Fig. 1

Generating creb3l1 mutant zebrafish. a Schematic representation of constructs expressing full-length zebrafish Creb3l (expressed in creb3l1+/+ fish), a fragment encoding amino acids 1-379 (1-379) that is generated in vivo by S2P cleavage, and the CRISPR/Cas9-generated TA fragment (expressed in creb3l1TA+/TA+ fish). Creb3l1 domains are represented by different colors and the sites cleaved by S1P and S2P are indicated. The 56 random amino acids at the C-terminus of the TA fragment are in green. b Exon 9 of the zebrafish creb3l1 gene was targeted (gRNA sequence is shown) to generate creb3l1TA+/TA+ fish with a +4 bp insertion (5 bp deletion and 9 bp insertion) that causes a frameshift mutation at amino acid 375 (T to I in the AST sequence, boxed in red). The creb3l1TA+/TA+ allele encodes a 375 amino acid fragment of Creb3l1 that contains the TA domain followed by 56 amino acids (in green) not found in Creb3l1. ccreb3l1 mRNA levels were assessed in creb3l1+/+ and creb3l1TA+/TA+ 7 dpf larvae. The creb3l1TA+/TA+ larvae show elevated levels of creb3l1 transcript, but the increase is not statistically significant when the dCq values are compared by an unpaired t-test. n = 4–5. Each data point represents a pool of ∼30 larvae. d HEK cells were mock transfected (lane NT: not transfected), or transfected with empty vector (lane EV), vector encoding wild-type full-length Creb3l1 (lane FL), vector encoding the TA+ fragment (lane TA+), or vector encoding the 1-397 construct (lane 1-379) shown in (a) (each construct tagged with HA at C-terminus). After 24 h, cells were lysed and the lysates analyzed by SDS-PAGE and Western blotting with anti-HA (to detect the construct) and anti-BIG2 (loading control). Bands of the appropriate MW were detected in the relevant lanes, but not in lanes containing untransfected cells or cells transfected with empty vectors, showcasing the specificity of the immunoblot. e–g HeLa cells were transfected with vectors encoding wild-type Creb3l1 (e), the 1-397 construct (f), or the TA+ fragment (g) shown in (a) (each construct tagged with HA at C-terminus), and after 24 h, cells were processed for double immune-fluorescence with anti-HA (to detect the construct) and anti-GM130 (Golgi marker), and stained with DAPI to mark the nuclei. e Wild-type full-length Creb3l1 localizes to ER, as indicated by its presence in ER continuous with the nuclear membrane (left panel, white arrows) and in anastomosing ER tubules in the cell periphery (right panel). N = 60 cells. f The 1-397 fragment is concentrated in the nucleus (left panel; right panel shows only the 1-397 signal), indicating efficient nuclear import. n = 25. g The TA+ fragment is detected in amorphous cytoplasmic foci (left panel, white arrows). In cells expressing higher levels of TA+, the protein is sequestered in cytoplasmic aggregates (right panel, white arrows). N = 60. Scale bars = 10 μm. hgolga2 expression in wild-type creb3l1+/+ and mutant creb3l1T+T/+ regenerates at 5 dpa. An unpaired t-test to compare the dCq values shows a significant decrease in golga2 transcript levels in creb3l1TA+/TA+ fish. p < 0.0001.