Download Masters change, slaves remain

What the papers say
Masters change, slaves remain
Patricia Graham,* Jill K. M. Penn, and Paul Schedl
Summary
Sex determination offers an opportunity to address many
classic questions of developmental biology. In addition,
because sex determination evolves rapidly, it offers
an opportunity to investigate the evolution of genetic
hierarchies. Sex determination in Drosophila melanogaster is controlled by the master regulatory gene, Sex
lethal (Sxl ). DmSxl controls the alternative splicing of a
downstream gene, transformer (tra), which acts with tra2
to control alternative splicing of doublesex (dsx). DmSxl
also controls its own splicing, creating an autoregulatory
feedback loop that ensures expression of Sxl in females,
but not males. A recent paper(1) has shown that in the
dipteran Ceratitis capitata later (downstream) steps in
the regulatory hierarchy are conserved, while earlier
(upstream) steps are not. Cctra is regulated by alternative
splicing and apparently controls the alternative splicing
of Ccdsx. However, Cctra is not regulated by CcSxl.
Instead it appears to autoregulate in a manner similar to
the autoregulation seen with DmSxl. BioEssays 25:1–4,
2003. ß 2002 Wiley Periodicals, Inc.
Introduction
The choice of sexual cell fate is a developmental decision akin
to the choice between becoming an epidermal cell or a neuron.
Thus sex determination offers a chance to investigate the
classical problems of developmental biology. How are choices
between possible cell fates made, maintained and implemented? Studies of sex determination in Drosophila melanogaster
have already made significant contributions to our understanding in this area. For example, analysis of the master
switch gene, Sex-lethal (Sxl ), has shown sexual identity is
remembered during the life cycle through a mechanism
involving alternative splicing and autoregulation.(2,3) Examination of sex determination in different species should add to
our knowledge of some of these topics and may make novel
contributions to others.
Sex determination shares some general features in all
species. An initial signal governing the choice of sex is transmitted through a regulatory cascade to activate the genes
ultimately required to produce the particular physiological and
behavioral phenotypes of the two sexes. In different species
Dept. Molecular Biology, Princeton University.
*Correspondence to: Patricia Graham, Dept. Molecular Biology,
Princeton University. NJ 08544-1041.
E-mail: [email protected]
DOI 10.1002/bies.10207
Published online in Wiley InterScience (www.interscience.wiley.com).
BioEssays 25:1–4, ß 2002 Wiley Periodicals, Inc.
the initial signals at the top of the regulatory hierarchy vary
considerably. For example, vertebrates use a male determining factor on the Y chromosome.(4 –6) Two well-characterized
model systems, Drosophila melanogaster and Caenorhabditis
elegans use the ratio of X chromosomes to autosomes
(reviewed in Ref. 7). Yet other species use autosomal factors
or environmental cues. Studying sex determination in different but related species will not only add to our understanding of decision making in development, but will also help
elucidate how regulatory hierarchies have evolved. Evidence
from studies comparing sex determination in different species
of dipteran insects reveals that the master genes at the top of
the regulatory hierarchy can change dramatically as new
species and genus evolve, while the slave genes at the bottom
of the hierarchy remain the same, carrying out essentially
identical functions from one species to the next. In this review,
we discuss some of what is known about sex determination in
Drosophila melanogaster and how this relates to what has
recently been learned about sex determination in Ceratitis
capitata.
Choice and memory in
Drosophila melanogaster
The initial signal and the mechanisms by which that signal are
translated into the female or male fate are well understood in
Drosophila melanogaster (Fig. 1A, for a recent review see
Ref. 8). The Sxl gene is turned on early in development in
females, while it remains off in males. The presence of Sxl
protein induces the productive splicing of mRNAs from the
downstream target gene transformer (tra). Tra protein expressed from the female tra mRNAs acts together with Tra2 (which
is expressed in both sexes) to activate the female splicing of
mRNAs from the gene doublesex (dsx).(9) The Dsxf protein
represses the transcription of genes required for male development and activates those required for female development.
When Sxl protein is absent, tra mRNA is spliced in a nonproductive pattern, which includes exon sequences containing
a stop codon. No Tra protein is expressed from the male tra
mRNA and as a consequence dsx mRNA is spliced in the male
pattern. The Dsxm protein translated from this mRNA represses female development and promotes male.
The choice of sexual identity early in development depends
upon a system that assesses the X chromosome to autosome
(X/A) ratio. The target for the X/A counting system is a special
Sxl establishment promoter Sxl-Pe. This promoter is turned on
for a brief period in pre-cellular blastoderm female (2X/2A)
BioEssays 25.1
1
What the papers say
Figure 1. Sex determination compared in Drosophila
melanogaster and Ceratitis capitata. The Ceratitis tra
gene has the role of ‘‘master regulator,’’ taking over the
position held by Sxl in Drosophila.
embryos, while it remains off in male (1X/2A) embryos. The
function of the Sxl proteins encoded by the Sxl-Pe mRNAs is to
activate the female-specific splicing of mRNAs expressed
from the Sxl maintenance promoter, Sxl-Pm. Sxl-Pm turns on
in both sexes at cellularization, just as Sxl-Pe is being shut
off in female embryos, and then remains on during the rest
of the life cycle. The female Sxl-Pe mRNAs lack the 3rd or male
exon, which contains multiple in-frame stop codons, and they
encode functional proteins that have two RRM-type RNAbinding domains. These Sxl proteins, in turn, direct the female
splicing of Sxl-Pm mRNAs, setting up a positive autoregulatory
feedback loop which ensures the continued production of Sxl
protein and thus memory of female identity. In males where
Sxl-Pe is never turned on, transcripts from Sxl-Pm are spliced
to include the translation terminating male exon and thus male
identity is set by default.
Choice and memory in Ceratitis capitata
The Sxl homologue has been isolated from several dipterans
including Ceratitis capitata(10–13) and is generally well conserved. Within the genus Drosophila, Sex-lethal is produced in
a sex-specific manner, which is consistent with the idea that
it retains its role as a master regulatory gene.(10) However, in
virtually every dipteran examined outside the genus Drosophila, Sex-lethal is not expressed in a sex-specific manner,
and has no apparent role in sex determination.(11,12) In fact,
although Sxl is present in Ceratitis, and has 69% identity and
79% similarity to DmSxl at the amino acid level, it is no longer
the master regulator of sexual identity. In this case, how is
sex determined in Ceratitis and what gene(s) controls and
maintains sexual identity?
In Ceratitis capitata, the X/A ratio is not used to determine
sexual fate. Instead, the presence of a Y-linked factor, M,
2
BioEssays 25.1
promotes male development while its absence leads to
female development(14) (Fig. 1B). Currently the M factor has
not been identified, and it is unknown how it promotes
maleness.
A recent paper(1) has shown that the Ceratitis homologue
of Dmtra is functionally conserved even though it has only
32–40% identity at the amino acid level. The first evidence of
functional conservation was the finding of sex-specific transcripts of Cctra. Furthermore, as in Drosophila, male-specific
tra transcripts contain multiple stop codons while the female
transcript encodes a SR-rich protein. To show that Cctra is
essential for female development, Pane et al. (2002) inhibited
Cctra function by injecting embryos with Cctra dsRNA, causing
the endogenous Cctra mRNA to be degraded. The injected XX
embryos developed into sexually transformed adult males
while the development of the XY embryos was unaffected.
Thus, despite the very poor sequence conservation, Cctra and
Dmtra are functionally similar.
Since the sexually transformed XX Ceratitis flies produced
predominantly male dsx RNA, it would appear that Cctra
controls sexual differentiation by regulating Ccdsx. Sequence
analysis of Ccdsx genomic DNA and cDNAs indicates that
there are Tra/Tra2-binding sites in positions similar to those in
Dmdsx, and that the Ccdsx mRNAs are structurally very
similar to those of Dmdsx.(12) Although it remains to be seen
whether there is a tra2 homologue in Ceratitis, this suggests
that the mechanism controlling the alternative splicing of dsx
mRNA may also be similar in the two species.
The dsRNA injections into XX embryos would be expected
to result in a transient loss of tra activity. Yet the adult animals
that develop from the embryos are transformed XX males and
RT-PCR analysis of the tra mRNAs from these animals reveals
that the message is spliced in the male pattern. One way to
explain how a transient loss of tra activity is sufficient to block
What the papers say
the female mode of tra splicing later in development, is that
Tra has a positive autoregulatory function and is required to
direct the female splicing of tra mRNA. In this case, a transient
loss of tra activity in XX embryos would be sufficient to induce
a permanent switch in sexual identity. Consistent with this
idea, examination of the Cctra gene revealed several putative
Tra/Tra2-binding sites in and around the male-specific exons.
If this model is correct, the role of Cctra in Ceratitis capitata
would be similar to that of Sxl in the Drosophilids: it would be at
the top of the sex determination hierarchy, directing female
sexual development by regulating downstream target genes,
and functioning to maintain female identity through its autoregulatory activity.
Although the initial signal and master regulatory genes vary
in different species, the target of these switch genes, the dsx
gene, appears to have both structural and functional conservation across a wide range of species. The dsx gene of
Megaselia scalaris shows both conserved structure and sexspecific splicing patterns.(15) dsx may also be structurally
and functionally conserved in Ceratitis capitata. Intriguingly,
this conservation may extend beyond dipterans as the dsx
homologue in Caenorhabditis elegans, male abnormal 3
(mab-3), also controls sexual cell fate.(16,17)
The role of Tra at the molecular level
Previous data indicate that SR-proteins such as Tra and Tra2
generally act to activate splice sites that would otherwise not
be used (reviewed in Ref. 18). This has been shown to be
the case for Drosophila Tra and Tra2 for the sex-specific
alternative splicing of dsx.(19) and another gene required for
mating behavior, fruitless (fru).(20) In Ceratitis, as mentioned
above, it has been proposed that Tra and Tra2 regulate dsx in
this manner also. Surprisingly, it appears that Tra and Tra2
may use a different mechanism to regulate the splicing of Cctra
mRNA. Based on the location of the putative Tra/Tra2-binding
sites in the Cctra transcript and the sequences of the sexspecifically alternatively spliced Cctra isoforms, Pane et al.(1)
have hypothesized that Tra and Tra2 block the use of the malespecific splice sites rather than activate a weaker splice site.
Although this is a new role for Tra and Tra2, it should be noted
that the context of a binding site can influence the activity of
splicing regulators. For example, in adenovirus, it has been
shown that SR proteins bind within an intron of the L1 transcriptional unit and are able to inhibit splicing by preventing the
recruitment of the U2 snRNP to the spliceosome.(21) When the
binding site is instead placed within an exon, the SR proteins
are able to activate the upstream splice site.
Beyond sex determination
Developmental decisions are often connected to one another.
Pathways regulating cell division ‘‘talk’’ with those regulating
differentiation to produce the correct number of the right type
of cells. Thus it is of interest to learn how related regulatory
hierarchies might be connected to one another. For example,
the process of sex determination is sometimes connected to
a related process called dosage compensation. In many species, the two sexes contain different numbers of sex chromosomes (as in humans where the male has one and the
female has two X chromosomes). Therefore expression of
genes on the sex chromosomes may need to be regulated to
compensate for the difference in dose between the sexes. This
compensatory process is called dosage compensation.
In Drosophila melanogaster, the processes of sex determination and dosage compensation are coupled by the multifunctional, master regulator Sxl. By contrast, in mammals,
dosage compensation is dependent upon Xist, a noncoding RNA, while sex determination is controlled by Sry.
In Drosophila, Sxl regulates dosage compensation in two
ways. First, in females DmSxl represses the translation of a
gene called male-specific lethal-2 (msl-2) which is required to
hyperactivate expression of X-linked genes in males.(22–24)
Second, DmSxl directly regulates expression of some X-linked
genes.(25,26) Because the processes of sex determination and
dosage compensation are coupled in Drosophila, changes at
the top of the sex determination hierarchy also cause sexspecific lethality rather than just sexual transformation.(8) One
side effect of this coupling might be that changes in the genes
at the top of the sex determination hierarchy are more difficult,
and thus the coupling might promote the evolutionary stability
of the hierarchy.
Dosage compensation in Ceratitis capitata has not been
formally investigated. However, the paucity of X-linked mutations and the observation that the Ceratitis X chromosome is
predominantly heterochromatic suggest that there may be
relatively few genes on the Ceratitis X chromosome. Thus
there may be little need for dosage compensation in this
species. This would not be a unique situation as Musca
domestica may have used the same solution to the problem of
a two-fold difference in gene dose between the sexes.(8) While
it seems likely that dosage compensation is absent in Ceratitis
capitata, if a system does exist, Tra apparently does not
control it, since the XX pseudomales that result from loss of Tra
are able to survive to adulthood. In fact, unlike Drosophila
pseudomales, the Ceratitis pseudomales are fertile, confirming that the Ceratitis Y chromosome does not carry additional
genes required for male development beyond the M factor.(27)
Conclusions
The results in this paper support the idea that genetic
hierarchies evolve from the ‘‘bottom up’’ as proposed by
Wilkins.(28) The slave gene at the bottom of the regulatory
hierarchy, dsx, shows both structural and functional conservation across many species. At higher levels in the hierarchy, the
regulatory genes can change from one species to the next.
Thus although tra has maintained its role in regulating dsx, Sxl
does not control tra. Instead, tra appears to have taken on the
BioEssays 25.1
3
What the papers say
role of the master switch gene that not only initiates the choice
of sexual identity, but also maintains that choice through
autoregulation.
One issue that has been left unresolved is what controls
the initial expression of tra in Ceratitis. Since maternal Cctra
mRNAs that are spliced in the female pattern have been
detected in unfertilized eggs, it is possible that protein translated from these mRNAs might be able to activate the initial
alternative splicing of the zygotic transcripts in females. In
males, the M factor might act to inhibit the maternal tra,
perhaps by preventing its translation or by blocking the activity
of the protein. In the future, it will be interesting to test this
model and determine the identity and function of the M factor.
In addition, it will be important to determine if the M factor
functions transiently early in development to control male
identity or whether it is present and is able to function throughout the entire lifecycle. Similarly, if Tra could be transiently
expressed in XY embryos at a time when the M factor is
no longer functional, the autoregulatory model predicts that
it would initiate the female pathway and sex transform
XY animals.
References
1. Pane A, Salvemini M, Bovi PD, Polito C, Saccone G. The transformer gene
in Ceratitis capitata provides a genetic basis for selecting and remembering the sexual fate. Development 2002;129:3715–3725.
2. Bell LR, Maine EM, Schedl P, Cline TW. Sex-lethal, a Drosophila
sex determination switch gene, exhibits sex-specific RNA splicing
and sequence similarity to RNA binding proteins. Cell 1988;55:1037–
1046.
3. Bell LR, Horabin JI, Schedl P, Cline TW. Positive autoregulation of sexlethal by alternative splicing maintains the female determined state in
Drosophila. Cell 1991;65:229–239.
4. Jacobs PA, Strong JA. A case of human intersexuality having a possible
XXY sex-determining mechanism. Nature 1959;183:302–303.
5. Ford CE, Jones KW, Polani PE, De Almeida JC, Briggs JH. A sexchromosome anomaly in a case of gonadal dysgenesis (Turner’s syndrome). Lancet 1959;1:711–713.
6. Welshons WJ, Russell LB. The Y chromosome as the bearer of male determining factors in the mouse. Proc Natl Acad Sci USA 1959;45:560–566.
7. Cline TW, Meyer BJ. Vive la difference:males vs females in flies vs
worms. Annu Rev Genet 1996;30:637–702.
8. Schutt C, Nothiger R. Structure, function and evolution of sex-determining
systems in Dipteran insects. Development 2000;127:667–677.
9. Hoshijima K, Inoue K, Higuchi I, Sakamoto H, Shimura Y. Control of
doublesex alternative splicing by transformer and transformer-2 in
Drosophila. Science 1991;252:833–836.
10. Bopp D, Calhoun G, Horabin JI, Samuels M, Schedl P. Sex-specific
control of Sex-lethal is a conserved mechanism for sex determination in
the genus Drosophila. Development 1996;122:971–982.
4
BioEssays 25.1
11. Meise M, Hilfiker-Kleiner D, Dubendorfer A, Brunner C, Nothiger R,
Bopp D. Sex-lethal, the master sex-determining gene in Drosophila, is
not sex- specifically regulated in Musca domestica. Development 1998;
125:1487–1494.
12. Saccone G, Peluso I, Artiaco D, Giordano E, Bopp D, Polito LC. The
Ceratitis capitata homologue of the Drosophila sex-determining gene
Sex-lethal is structurally conserved, but not sex-specifically regulated.
Development 1998;125:1495–1500.
13. Sievert V, Kuhn S, Paululat A, Traut W. Sequence conservation and
expression of the sex-lethal homologue in the fly Megaselia scalaris.
Genome 2000;43:382–390.
14. Willhoeft U, Franz G. Identification of the sex-determining region of the
Ceratitis capitata Y chromosome by deletion mapping. Genetics 1996;
144:737–745.
15. Kuhn S, Sievert V, Traut W. The sex-determining gene doublesex in
the fly Megaselia scalaris:conserved structure and sex-specific splicing.
Genome 2000;43:1011–1020.
16. Raymond CS, Shamu CE, Shen MM, Seifert KJ, Hirsch B, Hodgkin J,
Zarkower D. Evidence for evolutionary conservation of sex-determining
genes. Nature 1998;391:691–695.
17. Yi W, Zarkower D. Similarity of DNA binding and transcriptional
regulation by Caenorhabditis elegans MAB-3 and Drosophila melanogaster DSX suggests conservation of sex determining mechanisms.
Development 1999;126:873–881.
18. Lopez AJ. Alternative splicing of pre-mRNA:developmental consequences and mechanisms of regulation. Annu Rev Genet 1998;32: 279–
305.
19. Ryner LC, Baker BS. Regulation of doublesex pre-mRNA processing occurs by 30 -splice site activation. Genes Dev 1991;5:2071–
2085.
20. Heinrichs V, Ryner LC, Baker BS. Regulation of sex-specific selection of
fruitless 50 splice sites by transformer and transformer-2. Mol Cell Biol
1998;18:450–458.
21. Kanopka A, Muhlemann O, Akusjarvi G. Inhibition by SR proteins of
splicing of a regulated adenovirus pre-mRNA. Nature 1996;381:535–
538.
22. Bashaw GJ, Baker BS. The msl-2 dosage compensation gene of
Drosophila encodes a putative DNA-binding protein whose expression
is sex specifically regulated by Sex-lethal. Development 1995;121:3245–
3258.
23. Kelley RL, Solovyeva I, Lyman LM, Richman R, Solovyev V, Kuroda MI.
Expression of msl-2 causes assembly of dosage compensation regulators on the X chromosomes and female lethality in Drosophila. Cell
1995;81:867–877.
24. Zhou S, et al. Male-specific lethal 2, a dosage compensation gene of
Drosophila, undergoes sex-specific regulation and encodes a protein
with a RING finger and a metallothionein-like cysteine cluster. EMBO J
1995;14:2884–2895.
25. Bernstein M, Cline TW. Differential effects of Sex-lethal mutations on
dosage compensation early in Drosophila development. Genetics 1994;
136:1051–1061.
26. Kelley RL, Kuroda MI. Equality for X chromosomes. Science 1995;
270:1607–1610.
27. Schmidt R, Hediger M, Roth S, Nothiger R, Dubendorfer A. The Ychromosomal and autosomal male-determining M factors of Musca
domestica are equivalent. Genetics 1997;147:271–280.
28. Wilkins AS. Moving up the hierarchy: a hypothesis on the evolution of a
gentic sex determination pathway. Bioessays 1995;17:71–77.