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Dev Growth Differ
2018 Feb 01;602:112-120. doi: 10.1111/dgd.12421.
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Nanos3 of the frog Rana rugosa: Molecular cloning and characterization.
Kodama M
,
Yoshida M
,
Endo M
,
Kobayashi T
,
Oike A
,
Yasumasu S
,
Nakamura M
.
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Nanos is expressed in the primordial germ cells (PGCs) and also the germ cells of a variety of organisms as diverse as Drosophila, medaka fish, Xenopus and mouse. In Nanos3-deficient mice, PGCs fail to incorporate into the gonad and the size of the testis and ovary is thereby dramatically reduced. To elucidate the role of Nanos in an amphibian species, we cloned Nanos3 cDNA from the testis of the R. rugosa frog. RT-PCR analysis showed strong expression of Nanos3 mRNA in the testis of adult R. rugosa frogs, but expression was not sexually dimorphic during gonadal differentiation. In Nanos3-knockdown tadpoles produced by the CRISPR/Cas9 system, the number of germ cells decreased dramatically in the gonads of both male and female tadpoles before sex determination and thereafter. This was confirmed by three dimensional imaging of wild-type and Nanos3 knockdown gonads using serial sections immunostained for Vasa, a marker specific to germ cells. Taken together, these results suggest that Nanos3 protein function is conserved between R. rugosa and mouse.
F I G U R E 1 Number of germ cells
in embryos and tadpoles. Serial frozen
sections were prepared from embryos
and gonads of tadpoles at different
developmental stages and stained for
Vasa. The total number of germ cells was
counted under fluorescence microscopy.
The number of germ cells at different
stages is shown in the table on the right
(mean ± SE; n ≧ 3). Grey ( ♂ + ♀), white (
♂) and black ( ♀) columns indicate Vasa-positive
germ cells in; male plus female,
male, female, respectively . *p <.05
F I G U R E 2 Characterization of Rana
rugosa Nanos3. (a) Structure of Nanos3
protein. Numbers at the top of a column
indicate first and last amino acids and
the position of the zinc finger domain
(Zf-nanos)
is shown in grey for R. rugosa
Nanos3. (b) Comparison of amino acid
sequences of Zf-nanos
in R. rugosa with
other species of vertebrate. The predicted
amino acid sequence of R. rugosa Zf-nanos
was compared with other vertebrates
by alignment using Clustal W (http://
www.genome.jp/tools/clustalw/). Amino
acids identical among species are shown
in white letters on a black background.
Asterisks represent amino acids conserved
among all vertebrate species analyzed.
Acc No, of Nanos are as follows:
D. melanogaster nosA (NP_476658) and
nosB (NP_001262723); H. sapiens nos1
(NP_955631), nos2 (NP_001025032)
and nos3 (NP_001092092); M.
musculus nos1 (NP_848508), nos2
(NP_918953) and nos3 (NP_918948); O.
latipes nos1a (NP_001163808), nos1b
(NP_001153941), nos2 (NP_001153919)
and nos3 (NP_001116300); X. laevis
nos1 (NP_001081503); X. tropicalis
(NP_988857). (c) Phylogenetic tree of
Nanos families. The tree was constructed
by Clustal W with an unrooted program
(http//align.genome.jp/)
F I G U R E 3 Nanos3 expression. (a)
Nanos3 expression in various tissues. Total
RNA was prepared from eight different
tissues of adult frogs for PCR analysis.
The PCR products were electrophoresed
on 4.5% acrylamide gel and the gels were
stained in ethidium bromide to visualize
bands. (b) Nanos3 expression in the gonad
at various developmental stages. We
examined Nanos3 mRNA levels in male and
female gonads. Total RNA was prepared
from gonad/mesonephros complexes of
male (♂) and female (♀) tadpoles at St.25-1W
to -4W,
and from gonads at St. I, III,
and V
Nanos3 genepage may need to be disambiguated from LOC108710645.
F I G U R E 4 In situ hybridization analysis of Rana rugosa Nanos3 expression. In situ hybridization analysis was performed using whole tissues
and frozen sections (10 μm) from testes (a–e) and ovaries (f–j) of tadpoles at St. XV. Sections were stained for Vasa (d,i), and counterstained with
DAPI (e, j). Nanos3 mRNA expression was examined by whole-mount
(a and f) and in sections from whole-mount
tissues (b, c, g and h). a and f,
bar = 400 μm and others, bar = 10 μm
F I G U R E 5 Nanos3 gene mutagenesis.
(a) Targeting sites of Nanos3 mutagenesis.
The nucleotide sequence encoding the
Zf-nanos
domain is shown by a red box.
Numbers indicate nucleotide positions of
Rana rugosa Nanos3 cDNA (LC218725).
Two gRNA targeting sites are shown
at nucleotide positions 277–296, and
291–310 of the Nanos3 cDNA, as shown
in a dotted box. Nucleotide sequences
of the targeting sites are shown in a
magnified box with dotted lines. The
letters with green background indicate the
PAM. Amino acid sequences mutated in
targeting Nanos3 coding regions are shown
in Figure S1. (b) Fate of embryos injected
with gRNA/Cas9 mRNA. gRNA1/Cas9
mRNA (gRNA1) and gRNA2/Cas9 mRNA
(gRNA2) were injected into 521 and 686
fertilized eggs of R. rugosa, respectively.
The MMR buffer (None) was injected into
1,124 eggs as placebo control. Rates of
embryos appearing normal or abnormal
by St. 25-1W
are shown in columns with
numbers. (c) Appearance of tadpoles after
mutagenesis. Pictures show tadpole shape
after mutagenesis, developing normally or
abnormally in shape. Bar = 1 mm
F I G U R E 6 Immunohistology of Nanos3-KD
gonads. We prepared frozen sections (10 μm) from the Wt and Nanos3-KD
female (♀) and male
(♂) gonads of tadpoles at St. 25-1W
to -4W.
(a) Frozen sections were stained for Vasa (green) and counterstained with DAPI (blue; 1–12, 17–20),
and then subjected to Hematoxylin and Eosin (HE) staining (13–16 and 21–24). The magnified image of one Vasa-positive
cell is indicated by a
white arrowhead (10 and 18); the same cell is indicated by a black arrowhead in the figure showing HE staining (14 and 18). N, notochord and G,
gonad. Bar = 50 μm. (b) Frozen sections of tadpoles from Wt and Nanos3-KD
gonads of tadpoles at St. 25-4W
were stained for Vasa (26, 28, 30
and 32) and laminin (brown; 25, 27, 29 and 31). KD embryos in this figure were the same as the ones whose genome sequences are reported in
Figure S1. Gonads are indicated by dotted lines. G, gonad and O, ovarian cavity. Bar = 50 μm
F I G U R E 7 PGCs in Wt and Nanos3-KD
gonads. The total number of PGCs in the
Wt and Nanos3-KD
gonads was counted
under a fluorescent microscope after
staining frozen sections for Vasa. Wt male
( ♂, blue), Wt female ( ♀, red), Nanos3-KD
male ( ♂, light blue) and Nanos3-KD
female ( ♀, pink). Vertical bars indicate the
mean ± SE. *p < .05
