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Figure S1. the Identification of Two Type 1 Ca2+/Cam-Stimulated Adenylate Cyclases in Zebrafish and the Design of Morpholinos That Knockdown Their Expression

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Figure S1. the Identification of Two Type 1 Ca2+/Cam-Stimulated Adenylate Cyclases in Zebrafish and the Design of Morpholinos That Knockdown Their Expression Figure S1. The identification of two type 1 Ca2+/CaM-stimulated adenylate cyclases in zebrafish and the design of morpholinos that knockdown their expression. (A) Two close relatives of human ADCY1 (zADCY1a and zADCY1b) were identified and cloned in zebrafish. A sequence in ADCY1 corresponds to the known CaM-binding domain in mammalian ADCY1 (purple) and is highly conserved between human and zebrafish. Multiple morpholinos (MO) were designed to block the splicing of (B) ADCY1a and (C) ADCY1b pre-mRNAs. Morpholino target sequences (orange) correspond to junctions between introns (black lines) and exons (numbered blue boxes). RT-PCR between the primers indicated with black arrows was used to detect MO induced mRNA mis-splicing. Normal ADCY sequences are represented in black lettering and truncated sequences induced by misplicing in red. Stop codons are indicated with asterisks. Green ovals indicate the catalytic guanylate cyclase domains in ADCYs. The highest molecular weight bands generated by RT-PCR in the presence of control morpholinos arise from normally spliced transcripts, while lower molecular weight bands marked with arrowheads arise from morpholino induced mis-splicing. PCR products generated using primers recognizing EF1α in the same samples are shown below. (D-F) 36hpf embryos containing a Isl2b:GFP transgene were immunostained for GFP to visualize retinal ganglion cells (green). The expression of ADCY1 mRNAs was detected by in situ hybridization (red) with (D) a control probe, or with probes complementary to (E) ADCY1a or (F) ADCY1b. ADCY1b is expressed in the cell layer containing nascent retinal ganglion cells, while the expression level of ADCY1a is relatively low and most prominent in the ciliary margin of the eye. All photographs were taken identically and represent projections of Z series 5 μm thick. Anterior is to the top and all are viewed from the ventral side of the embryo. Scale bar: 50 μm. Figure S2. The topography of ipsilateral mis-projecting retinal axons is grossly normal in the tecta of zADCY8 morphants. zADCY8 morphants were fixed at 5dpf. Blue and red lipophilic dyes were injected either into the dorsal and ventral quadrants, or into the anterior and posterior quadrants of the retina. (A) A control preparation in which red dye traces retinal projections originating in the anterior retina and blue dye traces projections from the posterior retina. The contralateral tectum is boxed and axons are distributed in a normal topographic distribution. (B) Roughly the same topographic projection is established in the ipsilateral tectum (arrow) of a zADCY8 1 morphant embryo. (C) A control preparation in which red dye traces retinal projections originating in the dorsal retina and blue dye traces projections from the ventral retina. The contralateral tectum is boxed and axons are distributed in a normal topographic distribution. (D) A similar topographic projection is established in the ipsilateral tectum (arrow) of a zADCY8 morphant embryo. Scale bar: 100μm. Figure S3. Candidate midline retinal axon guidance cues and their receptors are expressed normally in zADCY8 morphants. (A-J') Control and zADCY8 morphant embryos were probed by in situ hybridization for potential midline guidance cues and their receptors. Control morphant Isl2b:GFP transgenic larvae at about 36hpf were compared to age matched zADCY8 morphant Isl2b:GFP transgenic larvae. (A-G') Ventral views or (H-J') dorsal views are shown of larvae reacted with Cyanine3-labeled tyromide to amplfy in situ signals (red) and immunostained for GFP to visualize retinal ganglion cells (green). The arrowheads in E' and G' indicates ipsilateral mis-projections. The arrows in H-J' indicate normal dorsal projections of retinal axons. In all cases, anterior is to the top. In situ probes included sonic hedgehog (shh), nkx2.2, semaphorin 3d (sema3d), neuropilin 1a (nrp1a), cxcr4b, robo2, slit2, slit1a, slit1b and slit3. Scale bar: 100μm. Figure S4. Knockdown of ADCY1b and ADCY8 synergize in the induction of ipsilateral retinal mis- projections. (A-E) Example images showing the typical brain and eye size of control, sema3a1, or ADCY morpholino treated zebrafish larvae. Embryos at the one to two-cell stage were micro-injected with the indicated morpholino, fixed at 5dpf, and images were taken with a dissecting scope. All images are lateral views with anterior to the right. The eyes of (B) sema3a1 and (C-E) ADCY morphants are smaller than that of control morphants. (F-J) Representative examples of retinal projections in control, sema3a1 and ADCY morpholino treated zebrafish larvae. Morpholino injected embryos were fixed at 5dpf and retinal axons were labeled with lipophilic dyes. All images are of dorsal views with anterior to the top. (F) In control morpholino treated larvae, all axons cross the midline and project contralaterally to the opposite tectum. Similarly, in (G) sema3a1 or (H) ADCY1a 2 morpholino treated larvae, all axons cross the midline and project contralaterally. However, in (I) ADCY1b morpholino treated larvae, some retinal axons fail to cross the midline and mis-project to ipsilateral tecta. (J) Knocking down ADCY1b and ADCY8 at the same time using a combination of morpholinos that by themselves have little effect induces ipsilateral misprojections in 28% of embryos. (I) The proportions of normal, weak ipsilateral (<50% axons mis-projected), and strong ipsilateral (>50% axons mis-projected) retinal projections induced by knocking down each target are indicated. The amount of each morpholino used per embryo is indicated within each column while the number of eyes examined in each condition are indicated above each column. Scale bar: 100μm. Table 1. ADCY sequences used to make in situ probes Sequences from the indicated Genebank accession numbers were used to probe for zebrafish ADCY1a, ADCY1b, and ADCY8. Table 2. Morpholino sequences used to knockdown ADCYs and the primer sequences used to perform RT-PCR to detect mis-spliced ADCY mRNAs. Sequences used to make morpholinos against zebrafish ADCY1a, ADCY1b, and ADCY8 are indicated along with the PCR primers that were used to monitor the slicing of their mRNA. 3 Figure S1 4 Figure S2 5 Figure S3 6 Figure S4 7 Table 1. ADCY sequences used to make in situ probes Gene name GeneBank accession number Corresponding part of nucleotides ADCY1a GU169394 1-1147; 1444-1976 ADCY1b GU169395 569-1562; 1504-3385 ADCY8 FJ472834 1-1108; 598-1802 Table 2. Morpholino sequences used to knockdown ADCYs and the primer sequences used to perform RT-PCR to detect mis-spliced ADCY mRNAs. Morpholino Morpholino Primer pair for RT-PCR to detect Expected size Name Sequence knockdown of truncated PCR product ZADCY1a 5'-CCTGACAATCAAAC Forw: 5'-CAAGATCTACATCCAGCGGCACG-3' 714-128=586bp MOE5 ACACAAACAAC-3' Rev: 5'-CAGGTCATGACGTTGGAGAAAGG-3' ZADCY1a 5'-CCAGACGTGATGAT The same as that for zADCY1a MOE5 714-142=572bp MOE7 GTGTTTGTACC-3' ZADCY1b 5'-AAACAAGATGCTGG Forw: 5'-GGAACTGCATCGAGGAGAGACTGC-3' 745-112=633bp MOE4 AAAACACACAC-3' Rev: 5'-GGTTTGATGTCTGACAGGATCAGACC-3' ZADCY1b 5'-GCACCTTCCTGAAA The same as that for zADCY1b MOE4 745-142=603bp MOE7 CCATCAGAGAC-3' ZADCY8 5'-AAGACAGAAATTAC Forw: 5'-TTTCTGGAGACACGCAGGTGCATCG-3' 577-112=465bp MOE3 CTCACGTTCTC-3' Rev: 5'-ATTCCCCCCGACTCCAGTTTGTTGG-3' ZADCY8 5'-AAGTGTGTTTACTT The same as that for zADCY8 MOE3 577-128=449bp MOE4 ACGTGTGCCAG-3' 8.
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