W plan apochromat 20x 1

P15 in utero experiments used Gsk3a^+/+Gsk3b^loxp/loxp control Gsk3a^−/−Gsk3b^loxp/loxp or Gsk3a^loxp/loxpGsk3b^loxp/loxp embryos. Images for the dendritic arbor reconstruction were acquired on a Zeiss LSM 7 MP multiphoton system using a W-PlanApochromat 20x/1.0 IR-corrected water immersion objective. On average, Z-stacks were acquired from 90 to 100 μm range with 0.65 μm steps. Dendritic reconstructions were performed using Neurolucida. A total of 15 control and 15 Gsk3-deleted cells from three mice each were reconstructed for dendritic analysis. Apical and basal dendrite polarity was quantified by generating dendrograms (Neurolucida) from reconstructed images. Sholl analysis calculated the number of dendrite intersections and dendritic lengths, Sholl quantification was conducted with an initial 15-μm somal radius and 20 μm concentric radial steps.

Larvae were anesthetized in 0.2 mg/mL ethyl-3-aminobenzoic acid ethyl ester (MESAB, Sigma-Aldrich E10521, St. Louis, MO) prior to confocal imaging except where noted. Larvae were mounted dorsal side-up (axial view) or lateral side-up (sagittal view) in 2% low-melting point agarose (Thermo Fisher Scientific 16520) in E3. Images were collected on a Zeiss LSM800 confocal microscope with a 20x water-immersion objective (Zeiss W Plan-Apochromat 20x/1.0). Images of tangential nucleus soma and axons were acquired in a lateral mount with an 80×80 μm imaging window. Stacks spanned ~30–40 μm, sampled every 1 μm. Images of nIII/nIV motor neurons were acquired in a dorsal mount with a 213×106 μm imaging window; stacks spanned approximately 90 μm, sampled every 1.5 μm. Images to validate nIII/nIV expression in a lateral mount were acquired using a 319×319 μm imaging window. Raw image stacks were analyzed using Fiji/ImageJ^{124 (link)}.

Embryos at desired time point were anaesthetized using 0.16 mg/ml tricaine to stop heartbeat and then embedded into a ∼ 1 mm inner diameter glass capillary filled with 1.5% low-melting agarose (LMA) in embryo medium (0.03% Instant Ocean salt into double distilled water). Once agarose polymerized, the capillary was placed in the sample holder and inserted into a microscope chamber filled with embryo medium containing tricaine, and the embedded sample was pushed out of the capillary for imaging at a chamber temperature of 28 °C. Images were taken on ZEISS Light-sheet Z.1 with W Plan-Apochromat 20x/1.0. Z-stack (thickness 3.83 μm and exposure time 60 ms), saved in the LSM format, and then processed using ZEN software (Zeiss). For each z-stack, maximum intensity projections were generated. Confocal microscopy was performed on the LSM800 (Zeiss) by mounting embryos in 1.5% LMA in glass-bottom dishes.