In insects, sex determination pathways involve three levels of master regulators: primary signals, which determine the sex; executors, which control sex-specific differentiation of tissues and organs; and transducers, which link the primary signals to the executors. The primary signals differ widely among insect species. In Diptera alone, several unrelated primary sex determiners have been identified. However, the doublesex (dsx) gene is highly conserved as the executor component across multiple insect orders. The transducer level shows an intermediate level of conservation. In many, but not all examined insects, a key transducer role is performed by transformer (tra), which controls sex-specific splicing of dsx. In Lepidoptera, studies of sex determination have focused on the lepidopteran model species Bombyx mori (the silkworm). In B. mori, the primary signal of sex determination cascade starts from Fem, a female-specific PIWI-interacting RNA, and its targeting gene Masc, which is apparently specific to and conserved among Lepidoptera. Tra has not been found in Lepidoptera. Instead, the B. mori PSI protein binds directly to dsx pre-mRNA and regulates its alternative splicing to produce male- and female-specific transcripts.
1. Introduction
The sex determination pathways in insects are diverse [1]. The diversity arises mostly from primary signals, whereas transducers and executors are more conserved [2][3]. In
The sex determination pathways in insects are diverse [2]. The diversity arises mostly from primary signals, whereas transducers and executors are more conserved [29,30]. In D. melanogaster
,
M. domestica
and
C. capitata
the primary sex determiner controls sex development via Tra-transduced signaling pathway, resulting in the alternative splicing of
dsx [4][5][6]. The transducer gene usually encodes a splicing factor, and manipulation by the primary signals therefore exists or functions as a sex determiner only in one sex. The products of the executor gene
[5,16,17]. The transducer gene usually encodes a splicing factor, and manipulation by the primary signals therefore exists or functions as a sex determiner only in one sex. The products of the executor gene dsx act downstream of these switches. There are female- and male-specific isoforms of DSX, which control sexual development in most insect species [7]. However, in the silkworm, the ortholog of
act downstream of these switches. There are female- and male-specific isoforms of DSX, which control sexual development in most insect species [31]. However, in the silkworm, the ortholog of tra
does not exist, and no
dsx cis-regulatory element binding sites are found, which indicates the sex determination pathways are markedly different from those seen in flies [8][9].
cis-regulatory element binding sites are found, which indicates the sex determination pathways are markedly different from those seen in flies [20,32].
In the lepidopteran model species
B. mori
, the primary signal is the PIWI-interacting RNA (piRNA)
Fem, which is derived from the W chromosome [10].
, which is derived from the W chromosome [24]. Fem
silences a gene unique to Lepidoptera,
Masc
, which is essential for both male determination and repression of the vital process of dosage compensation of Z-linked genes during embryogenesis (
) [11][12][13][14][15]. In recent years, sex-determined factors encoded by
) [21,26,33,34,35]. In recent years, sex-determined factors encoded by PSI
,
Znf-2, Siwi
and
Gtsf1 have been identified along with preliminary established genetic cascade of the sex determination in the silkworm [16][17][18][13][19].
have been identified along with preliminary established genetic cascade of the sex determination in the silkworm [25,27,28,33,36].
2. Feminizers, the Primary Signals
Research on sex determination factors in the silkworm began in 1933 when Hasimoto hypothesized that the F-factor originates from the W chromosome [20]. Subsequent genetic studies showed that one copy of the W chromosome is sufficient to determine femaleness [21]. However, in part due to the various transposable elements, their remnants, and simple repeats on W chromosome, the identity of the F-factor was a mystery [22][23]. Only recently was the F-factor identified when researchers used new generation sequencing technologies, bioinformatics and focused on non-coding RNAs (such as microRNAs, long non-coding RNAs and piRNAs). The F-factor is a single piRNA derived from a piRNA precursor named
Research on sex determination factors in the silkworm began in 1933 when Hasimoto hypothesized that the F-factor originates from the W chromosome [39]. Subsequent genetic studies showed that one copy of the W chromosome is sufficient to determine femaleness [40]. However, in part due to the various transposable elements, their remnants, and simple repeats on W chromosome, the identity of the F-factor was a mystery [41,42]. Only recently was the F-factor identified when researchers used new generation sequencing technologies, bioinformatics and focused on non-coding RNAs (such as microRNAs, long non-coding RNAs and piRNAs). The F-factor is a single piRNA derived from a piRNA precursor named Fem
, which is transcribed from the W chromosome.
Fem functions as the primary determinant of female sex in the silkworm [10][24][25][26][27][28].
functions as the primary determinant of female sex in the silkworm [24,43,44,45,46,47].
piRNAs are a class of small RNAs of 24–31 nucleotides in size. They are produced from transposons and from discrete genomic loci called piRNA clusters. The piRNAs guide PIWI proteins to target transcripts [29][30]. In flies, piRNAs and PIWI proteins mainly function in germ cells during gametogenesis to suppress transposable elements, which are selfish genomic elements that are able to jump around the genome [29][30]. It is fascinating that the
piRNAs are a class of small RNAs of 24–31 nucleotides in size. They are produced from transposons and from discrete genomic loci called piRNA clusters. The piRNAs guide PIWI proteins to target transcripts [48,49]. In flies, piRNAs and PIWI proteins mainly function in germ cells during gametogenesis to suppress transposable elements, which are selfish genomic elements that are able to jump around the genome [48,49]. It is fascinating that the Fem
-derived piRNA participates in sex determination, which is a somatic cell fate event in the silkworm. The piRNA pathway is not well characterized in vivo, and as a result how
Fem functions at the molecular level is still a puzzle [31]. piRNAs have no enzyme activity, and instead they assemble into piRNA-induced silencing complexes (piRISCs) with the PIWI proteins such as Siwi and Argonaute RISC Catalytic Component 3 (Ago3) identified in the silkworm [24]. After loading onto PIWI proteins, piRNAs are produced by a unique biogenesis pathway called the “ping-pong” cycle. The ping-pong cycle is a posttranscriptional gene-silencing mechanism in which RNAs degraded by piRNA-guided transcript silencing provide substrates for additional piRNA production [32]. We have discovered that SIWI is crucial for feminizing the silkworm, whereas Ago3 mutants display no phenotype involved in sex determination [16]. Our studies suggest that SIWI is dominant during
functions at the molecular level is still a puzzle [50]. piRNAs have no enzyme activity, and instead they assemble into piRNA-induced silencing complexes (piRISCs) with the PIWI proteins such as Siwi and Argonaute RISC Catalytic Component 3 (Ago3) identified in the silkworm [43]. After loading onto PIWI proteins, piRNAs are produced by a unique biogenesis pathway called the “ping-pong” cycle. The ping-pong cycle is a posttranscriptional gene-silencing mechanism in which RNAs degraded by piRNA-guided transcript silencing provide substrates for additional piRNA production [51]. We have discovered that SIWI is crucial for feminizing the silkworm, whereas Ago3 mutants display no phenotype involved in sex determination [25]. Our studies suggest that SIWI is dominant during Masc
mRNA silencing via
Fem
-piRNA, whereas Ago3 have minor effects on
Fem
piRNA processing.
The demonstration that piRNAs have a function in sex determination in the silkworm prompted us try to understand the intricate biogenesis of PIWI-interacting RNAs. The sex determination cascades, as well as piRNA pathways, are distinct in
D. melanogaster
and
B. mori [31]. The piRNA biogenesis pathway consists of a list of components which are specialized for their processing [33]. However, several key elements of the piRNA pathway in the fly, such as Yb, Rhino, Deadlock and Cutoff, which are crucial for piRNA transcription initiation and piRNA processing, are absent in the silkworm [33]. We have reported that a conserved component of the piRNA pathway called Gtsf1 is involved in female sex determination in the silkworm [18]. In
[50]. The piRNA biogenesis pathway consists of a list of components which are specialized for their processing [52]. However, several key elements of the piRNA pathway in the fly, such as Yb, Rhino, Deadlock and Cutoff, which are crucial for piRNA transcription initiation and piRNA processing, are absent in the silkworm [52]. We have reported that a conserved component of the piRNA pathway called Gtsf1 is involved in female sex determination in the silkworm [28]. In Drosophila, Gtsf1 is required for female fertility and interacts with PIWI via its C-terminal end; and it is also essential for piRISCs-induced transposon silencing but not for piRNA biogenesis [34][35]. In the silkworm Gtsf1 is not only necessary for transposon silencing but also for piRNA biogenesis [18]. In addition, our co-immunoprecipitation experiments suggested that Gtsf1 interacts with SIWI. Such an interaction was also observed in
, Gtsf1 is required for female fertility and interacts with PIWI via its C-terminal end; and it is also essential for piRISCs-induced transposon silencing but not for piRNA biogenesis [53,54]. In the silkworm Gtsf1 is not only necessary for transposon silencing but also for piRNA biogenesis [28]. In addition, our co-immunoprecipitation experiments suggested that Gtsf1 interacts with SIWI. Such an interaction was also observed in Drosophila [18][34][35]. Interestingly, not every component from the piRNA pathway participates in feminization. For instance, Maelstrom (Mael) is essential for spermatogenesis and oocyte development in
[28,53,54]. Interestingly, not every component from the piRNA pathway participates in feminization. For instance, Maelstrom (Mael) is essential for spermatogenesis and oocyte development in Drosophila as it is involved in piRNA-mediated silencing of transposable elements [36]. However, depletion of
as it is involved in piRNA-mediated silencing of transposable elements [55]. However, depletion of Mael in the silkworm leads to spermatogenesis defects but does not affect sexual development [37]. The
in the silkworm leads to spermatogenesis defects but does not affect sexual development [56]. The poly(A)-specific ribonuclease-like domain-containing
(
Pnldc1
) is necessary for piRNA maturation in silkworms, and mutation of
Pnldc1 leads to abnormalities in nuclei of cells in eupyrene sperm bundles but not cells of other organs [38][39]. We have generated transgenic lines using CRISPR-Cas9 technology with mutations in
leads to abnormalities in nuclei of cells in eupyrene sperm bundles but not cells of other organs [57,58]. We have generated transgenic lines using CRISPR-Cas9 technology with mutations in Zucchini
and
Papi
, which encode enzymes required for 3’-end processing of piRNAs, in
Tdrd12
and
Tudor-SN
, which are involved in ping-pong cycling, and in
Yu
,
Armi
and
Mino
, which encode chaperonins that function during piRNAs biogenesis, but none of these lines exhibit any obvious phenotypes (unpublished data). However, all these genes are crucial for piRNA processing in
Drosophila,
and their mutation will cause sterility, whereas our results indicate that the piRNA processing pathway is also different between silkworms and flies, and the processing of the primary signal
Fem-piRNA is independent of those elements [33]. Furthermore, we do not understand how
-piRNA is independent of those elements [52]. Furthermore, we do not understand how Fem
transcription is activated, and it is possible that piRNAs other than
Fem
or non-coding RNAs from W are involved in sex determination. If other non-coding RNAs from W are involved in the female sex determination of
B. mori
, their role is expected to be minor if compared with Fem. Indeed,
Fem
repression is sufficient to cause masculinization of
dsx
splicing in embryos.
3. Masculinizers, the Possible Transducers
Masc
is the target gene of the primary signal mediated by
Fem [10]. Evolutionary analysis revealed that
[24]. Evolutionary analysis revealed that Masc is conserved among the species in Lepidoptera but is not found in other insects, indicating that the sex determination pathway in Lepidoptera is likely distinct [40]. In the silkworm,
is conserved among the species in Lepidoptera but is not found in other insects, indicating that the sex determination pathway in Lepidoptera is likely distinct [18]. In the silkworm, Masc is located on the Z chromosome [10]. It encodes a CCCH-tandem zinc-finger protein and controls both masculinization and dosage compensation [41]. Recent studies have demonstrated that
is located on the Z chromosome [24]. It encodes a CCCH-tandem zinc-finger protein and controls both masculinization and dosage compensation [59]. Recent studies have demonstrated that Masc
is also required for masculinization in multiple lepidopterans including
Trilocha varians
,
Ostrinia furnacalis
and
Agrotis ipsilon [11][14][15]. Besides, it is interesting that in
[21,34,35]. Besides, it is interesting that in O. furnacalis
,
Wolbachia
-induced male-specific lethality is also caused by a failure of dosage compensation via suppression of
Masc [42]. However, it remains to be determined whether Masc functions as masculinizer among all the species in the Lepidoptera order. A novel splice variant of
[60]. However, it remains to be determined whether Masc functions as masculinizer among all the species in the Lepidoptera order. A novel splice variant of Masc
(
Masc-S
), which lacks the intact sequence of the cleavage site for
Fem
-piRNA, encodes a C-terminal truncated protein that has been identified in both sexes. The variant of
Masc
,
Masc-S, participates in female genital development in the silkworm [43]. Moreover, there is still a gap between Masc and
, participates in female genital development in the silkworm [61]. Moreover, there is still a gap between Masc and dsx
, and further investigations that identify the direct targets of Masc will be able to clarify its role.
In
Drosophila
females, female-specific
dsx is promoted by Tra and Tra-2 as trans-acting splicing activators [4][2] (
is promoted by Tra and Tra-2 as trans-acting splicing activators [5,29] ( E). In
E). In
Bombyx
, lacking
tra
ortholog, the sex-specific splicing regulation of the
dsx
gene is different (
F). A splicing factor might have replaced the function as Tra in
F). A splicing factor might have replaced the function as Tra in
transformer-less species during evolution [44][45][46]. PSI is a KH-domain RNA-binding splicing factor that regulates tissue-specific alternative splicing of P-element transposon transcripts to restrict transposition activity to germ-line tissues and that influences development and mating behavior in flies [47][48][49][50][51][52]. Though PSI is not involved in the sex determination pathway in
-less species during evolution [9,62,63]. PSI is a KH-domain RNA-binding splicing factor that regulates tissue-specific alternative splicing of P-element transposon transcripts to restrict transposition activity to germ-line tissues and that influences development and mating behavior in flies [64,65,66,67,68,69]. Though PSI is not involved in the sex determination pathway in Drosophila
, it is a key auxiliary factor in silkworm male sex determination. PSI binds to exon 4 of
dsx pre-mRNA and facilitates its male-specific splicing [19]. Genetic evidence, by using a transgenic CRISPR/Cas9 system, has shown that the mutation of
pre-mRNA and facilitates its male-specific splicing [36]. Genetic evidence, by using a transgenic CRISPR/Cas9 system, has shown that the mutation of PSI will cause partial male-to-female defects, whereas it has been demonstrated that it is indispensable for masculinization in the silkworm [13]. It is still unclear that
will cause partial male-to-female defects, whereas it has been demonstrated that it is indispensable for masculinization in the silkworm [33]. It is still unclear that PSI
affects only males, however, as it is present in both sexes. Besides, according to published reference genomes,
PSI is present in multiple lepidopterans, but whether it is involved in sex determination still remains to be identified [53]. Recently, it was reported that a
is present in multiple lepidopterans, but whether it is involved in sex determination still remains to be identified [70]. Recently, it was reported that a Znf-2
mutant has a phenotype similar to that of the
PSI loss-of-function mutant [17].
loss-of-function mutant [27]. Znf-2
encodes a CCCH-type zinc finger protein and contributes to male-specific splicing of
dsx in silkworm cell lines [54]. The
in silkworm cell lines [71]. The Znf-2
mutant males have feminized external genitalia, and the female isoform of
dsx
is detected in males, indicating that
Znf-2 is essential for male sexual differentiation [17]. In addition, larval development is delayed and body size diminished in
is essential for male sexual differentiation [27]. In addition, larval development is delayed and body size diminished in PSI
and
Znf-2
mutant males, demonstrating that these factors have functions in development in the silkworm.
Figure 12.
Comparison of the sex determination cascade between
Drosophila melanogaster
(
A
,
C
,
E
) and
Bombyx mori
(
B
,
D
,
F
). In female
D. melanogaster
, the dosage of several X-linked signal element proteins regulates the transcription initialization of
Sex-lethal
(
Sxl
). An autoregulatory feedback loop ensures continuous expression. Methyltransferase complex components also regulate the precise splicing of
Sxl
(
A) [55][56][57][58]. The
Sxl
protein (SXL) directly binds to
transformer
(
tra
) pre-mRNA and regulates its alternative splicing.
Male-specific-lethal 2 (Msl2)
is inhibited at the mRNA level (
C
). Functional
transformer
protein (TRA) with
transformer-2
protein (TRA2) together regulate
doublesex
(
dsx
) female-specific alternative splicing (
E) [4]. The female-specific DSX (DSXF) interacts with
) [5]. The female-specific DSX (DSXF) interacts with Intersex protein (IX) to promote female development [59]. In female
protein (IX) to promote female development [86]. In female B. mori
, the
Fem
inizer piRNA (
Fem
) derived from the female W chromosome is postulated to interact with the protein
Silkworm-PIWI
(SIWI) and
gametocyte-specific factor 1
(GTSF1) to form the piRNA-induced silencing complexes that silences
Masculinizer
(
Masc
) at the mRNA level (
B) [10][16][18]. In male
B. mori
, the
Masc
protein (MASC) increases the expression of
Bombyx mori dsx RNA-binding protein 3
(RxRBP3) (
D
). RxRBP3, together with the protein of
P-element somatic inhibitor
(PSI), regulates the male-specific alternative splicing of
dsx
(
F) [12][60][61][62]. The male-specific DSX (DSXM) promotes male development [63][64].
) [26,72,87,96]. The male-specific DSX (DSXM) promotes male development [37,38].
There is strong evidence supporting that Masc, PSI and Znf-2 are indispensable for masculinization, but where and how exactly these factors function in the sex determination cascade is not known. These questions may be answered by dissection of the functional relationships among Masc, PSI and Znf-2, and by identification of additional players in the sex determination pathway. Recent studies have identified components as BxRBP1, BxRBP2 and BxRBP3 (
Bombyx mori dsx
RNA-binding proteins) are
dsx
pre-mRNA binding protein by using the strategy of yeast three-hybrid screening. It is interesting that the alternatively transcribed
BxRBP3
isoforms are regulated by Masc and physically interact with PSI, but whether
BxRBP3 is the transducer remains to be verified via genetic methods [60] (
is the transducer remains to be verified via genetic methods [72] ( D,F). However, based on the observation that females can be completely reversed to males when the signal transducer
D,F). However, based on the observation that females can be completely reversed to males when the signal transducer
tra is knocked-down or knocked-out in Diptera, we speculate that the transducer in the silkworm either has not yet been identified or may act very differently in the sex determination cascade [65][66][67][68][69].
is knocked-down or knocked-out in Diptera, we speculate that the transducer in the silkworm either has not yet been identified or may act very differently in the sex determination cascade [19,73,74,75,76].
4. Doublesex, the Executor
DSX protein sex-specific isoforms are conserved in insects and act as the downstream regulator in the sex determination pathway [7]. DSX controls sexual dimorphic development in insects, and mutation of sex-specific isoforms will cause male or female reproductive defects, respectively; hence, it has been targeted in development of sterile insect biotechnology [70][71][72][73]. As in Diptera, also in Lepidoptera, the
DSX protein sex-specific isoforms are conserved in insects and act as the downstream regulator in the sex determination pathway [31]. DSX controls sexual dimorphic development in insects, and mutation of sex-specific isoforms will cause male or female reproductive defects, respectively; hence, it has been targeted in development of sterile insect biotechnology [77,78,79,80]. As in Diptera, also in Lepidoptera, the dsx pre-mRNA is alternatively spliced to encode transcription factors that contain a common N-terminal domain and a sex-specific C-terminal domain [74]. The N-terminal domain possesses a DNA-binding motif that mediates the binding of DSX to target genes, whereas the C-terminal domain is crucial for DSX regulation of sexual development [63][75]. Disruptions of functions of certain isoforms of DSX lead to either sex-specific sexual-dimorphic defects or intersexual phenotypes in multiple Lepidoptera insects including
pre-mRNA is alternatively spliced to encode transcription factors that contain a common N-terminal domain and a sex-specific C-terminal domain [81]. The N-terminal domain possesses a DNA-binding motif that mediates the binding of DSX to target genes, whereas the C-terminal domain is crucial for DSX regulation of sexual development [37,82]. Disruptions of functions of certain isoforms of DSX lead to either sex-specific sexual-dimorphic defects or intersexual phenotypes in multiple Lepidoptera insects including B. mori
,
A. ipsilon
,
Plutella xylostella
and
H. cunea [64][76][77][78].
In
Drosophila
, the transcription cofactor Intersex (Ix) functions together with the female-specific product of
dsx, DSXF, to implement female sexual differentiation [59] (
, DSXF, to implement female sexual differentiation [86] ( E). However, depletion of
E). However, depletion of
ix
in the silkworm does not affect gonad development or splicing of the
dsx
pre-mRNA in either sex, suggesting that
ix functions differently in silkworms than in flies [61]. The transcription factor Fruitless (Fru) plays roles in sexual behaviors and is regulated by the sex determination cascade in flies [79]. A recent study in the silkworm demonstrated that Fru is also affected by the upstream sex-determining factors and acts together with DSX to regulate both mating and courtship behavior [80].
functions differently in silkworms than in flies [87]. The transcription factor Fruitless (Fru) plays roles in sexual behaviors and is regulated by the sex determination cascade in flies [7]. A recent study in the silkworm demonstrated that Fru is also affected by the upstream sex-determining factors and acts together with DSX to regulate both mating and courtship behavior [88].
Although DSX exists in the majority of insect species, the mechanisms by which it functions in sex determination and subsequent developmental processes are poorly understood in most insect species. Only a few genes directly controlled by DSX have been identified, although numerous targets of DSX have been predicted and analyzed on the genome-wide scale in
Drosophila [81][82]. It remains to be determined whether the events downstream of DSX were conserved among insects during evolution [83].
[89,90]. It remains to be determined whether the events downstream of DSX were conserved among insects during evolution [91].
