statistics and 95% confidence intervals. These should be reported for all key questions and not only when the p-value is less than 0.05.
Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: (For large datasets, or papers with a very large number of statistical tests, you may upload a single table file with tests, Ns, etc., with reference to sections in the manuscript.)

Group allocation
• Indicate how samples were allocated into experimental groups (in the case of clinical studies, please specify allocation to treatment method); if randomization was used, please also state if restricted randomization was applied • Indicate if masking was used during group allocation, data collection and/or data analysis Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: Additional data files ("source data") • We encourage you to upload relevant additional data files, such as numerical data that are represented as a graph in a figure, or as a summary table • Where provided, these should be in the most useful format, and they can be uploaded as "Source data" files linked to a main figure or table • Include model definition files including the full list of parameters used • Include code used for data analysis (e.g., R, MatLab) • Avoid stating that data files are "available upon request" Please indicate the figures or tables for which source data files have been provided: The types of statistical analysis used in this study is reported in the Materials in Methods section under the subheading Statistical analysis as well as in each figure legend.
LKB1 F/F mice from the same litters were used as littermate controls, while LKB1 F/F Chx10-Cre or Cx57-iCre were used as experimental animals. AMPKα1 F/F α2 F/F mice from the same litters were used as littermate controls, while AMPKα1 F/F α2 F/F Chx10-Cre were used as experimental animals. This is detailed within the Materials and Methods section.
Source data files are included for figures 1, 2, 3, and 4, as well as figure 1-figure supplement 2.