Fig 2: Gross comparison of ocular development in wild-type FVB/N mice and P0-3.9GFPCre, Le-Cre, and MLR10 transgenic mice. All genotypes exhibit grossly normal appearing eyes and lenses at all stages examined, with the exception of mice homozygous for the Le-Cre transgene (Le-CreHomo). Homozygous Le-Cre mice (middle column) exhibit externally obvious microphthalmia and small lenses at 3 weeks after birth (P21). Homozygous Le-Cre lenses exhibit consistent size reduction with evidence of fiber cell disorganization and nuclear retention that progressively worsens with age. As all mice were maintained on an FVB/N inbred background, all P21 eye sections exhibit photoreceptor degeneration from rd1 homozygous mutation [57]

Fig 3: DAGLB mediates the main 2-AG synthesis in the nigral DANs.a Diagram of AAV-mediated CRISPR/saCas9 gene targeting vector and the sequence of Dagla and Daglb sgRNAs. b Western blot of DAGLB in cultured cortical and hippocampal neurons transfected with control, Dagla, or Daglb KD AAV vectors in combination with Cre-expressing AAV vectors. Actin was used as a loading control. Four independent experiments were performed. c, d Bar graph quantifies DAGLB (c) and DAGLA (d) protein levels in four independent experiments. Data were presented as mean ± SEM. One-way ANOVA, multiple comparisons, adjusted ****p < 0.0001. e Schematic illustrates fiber photometry recording of 2-AG signals in the SN of DAN-control and DAN-Daglb KD mice. DLS: dorsolateral striatum. f Cartoon of eCB sensor eCB2.0. g Co-staining of eCB2.0, tdT and TH in the midbrain sections of DAN-Daglb KD mice. Scale bar: 100 μm. More than five independent experiments were performed. h Sample photon counts of eCB2.0 (wavelength: 500–540 nm) and tdT (wavelength: 575–650 nm) emission immediately before and 120 min after JZL184 (16 mg/kg) administration. i Time course of eCB2.0 signals in the SN of DAN-control [n = 4, 2 Male(M)/2 Female(F)] and DAN-Daglb KD (n = 4, 2M/2F) mice before and after JZL184 (16 mg/kg) treatment. Data were presented as mean ± SEM. Sidak’s multiple comparisons, adjusted ***p = 0.0001. ****p < 0.0001. j Dose response of eCB2.0 signals 120 min after JZL administration at 0 (vehicle only), 4, 16, and 40 mg/kg. n = 4 mice per group. Data were presented as mean ± SEM. Sidak’s multiple comparisons, adjusted ***p = 0.0003. ****p < 0.0001. k LC-MS/MS quantification of 2-AG in the SNc of DAN-control (n = 9) and DAN-Daglb KD (n = 12) mice. Data were presented as mean ± SEM. Unpaired t test, two-tailed, *p = 0.02.

Fig 4: DNA constructs used to make Cre-expressing transgenic mouse lines. Black arrows represent the transcription start sites for the mouse Pax6 P0 promoter (blue in the Le-Cre and P0-3.9GFPCre constructs) or the mouse αA-crystallin promoter (light blue in the MLR39 and MLR10 constructs). The pink and green boxes represent CRE and GFP coding sequences, respectively. The thin black line within the αA-crystallin promoter in the MLR10 construct represents the engineered Pax6 binding site. The orange EE and yellow RP boxes represent the Pax6 ectodermal enhancer element and the retina/pancreas enhancer elements, respectively. The IRES (white box in the Le-Cre construct) stands for internal ribosome entry site, and nlsCre stands for Cre recombinase with an added nuclear localization sequence. There is approximately 400 bp of mouse Pax6 sequence from the first intron included at the 3′ end of the Le-Cre transgenic construct, following the GFP coding sequence

Fig 5: Differential gene expression in hemizygous newborn Le-Cre and P0-3.9GFPCre lenses. The expression of transcripts expressed in newborn Le-Cre and P0-3.9GFPCre lenses were compared to newborn lens transcripts from the background FVB/N strain mice by RNA-Seq analysis. A Venn diagram (a) illustrates the total number of genes differentially expressed by the transgenic lenses versus the FVB/N lenses while the heatmap (b) lists all of the transcripts differentially expressed with red indicating overexpression and blue indicating underexpression. The Le-Cre lenses differentially expressed 16 genes while the P0-3.9GFPCre lenses differentially expressed only three genes. Both transgenic lenses overexpressed CRE and Hspb1 transcripts. The P0-3.9GFPCre lenses uniquely underexpressed Usp11. Many of the overexpressed transcripts in the Le-Cre lenses (Cdkn1a, Hmox1, Phlda3, and Trp53inp1) are associated with apoptosis and/or stress response (Hspb1, Ephx1)