Effects of NDRG1 family proteins on photoreceptor outer segment morphology in zebrafish

Rods and cones are functionally and morphologically distinct. We previously identified N-myc downstream-regulated gene 1b (ndrg1b) in carp as a cone-specific gene. Here, we show that NDRG1b and its paralog, NDRG1a-1, contribute to photoreceptor outer segment (OS) formation in zebrafish. In adult zebrafish photoreceptors, NDRG1a-1 was localized in the entire cone plasma membranes, and also in rod plasma membranes except its OS. NDRG1b was expressed specifically in cones in the entire plasma membranes. In a developing retina, NDRG1a-1 was expressed in the photoreceptor layer, and NDRG1b in the photoreceptor layer plus inner nuclear layer. Based on our primary knockdown study suggesting that both proteins are involved in normal rod and cone OS development, NDRG1a-1 was overexpressed or NDRG1b was ectopically expressed in rods. These forced-expression studies in the transgenic fish confirmed the effect of these proteins on the OS morphology: rod OS morphology changed from cylindrical to tapered shape. These taper-shaped rod OSs were not stained with N,N’-didansyl cystine that effectively labels infolded membrane structure of cone OS. The result shows that rod OS membrane structure is preserved in these taper-shaped OSs and therefore, suggests that tapered OS morphology is not related to the infolded membrane structure in cone OS.


Identification of three NDRG1 family mRNAs
To detect the expression of zebrafish ndrg1 family mRNAs, coding sequence of each of the known zebrafish orthologs (NM_001128353.1 for ndrg1a-1, NM_213348.3 for ndrg1a-2, and NM_200692.2 for ndrg1b) was amplified by reverse transcription (RT)-PCR. First-strand cDNA was synthesized from zebrafish adult eye total RNA by reverse transcription using SuperScript III reverse transcriptase (Invitrogen) with an oligo(dT)18 primer. Primer sets used for PCR to obtain full-length NDRG1a family genes are listed in Supplementary Table S1. The PCR products were cloned into pGEM-T-Easy vector (Promega) and amplified.

Generation of antisera
Partial peptides or a whole protein (Fig. S1c) was used to raise antisera. To raise the antiserum against NDRG1a-1 protein and that against NDRG1a-2 protein, we prepared two partial peptides of NDRG1a-1 (Met1-Ala17) and NDRG1a-2 (Met1-Lys30), respectively. NDRG1a-1 and NDRG1a-2 are different only in these sequences. We also prepared a peptide of a common sequence in both NDRG1a-1 and NDRG1a-2 at their C-terminal 54 amino acids. These peptides were expressed as N-terminally GST-fused ones in E. coli BL21 (DE3). For the expression of these peptides, a DNA sequence corresponding to each peptide was PCR-amplified with a primer set containing EcoRI site in the forward primer and SalI site in the reverse primer, subcloned into a pGEM-T-Easy vector and amplified in E. coli XL1 Blue. After the sequence was verified, the vector was cut with EcoRI and SalI. Then the insert was ligated to the corresponding sites of pGEX-5X-1 (GE Healthcare) to obtain an N-terminally GST-fused product. Each plasmid was transformed into E. coli BL21 (DE3) for expression. To raise antiserum against NDRG1b protein, we prepared N-terminally GST-fused NDRG1b whole protein similarly as the above peptides. In case when a DNA used for expression of a partial peptide or NDRG1b protein contains a restriction site for NcoI, XhoI or SalI, we replaced the corresponding triplet with that coding the same amino acid but consisting of a different set of a triplet (silence mutation) to obtain a proper product.
A suspension of E. coli expressing the GST-fused partial peptides or a whole protein was sonicated, and the supernatant was collected and purified according to the manufacturer's instruction. A peptide or a protein was mixed with Freund's Adjuvant (Sigma-Aldrich) and about 100-200 µg of each peptide or a protein was used to immunize ddY mice.
For this, each of cDNA of NDRG1 family proteins was ligated to EcoRI/SalI sites of pMAL-C2E (New England Biolabs). As a control, we also obtained MBP by introducing a stop codon immediate downstream of DDDDK, a linker to the protein to be expressed. All of the proteins were expressed in E. coli BL21 (DE3) and purified according to the manufacturer's protocol. These proteins were then subjected to size-fractionation and the largest bands were collected and stored at -80°C until use.
NDRG1a-2 was prepared by amplifying the cDNA by PCR containing NcoI and XhoI sites, inserted into the corresponding sites in pET-16b (Novagen). Subsequently this protein was expressed in E. coli BL21 (DE3).
Selectivity of each of antiserum used was examined with immunoblot as shown in Supplementary Fig. 1d.
In addition to this, we also confirmed the selectivity of anti-NDRG1a-1 antiserum and that of anti-NDRG1b antiserum immunohistochemically by checking the selective reduction of the corresponding protein expression level in fish injected with morpholino(s) against NDRG1a-1 and NDRG1b, respectively.
Selectivity of NDRG1a-2 antiserum was also confirmed from the fact that it did not show the signal in wildtype rods ( Fig. 1c and d, and Supplementary Fig. S2b) but did show it in rods where NDRG1a-2 was forced to be expressed (Fig. 4d).
To examine the expression of NDRG1 family proteins with immunoblot in the zebrafish retina, typically ~20 sheets of adult zebrafish retinas were collected. The fish had been dark-adapted overnight, anesthetized and decapitated in ice-chilled water in the dark. Then, both eyes were detached and retinas were enucleated under a stereomicroscope in dim light. The retinas were solubilized by homogenization in sample buffer (50 mM Tris-HCl, pH 6.8, 2% [w/v] SDS, 10% [w/v] glycerol, 0.0025% bromophenol blue, 1.25% [v/v] ² -mercaptoethanol), heated for 10 mins at 95°C, chilled on ice, snap-frozen and stored at -80°C until use.
Immunoblot analysis was made as described previously 1 .

Immunohistochemistry and immunocytochemistry
Expression of NDRG1 family proteins in the retina and their subcellular localization were examined basically according to the method reported previously 2 . Light-adapted zebrafish adult eyes were fixed with a solution containing 4% paraformaldehyde in phosphate-buffered saline (PBS; 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 140 mM NaCl, 2.7 mM KCl, pH 7.3) for an hour at 4°C. Then the solution was replaced sequentially with PBS containing 10% sucrose (10% sucrose/PBS), 20% sucrose/PBS for at least 2 hrs each and subsequently 30% sucrose/PBS overnight. Finally the tissues were immersed and embedded overnight in a mixture of OCT compound (Tissue tech) and 30% sucrose/PBS at a 1:1 ratio, and stored at -80°C until use.
For larvae, eyes were prepared in the same way as adult eyes except that larval whole bodies at 48 hours postfertilization (hpf), 54 hpf, 60 hpf and 72 hpf were fixed for 13.5 hrs, while at 96 hpf and 6 days postfertilization (dpf) they were fixed for 18 hrs and 24 hrs, respectively.
The fixed eyes cryosectioned at 10 µm thickness were placed on a MAS-coated slide (Matsunami, Osaka, Japan) and dried for 2.5 hrs. After washing out the resin with PBS containing 0.5% Triton X-100 (PBST) three times, the sections were treated overnight at 4°C with a blocking reagent (5% normal goat serum in PBST).
After the sections were washed with PBST three times, the samples were incubated overnight at 4°C with antiserum we prepared and when necessary, with antibody commercially available, in the blocking reagent.
Primary antisera and antibodies used in this study were as follows: specific anti-NDRG1a-1 antiserum Expression of each NDRG1 protein in single isolated cells was examined basically according to the method reported previously 3 . Rods and cones isolated from the retina (see Methods) were attached to a glass slide by centrifugation (300 rpm, 2 min; Cytopro, ELITech). The cells were fixed with 100% methanol for 2.5 min at room temperature and treated for another 1 min with Ringer's solution (119.9 mM NaCl, 2.6 mM KCl, 0.5 mM CaCl 2 , 0.5 mM MgCl 2 , 0.5 mM MgSO 4 , 1 mM NaHCO 3 , 16 mM glucose, 0.5 mM NaH 2 PO 4 , 4 mM HEPES, pH 7.5) containing 0.005% Triton X-100 and 5% normal goat serum for permeabilization and blocking, respectively. Other procedures were the same as the immunostaining of the retina in the eye as stated above except that Ringer's solution was used instead of PBST.
The eye sections or isolated cells were observed using an inverted confocal microscope (LSM510META, Zeiss) with plan neofluar 20x/NA 0.5 and C-APO 40x/NA 1.2 water immersion objective lenses.

Analysis of temporal expression patterns of NDRG1 family genes and proteins
Forty zebrafish larvae were collected each time at ~12 hour intervals from 1 hpf to 96 hpf, snap-frozen in liquid nitrogen and stored at -80°C. Frozen larvae were homogenized with 250 µL of TRI Reagent (Sigma-Aldrich) and total RNA was isolated according to the manufacture's instruction. Unfertilized eggs were also used. A total of 1 µg of RNA was primed with an oligo(dT)18 primer and reverse-transcribed with SuperScript III RTase (Thermo Fisher Scientific) or water as a negative control at 42°C for an hour and cDNAs were stored at -80°C until use. RT-PCR was performed using 30 ng of cDNA templates at 94°C for 30 sec, then at 58°C for 30 sec and finally at 72°C for 40 sec. The reactions were repeated for 35 cycles. Primers used for the RT-PCR were listed in Supplementary Table S2.

Generation of transgenic zebrafish
Silence mutations were introduced by site-directed mutagenesis prior to construction of an expression vector if each gene had NcoI, XhoI, NotI and/or SalI restriction site in its coding sequence. To identify cones readily, mCherry was expressed in cones in the membrane-bound and prenylated form in some of the studies.
The construct, mCherry-HrasCAAX, where CAAX is a prenylation sequence, was produced by the Tol2kit 4 .
First, mCherry sequence was amplified from pmCherry-N1 (Clontech) by the primer sets in which HrasCAAX sequence was contained in the reverse primer and mCherry-HrasCAAX was subcloned into pGEM-T-Easy vector to amplify. The insert was excised with NcoI and NotI, and ligated to the downstream of a vector containing a promotor region of cone specific transducin α subunit (T±CP) 5 . NDRG1 family proteins were overexpressed (NDRG1a-1) or ectopically expressed (NDRG1a-2 and NDRG1b) in zebrafish rods. As a control, zebrafish expressing unmodified mCherry in rods was produced. To establish these transgenic lines, coding sequence of each protein was amplified by PCR with the primer sets containing NcoI site in a forward primer and XhoI site in a reverse primer, and subcloned into pGEM-T-Easy vector to amplify. The inserts were then excised with NcoI and NotI, and ligated to the downstream next to the rhodopsin promoter in a pCR2.1-TOPO vector (Invitrogen) 6  Medium containing 1-phenyl-2-thiourea, and those larvae (F 1 ) carrying the transgene heterologously (+/-) were selected at 3 dpf and further raised until adult. Transgenic zebrafish lines were maintained by selecting these heterologous fish after crossing them with wildtype zebrafish.

Image analysis and statistical analysis
Acquired images shown in Supplementary Fig. S3 were analyzed with ImageJ (Rasband, W.S., ImageJ, National Institutes of Health, Bethesda, MD). Signal intensity of each pixel in the image was converted to an 8-bit representation. Immunopositive area was surrounded with a line manually by eye to determine the size of the area and also the total signal intensity in this area. To determine the concentration of the pigment, the average intensity of an opsin signal was obtained with dividing the total signal intensity by corresponding immunopositive area. Data are presented as mean ± standard deviation, and levels of significance (P value, Student's t-test) are indicated in the figure legends.

N, N'-Didansyl cystine staining
To examine whether rods with tapered OS show the OS similar to that of a cone, we stained the rods and cones with N,N'-didansyl cystine (DDC, Sigma-Aldrich) which has been shown to label cone OS much more effectively than rod OS 8