The sexual development pathway

Although both mammals and fruit flies produce XX females and XY males, their chromosomes achieve these ends using very different means. The sex-determining mechanisms in mammals and in insects such as Drosophila are very different. In mammals, the Y chromosome plays a pivotal role in determining the male sex. Thus, XO mammals are females, with ovaries, a uterus, and oviducts (but usually very few, if any, ova). In Drosophila, sex determination is achieved by a balance of female determinants on the X chromosome and male determinants on the autosomes. Normally, flies have either one or two X chromosomes and two sets of autosomes. If there is but one X chromosome in a diploid cell (1X:2A), the fly is male. If there are two X chromosomes in a diploid cell (2X:2A), the fly is female (Bridges 1921, 1925). Thus, XO Drosophila are sterile males. In flies, the Y chromosome is not involved in determining sex. Rather, it contains genes active in forming sperm in adults. Table 17.1 shows the different X-to-autosome ratios and the resulting sex.

Table 17.1

Ratios of X chromosomes to autosomes in different sexual phenotypes in Drosophila melanogaster.

In Drosophila, and in insects in general, one can observe gynandromorphs—animals in which certain regions of the body are male and other regions are female (Figure 17.15). This can happen when an X chromosome is lost from one embryonic nucleus. The cells descended from that cell, instead of being XX (female), are XO (male). Because there are no sex hormones in insects to modulate such events, each cell makes its own sexual “decision.” The XO cells display male characteristics, whereas the XX cells display female traits. This situation provides a beautiful example of the association between insect X chromosomes and sex.

Figure 17.15

Gynandromorphs. (A) Gynandromorph of D. melanogaster in which the left side is female (XX) and the right side is male (XO). The male side has lost an X chromosome bearing the wild-type alleles of eye color and wing shape, thereby allowing the expression (more...)

Any theory of Drosophila sex determination must explain how the X-to-autosome (X:A) ratio is read and how this information is transmitted to the genes controlling the male or female phenotypes. Although we do not yet know the intimate mechanisms by which the X:A ratio is made known to the cells, research in the past two decades has revolutionized our view of Drosophila sex determination. Much of this research has focused on the identification and analysis of the genes that are necessary for sexual differentiation and the placement of those genes in a developmental sequence. Several genes with roles in sex determination have been found. Loss-of-function mutations in most of these genes—Sex-lethal (Sxl), transformer (tra), and transformer-2 (tra2)—transform XX individuals into males. Such mutations have no effect on sex determination in XY males. Homozygosity of the intersex (ix) gene causes XX flies to develop an intersex phenotype having portions of male and female tissue in the same organ. The doublesex (dsx) gene is important for the sexual differentiation of both sexes. If dsx is absent, both XX and XY flies turn into intersexes (Baker and Ridge 1980; Belote et al. 1985a). The positioning of these genes in a developmental pathway is based on (1) the interpretation of genetic crosses resulting in flies bearing two or more of these mutations and (2) the determination of what happens when there is a complete absence of the products of one of these genes. Such studies have generated the model of the regulatory cascade seen in Figure 17.16.

Figure 17.16

Proposed regulation cascade for Drosophila somatic sex determination. Arrows represent activation, while a block at the end of a line indicates suppression. The msl loci, under the control of the Sxl gene, regulate the dosage compensatory transcription (more...)

The sex-lethal gene as the pivot for sex determination

Interpreting the x:a ratio

The first phase of Drosophila sex determination involves reading the X:A ratio. What elements on the X chromosome are “counted,” and how is this information used? It appears that high values of the X:A ratio are responsible for activating the feminizing switch gene Sex-lethal (Sxl). In XY cells, Sxl remains inactive during the early stages of development (Cline 1983; Salz et al. 1987). In XX Drosophila, Sxl is activated during the first 2 hours after fertilization, and this gene transcribes a particular embryonic type of Sxl mRNA that is found for only about 2 hours more (Salz et al. 1989). Once activated, the Sxl gene remains active because its protein product is able to bind to and activate its own promoter (Sánchez and Nöthiger 1983).

This female-specific activation of Sxl is thought to be stimulated by “numerator proteins” encoded by the X chromosome. These constitute the X part of the X:A ratio. Cline (1988) has demonstrated that these numerator proteins include Sisterless-a and Sisterless-b. These proteins bind to the “early” promoter of the Sxl gene to promote its transcription shortly after fertilization.

The “denominator proteins” are autosomally encoded proteins such as Deadpan and Extramacrochaetae. These proteins block the binding or activity of the numerator proteins (Van Doren et al. 1991; Younger-Shepherd et al. 1992). The denominator proteins may actually be able to form inactive heterodimers with the numerator proteins (Figure 17.17). It appears, then, that the X:A ratio is measured by competition between X-encoded activators and autosomally encoded repressors of the promoter of the Sxl gene.

Figure 17.17

The differential activation of the sxl gene in females and males. (A) In wild-type Drosophila with two X chromosomes and two sets of autosomes (2X:2A), the numerator proteins encoded on the X chromosomes (sis-a, sis-b, etc.) are not all bound by inhibitory (more...)

Maintenance of sxl function

Shortly after Sxl transcription has taken place, a second, “late” promoter on the Sex-lethal gene is activated, and the gene is now transcribed in both males and females. However, analysis of the cDNA from Sxl mRNA shows that the Sxl mRNA of males differs from sxl mRNA of females (Bell et al. 1988). This difference is the result of differential RNA processing. Moreover, the Sxl protein appears to bind to its own mRNA precursor to splice it in the female manner. Since males do not have any available Sxl protein when the late promoter is activated, their new Sxl transcripts are processed in the male manner (Keyes et al. 1992). The male Sxl mRNA is nonfunctional. While the female-specific Sxl message encodes a protein of 354 amino acids, the male-specific Sxl transcript contains a translation termination codon (UGA) after amino acid 48. The differential RNA processing that puts this termination codon into the male-specific mRNA is shown in Figures 17.17B and 17.18. In males, the nuclear transcript is spliced in a manner that yields eight exons, and the termination codon is within exon 3. In females, RNA processing yields only seven exons, and the male-specific exon 3 is now spliced out as a large intron. Thus, the female-specific mRNA lacks the termination codon.

Figure 17.18

The pattern of sex-specific RNA splicing in three major Drosophila sex-determining genes. The pre-mRNAs are located in the center of the diagram and are identical in both male and female nuclei. In each case, the female-specific transcript is shown at (more...)

The protein made by the female-specific Sxl transcript contains two regions that are important for binding to RNA. These regions are similar to regions found in nuclear RNA-binding proteins. Bell and colleagues (1988) have shown that there are two targets for the female-specific Sxl protein. One of these targets is the pre-mRNA of Sxl itself. The second is the pre-mRNA of the next gene on the pathway, transformer.

WEBSITE

17.9 Other sex determination proteins inDrosophila. Sex-lethal does not work alone, but in concert with several other proteins whose presence is essential for its function. Many of these proteins have other roles during development. http://www.devbio.com/chap17/link1709.shtml

The transformer genes

The Sxl gene regulates somatic sex determination by controlling the processing of the transformer (tra) gene transcript. The tra message is alternatively spliced to create a female-specific mRNA as well as a nonspecific mRNA that is found in both females and males. Like the male sxl message, the nonspecific tra mRNA contains a termination codon early in the message, making the protein nonfunctional (Boggs et al. 1987). In tra, the second exon of the nonspecific mRNA has the termination codon. This exon is not utilized in the female-specific message (see Figure 17.18). How is it that the females make a different transcript than the males? The female-specific protein from the Sxl gene activates a female-specific 3´ splice site in the transformer pre-mRNA, causing it to be processed in a way that splices out the second exon. To do this, the Sxl protein blocks the binding of splicing factor U2AF to the nonspecific splice site by specifically binding to the polypyrimidine tract adjacent to it (Figure 17.19; Handa et al. 1999). This causes U2AF to bind to the lower-affinity (female-specific) 3´ splice site and generate a female-specific mRNA (Valcárcel et al. 1993). The female-specific tra product acts in concert with the product of the transformer-2 (tra2) gene to help generate the female phenotype.

Figure 17.19

Stereogram showing binding of tra pre-mRNA by the cleft of the Sxl protein. The bound 12-nucleotide RNA (GUUGUUUUUUUU) is shown in yellow. The strongly positive regions are shown in blue, while the scattered negative regions are in red. It is worth crossing (more...)

Doublesex: The switch gene of sex determination

The doublesex (dsx) gene is active in both males and females, but its primary transcript is processed in a sex-specific manner (Baker et al. 1987). This alternative RNA processing appears to be the result of the action of the transformer gene products on the dsx gene (see Figure 5.31). If the Tra2 and female-specific Tra proteins are both present, the dsx transcript is processed in a female-specific manner (Ryner and Baker 1991). The female splicing pattern produces a female-specific protein that activates female-specific genes (such as those of the yolk proteins) and inhibits male development. As discussed in Chapter 5, if functional Tra is not produced, a male-specific transcript of dsx is made. This transcript encodes an active protein that inhibits female traits and promotes male traits.

The functions of the Doublesex proteins can be seen in the formation of the Drosophila genitalia. Male and female genitalia in Drosophila are derived from separate cell populations. In male (XY) flies, the female primordium is repressed, and the male primordium differentiates into the adult genital structures. In female (XX) flies, the male primordium is repressed, and the female primordium differentiates. If the dsx gene is absent (and thus neither transcript is made), both the male and the female primordia develop, and intersexual genitalia are produced. Similarly, in the fat body of Drosophila, activation of the genes for egg yolk production is stimulated by the female Dsx protein and is inhibited by the male Dsx protein (Schüpbach et al. 1978; Coschigano and Wensink 1993; Jursnich and Burtis 1993).

According to this model (Baker 1989), the result of the sex determination cascade comes down to what type of mRNA is going to be processed from the dsx transcript. If the X:A ratio is 1, then Sxl makes a female-specific splicing factor that causes the tra gene transcript to be spliced in a female-specific manner. This female-specific protein interacts with the Tra2 splicing factor to cause the doublesex pre-mRNA to be spliced in a female-specific manner. If the doublesex transcript is not acted on in this way, it will be processed in a “default” manner to make the male-specific message.