Wang et al. 10.1073/pnas.0609708104. |
Fig. 7. Structure of the Smed-nanos gene and protein. (A) Exon--intron map of Smed-nanos. The five exons are depicted by rectangles (shaded, ORF; unshaded, UTR); the four introns are depicted by lines, with the nucleotide size listed below. Alternative splicing leads to the 15-nucleotide insertion at the beginning of the second exon. (B) Alignment of conserved C-terminal region of selected NANOS family members: mouse (NANOS1_Mouse, BAC76003), Human (NANOS1_Human, AAL36982), Xenopus (XCAT-2, CAA51067), S. mediterranea (Smed-NANOS), and Drosophila (NANOS, AAA28715). NANOS zinc finger RNA-binding motif is indicated by the black line; asterisks indicate the most conserved C and H residues in the motifs. (C) Northern blot of total RNA from sexual and asexual strains of S. mediterranea. Smed-nanos RNA is detected in both strains; rRNA is shown as a loading control.
Fig. 8. Expression of Smed-nanos and germinal histone H4 in newly hatched animals. (A and B) Expression of germinal histone H4 (A) and anosmin-1 (AY066061) (B) in day 1 (D1) hatchlings. (C--E) In situ hybridization to Smed-nanos in D1 (C), D3 (D), and D7 (E) hatchlings. (Inset D, and F) High-magnification differential interference contrast microscopic views of the Smed-nanos-positive clusters observed in D and E, respectively. Total numbers of Smed-nanos-positive animals are: 4 of 38 on D1; 17 of 21 on D3; and 9 of 9 on D7. [Scale bar, 500 mm (A--C and E); 40 mm (D and F).] (G and H) Whole-mount in situ hybridization to Smed-nanos in D3 hatchlings. (G) Nonirradiated control. Five of five animals have Smed-nanos-positive cells. (H) Animals irradiated on D1. None of the 13 animals has Smed-nanos-positive cells. (Scale bar, 500 mm.) (I and J) Whole-mount FISH to germinal histone H4 in D1 (I) and D3 hatchlings (J). One of seven D1 hatchling and 4/6 D3 hatchlings have germinal histone H4-positive dorsal clusters (indicated by arrows). Images show the tail region, anterior is to the upper left. (Scale bar, 100 mm.)
Fig. 9. Inhibition of gene expression in the testes of S. mediterranea hermaphrodites by ingestion of bacterially expressed dsRNA. (A--C) Control animals showed normal testes expression of Smed-nanos (EF035555), T-plastin (DN311193), and Smedwi-2 (DQ186986), respectively (n = 5 for each treatment). (D--F) Double-stranded RNA-fed animals showed dramatically decreased expression of targeted genes in the testes (n = 5 for each treatment). (Scale bar, 1 mm.) (G and H) Transverse cryosections (20 mm) of animals in Fig. 4 I and J at pharyngeal region (ph). Control animal (G) has normal testes morphology with the compacted chromatin of the spermatozoa easily visible by DAPI staining (arrows). No testes structures were detected in Smed-nanos RNAi animal (H). (Scale bar, 60 mm.)
Fig. 10. Presumptive germ cell clusters in asexual planarians visualized by Smed-nanos and germinal histone H4 FISH and double labeling with germinal H4 FISH and the neoblast marker, anti-SMEDWI -1. (A) In situ hybridization to Smed-nanos (n = 10). (B) In situ hybridization to germinal histone H4 (n = 8); the germinal histone H4-positive cell clusters and the somatic neoblast staining were eliminated after g-irradiation. (Scale bars, 200 mm.) (A and B Insets) High-magnification views of the boxed areas. (Inset scale bars, 40 mm.) (C--E) Whole-mount fluorescent in situ to germinal histone H4 and immunofluorescence with anti-SMEDWI-1. (C) germinal histone H4 mRNA was detected in mesenchymal cells behind or surrounding the photoreceptors. (D) Immunofluorescence detected SMEDWI-1 protein in mesenchymal cells both behind and in front of the photoreceptors, the latter of which are in the process of differentiation. (E) Overlay of C and D. (Scale bar, 200 mm.) Orientation of all panels: anterior to upper left.
SI Methods
Isolation of Smed-nanos.
Early in the Schmidtea mediterranea genome project we queried the available whole-genome shotgun reads (Washington University Genome Sequencing Center, St. Louis, MO) by tblastn using the mouse NANOS1 (Q80WY3) and Drosophila NANOS (P25724) sequences and obtained a single read (TI 314542315) containing the sequence predicted to encode the Nanos-zinc finger motif. We designed primers corresponding to this highly conserved motif to amplify the transcript from a directionally cloned cDNA library in pBluescript II SK(+) generated from S. mediterranea juvenile hermaphrodites (1). The gene-specific primers used were:5'- TTGATTGTATGAGCAAAGTCACC-3' (reverse outer),
5'-CACAATGGGCATGTATAATTACG-3' (reverse inner),
5'- GGAAGCATGGCCTGAAAAGC-3' (forward outer), and
5'- TTCGCAAAGAGAGTCATATTGAAC-3' (forward inner).
These primers were used in combination with standard primers M13 forward and reverse and modified primers to the T7 and T3 promoters
5'-CGCGTAATACGACTCACTATAGGG-3' and
5'-GCTATGACCATGATTACGCCAAGC-3', respectively.
A ~470-bp (forward) and a ~408-bp (reverse) fragment were obtained from the second round of amplification and were gel-purified by using Qiaex II resin (Qiagen, Valencia, CA), cloned into pCR II T-A cloning vector, and transformed into Oneshot TOP10F' competent cells (Invitrogen, Carlsbad, CA). Clones were checked for inserts by colony PCR; plasmid DNA was then purified by miniprep (Wizard Plus SV minipreps, Promega, Madison, WI). Inserts were sequenced by using the standard Big Dye 3.1 sequencing reaction (Applied Biosystems, Foster City, CA) and were analyzed by using Sequencher 4.6 (Gene Codes Corporation, Ann Arbor, MI).
To generate a riboprobe, a modified M13 forward 5'-gcgaattcATAATTCTACATTGAC-3' and T7 5'-gcgaattcCATATAACATTGATTC-3' combined with the gene-specific reverse primers were used to reamplify Smed-nanos from cDNA. The PCR product was digested with EcoRI and BamHI and cloned into pBluescript II SK(+) (Invitrogen) and verified by sequencing. To obtain the full-length Smed-nanos sequence, additional rounds of 5' amplification from the cDNA library yielded a 579-bp fragment containing the remaining 5' sequence that matches the genomic sequence [supporting information (SI) Fig. 7].
Northern Blot Analysis.
Total RNA (5 mg) was separated in a formaldehyde/agarose gel. After electrophoresis, RNA was capillary transferred to Hybond N+ nylon membrane (Amersham Pharmacia, Piscataway, NJ) and UV cross-linked to the membrane by using a Spectrolinker XL-1500 (Spectronics Corporation, Westbury, NY). The digoxigenin-labeled RNA probe contained 678 bp, corresponding to the Smed-nanos gene. Hybridizations were carried out in DIG Easy Hyb (Roche, Indianapolis, IN) for 18 h at 68°C. The blot was washed twice in 2´ SSC/0.1% SDS at room temperature and twice in 0.5´ SSC/0.1% SDS 68°C. The hybridized probe was detected as described in ref. 2.Fluorescent in Situ Hybridization.
Whole-mount in situ hybridization was performed as described in ref. 1. After blocking, samples were incubated with 1:100 anti-Dig-POD (Roche) overnight at 4°C. After antibody incubation, samples were then washed five times for 1 h with MABT (100 mM maleic acid/150 mM mM NaCl/0.1% Tween 20, pH 7.5) followed by two 10-min washes with PBS. Samples were developed with 1:1,000 FITC-tyramide generated by using fluorescein mono N-hydroxysuccinimide-ester (46100; Pierce Chemical Co., Rockford, IL) and tyramide (T-2879; Sigma, St. Louis, MO) in the presence of 0.001% H2O2 in PBST (0.01% Tween 20) for 10-20 min. After developing, samples were washed with PBST for 1-2 days and then mounted with Vectashield (Vector Laboratories, Burlingame, CA). Samples were imaged with an AxioCam MRm mounted on a SteREO Lumar.V12 microscope (Zeiss, Thornwood, NY) and a CoolSnap HQ (PhotoMetrics, Huntington Beach, CA) camera mounted on CARV confocal/ Nikon TE2000 microscope. Within a given experiment (e.g., those shown in Fig. 3C and SI Fig. 8 I and J), samples were developed in fluorescent substrate for the same times and imaged using identical exposure conditions. A detailed protocol for FITC-tyramide synthesis is available from http://xenbase.org/methods.1. Zayas RM, Hernandez A, Habermann B, Wang Y, Stary JM, Newmark PA (2005) Proc Natl Acad Sci USA 102:18491-18496.
2. Zayas RM, Bold TD, Newmark PA (2005) Mol Biol Evol 22:2048-2054.