DEV011262 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Expression of hypothalamic genes relative to DA neurons. In situ hybrydization with RNA probes directed against fezl (a,b), otpb (c), pacab1 (e,f,h) and pac1 (g) followed by immunostaining with an anti-tyrosine hydroxylase (TH) antibody. (D) Sagittal paraffin sections (6 µM) of an embryo which was subjected to in situ hybridization with an otpb- directed probe followed by anti-TH immunohistochemistry. HB, hindbrain; hpf, hours post fertilization; Hyp, hypothalamus; MB, midbrain; NPO, neurosecretory preoptic area; PT, posterior tuberculum; Tel, telencephalon.

• Supplemental Figure S2 -

  Fig. S2. In situ hybridization with pac1, isotocin (it) and fezl RNA probes. (A,B) High resolution micrographs (lateral view) of embryos which were subjected to in situ hybridization with RNA probes directed against pac1 followed by anti-TH immunohistochemistry. (C) Double in situ hybridization with RNA probes directed against isotocin (it) and fezl (ventral view). (D,E) Wild type (WT; D) and tof^m808 (E) mutant with antisense fezl probe followed by anti-TH immunohistochemistry.

• Supplemental Figure S3 -

  Fig. S3. Antisense morpholinos to otpb, pac1 and pacap1b affect dopamine transporter (DAT)-positive DA neurons. (a-c,E,F) Embryos were injected with either a buffer solution (WT; A,E), otpb morpholino (otpbMO; B,F) or pac1 morpholino (pac1MO; c) and were thereafter subjected to in situ hybridization with either a dopamine transporter (dat)-directed probe (A-C; at 52 hpf) or dat+prolactin (PRL)-directed probes (E,F; at 72 hpf). The two prominent clusters of dopaminergic (DA) neurons (Gr. 2 and Gr. 3-6), are indicated. (d,G) Histograms showing the effects of otpb, pac1 and pacap1b morpholinos on average cell counts of DAT⁺ dopaminergic (DA) and isotocinergic (IT) neurons. Bars indicate the standard deviation. The number of embryos (n) is shown below. To represent unilateral cell number (as shown in the micrographs), neurons were counted on both sides of the brain and the total number was divided by two. Scale bar: 50 µm.

• Supplemental Figure S4 -

  Fig. S4. PACAP1b affects Otp protein levels. (a-D) Embryos were injected with either a buffer solution (WT, a,C) or pacap1b morpholino (pacap1bMO; B,D) and were thereafter (52 hours post injection) subjected to either in situ hybridization with an otpb-directed probe (a,B), or to immunohistochemistry with an anti-Otp antibody (C, D). The activity of pacap1bMO in each embryo was then evaluated by anti-tyrosine hydroxylase (TH) immunostaining. (E) Nuclear-enriched protein extracts from mock (WT), pac1MO and pacap1bMO injected embryos were subjected to western blot analysis with either anti-Otp antibody or anti-Rcc1 (internal control for a ubiquitous nuclear protein). (F) Specificity of the anti-Otp antibody. HEK293 cells were transfected with a cDNA encoding to the Otpb protein and monolayers were cultured for 24 hours before protein extraction and western blot. Specific immunoreactivity against a major protein band of ∼50 kDa was demonstrated by peptide antibody displacement. PVDF filters containing the fractionated cell lysate were then subjected to immunobloting (IB) with an affinity-purified anti-Otp antibody, which was either pre-incubated (+) with a synthetic Otp peptide or not (−). Ab, antibody; IB, immunoblot. Scale bars: (a,B) 100 µm; (C,D) 50 µm.

• Supplemental Figure S5 -

  Fig. S5. Effects of otpa antisense RNA probes and otpb mRNA. Wild-type (WT; a) embryos and their too few^m808 (tof^m808) mutant siblings (B) were fixed at 52 hours post fertilization and subjected to whole-mount in situ hybridization with antisense RNA probes directed against otpa followed by immunostaining with an antibody directed against tyrosine hydroxylase (TH). Black arrow heads mark deficient otpa expression domains. (C-F) Embryos were injected with either a buffer solution (Control, C,E) or otpb mRNA (E,F) and were thereafter (48 hours post injection) subjected to either in situ hybridization with probes directed to either pac1 (C,D) or pacap1b (E,F). HB, hindbrain; PT, posterior tuberculum; Tel, telencephalon. Scale bars, 50 µm.

• Supplemental Figure S6 -

  Fig. S6. Effects of fezl-directed splice-blocking morpholinos. (a) Gel electrophoresis image shows defects in the splicing process following injection of splice-blocking morpholinos (MO; denoted sp1 and sp3). Blue arrows point to the correctly spliced mRNA, whereas red arrows indicate altered splicing products. (b) Table and scheme summarizing fezl gene structure, binding sites of morpholinos and PCR oligonucleotides and the expected mRNA size in the control and mortholino (splice-blocked) injected embryos.

• Supplemental Figure S7 -

  Fig. S7. Effects of otpb-directed splice-blocking morpholino. (a) Gel electrophoresis image shows defects in the splicing process following injection of otpb morpholino (MO; denoted sp2). Blue arrows point to the correctly spliced mRNA, whereas red arrows indicate altered splicing products. (b) Table and scheme summarizing otpb gene structure, binding sites of morpholino and PCR oligonucleotide and the expected mRNA size in the control and morpholino (splice-blocking) injected embryos.

• Supplemental Figure S8 -

  Fig. S8. Identification of PAC1 translation start site. (a,b) partial DNA and protein sequences of PAC1. In order to reveal the ATG translation start site of PAC1, we performed 5′ rapid amplification of cDNA ends (RACE). Sequencing of the resulting RACE fragment (a) followed by BLAT analysis against the genome database revealed the PAC1 translation start site. This new sequence (marked in yellow) is encoded by previously not annotated exons 1,2 and part of exon 3 and is translated to a 56 amino acid stretch (B, marked in yellow), which is highly homologous to the N-terminal protein sequence of other species as well as to another gene family member from zebrafish. The novel and previously annotated translation start sites of this copy of PAC1 gene are underlined.

• Supplemental Figure S9 -

  Fig. S9. Pulse-chase analysis of Otp protein stability. PC12 cells were incubated with methionine-free medium for 2 hours followed by pulse-labeling with medium containing radio-labeled ³⁵Smethionine (at 100 µCi/ml) for a period of 40 minutes. Thereafter, cells were chased with cold methionine and cyclohehimide (CHX, at 50 µg/ml) for the indicated time periods, in the presence or absence of synthetic PACAP₃₈ peptide. Protein extracts were made at the indicated times and equal amounts of total protein were subjected to immunoprecipitation with an anti-Otp antibody followed by gel electrophoresis, autoradiography and densitometry.