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Modern Genetic Analysis Chapter 16. The Genetic Basis of Development
Drosophila Sex Determination: Every Cell for Itself

Every cell lineage in Drosophila makes its own sexual decision. One of the best ways to demonstrate this is by analyzing XX-XY mosaic flies; that is, individual flies containing a mixture of XX and XY cells. Such mosaics show a mixture of male and female phenotypes, according to the genotype of each individual cell. The interpretation of this difference is that every cell in Drosophila independently determines its sex.

Phenotypic Consequences of Different X Chromosome to Autosome Ratios

In Drosophila, the chromosomal basis of sex determination is due to the ratio of X chromosomes to sets of autosomes. Recall that, in Drosophila, n=4: one sex chromosome and three different autosomes. Hence, one autosomal set, which we shall represent as A, comprises the three different autosomes and, in a diploid fly, A=2. The effect of this X:A ratio can best be seen by examining sex chromosome aneuploids (Table 16-2). A normal 2X Drosophila diploid (XX AA) has an X:A ratio of 1.0 and is phenotypically female. An XY diploid (XY AA) has an X:A ratio of 0.5 and is male; an XO diploid also is male (although sterile). Triploids with three X chromosomes (XXX AAA) are females, those with one X (XYY AAA) are male, and those with two X's (XXY AAA) are "in between" (intersexes).

The Basics of the Regulatory Pathway

Let's look first at an overview of the pathway (Figure 16-3). The X:A ratio in the early embryo establishes whether a fly will become male or female. This directive for establishing the sexual phenotype is carried out by a master regulatory switch and several downstream sex-specific genes. The "off" position of the switch produces the male pathway of determination, whereas the "on" position shunts cells into the female mode of sexual determination. The pathway is initiated by differential transcription, the direction of the switch is maintained, and the decision is propagated by differential RNA splicing. The default mode of the pathway culminates in the production of male-specific transcription factors, whereas the alternative shunt culminates in the production of female-specific transcription factors.

The Regulatory Switch

Through genetic analysis, we know that the regulatory switch is the activity of a gene called Sxl (Sex lethal). In a fly with an X:A ratio of 1, SXL protein is synthesized and the fly develops as a female. In a fly with an X:A ratio of 0.5, no functional SXL protein is produced, and consequently, the fly develops as a male.

Setting the switch in the on or off position.

The X:A ratio sets in motion the sex determination pathway by an interaction the protein products of a series of X-chromosomal, zygotically expressed numerator genes and autosomal, maternally and zygotically expressed denominator genes. At least some of the numerator and denominator genes encode transcription factors of a type called basic helix-loop-helix (bHLH) proteins. bHLH proteins are known to function as transcription factors only when two bHLH monomers complex to form a dimeric protein. For our purposes here, we shall use NUM to designate the X chromosomeencoded bHLH numerator proteins and DEM to indicate the autosomally encoded bHLH denominator proteins.

These transcription factors have only one role: in a narrow time window in the early Drosophila embryoroughly 2 to 3 hours after fertilizationthey determine if the Sxl regulatory switch gets flipped on. The Sxl gene is essentially a "toggle switch" that is permanently locked into an "on" position in females or an "off" position in males (Figure 16-4a on the following page). To set the Sxl switch in the "on" position, the level of the active X:A NUM transcription factors must be high (owing to an X:A ratio of 1.0). With high (female) levels, the X:A transcription factors present in the early embryo bind to enhancers of the Sxl gene, activating its transcription from the Sxl early promoter. The transcript made from the early promoter then produces active SXL protein.

In contrast, if the levels of the NUM factors are too low, as is the case when the X:A ratio is 0.5, then there is insufficient transcription factor to activate Sxl transcription and no SXL protein is made.

The NUM proteins very likely measure the X:A ratio by competing for dimer formation with the DEM proteins to form active NUM transcription-factor dimeric protein complexes (Figure 16-4b). Although we do not know for sure how this works, here is a plausible mechanism.
The NUM monomers have a sequence-specific DNA-binding site, whereas the DEM proteins lack any DNA-binding site.
The DNA-binding site of NUM recognizes an enhancer sequence that regulates transcription from the promoter of the Sxl regulatory switch gene. As we will see in the next section, transcription from this promoter is required for establishment of Sxl gene expression in the early embryo.
Both NUM and DEM polypeptides are synthesized at levels proportionate to the number of copies of each bHLH-encoding numerator or denominator gene in the cell. In this way, embryos with an X:A ratio of 1.0 have twice as much NUM polypeptide per cell as do embryos with an X:A ratio of 0.5. In contrast, regardless of the X:A ratio, these cells have the same level of DEM.
All possible combinations of dimers can form, in proportion to the relative concentrations of NUM and DEM monomers in a cell: NUMNUM homodimers, NUMDEM heterodimers, and DEMDEM homodimers.
To be an active transcription factor, both subunits of a bHLH dimer must possess sequence-specific DNA-binding sites. This is true only for NUM homodimers. In a sense, then, when present in the same dimer, the DEM monomers are inhibitory to the transcription-factor activity of the NUM subunits.

The outcome of this scenario is that the higher the NUM:DEM ratio, the more active NUMNUM transcription factor will be present in a cell. Thus, in early embryos with an X:A ratio of 1.0, we can expect that much more active numerator transcription factor will accumulate than in embryos with an X:A ratio of 0.5.

MESSAGE
Proteinprotein interactions, such as competition between normal and inhibitory subunits for dimer formation, can be triggers for controlling developmental switches.

Maintaining the switch in a stable position.

The Sxl gene has two promoters. The early promoter is the only one that is activated by the NUMNUM transcription factors. The early promoter (PE) is only active early in embryogenesis. Later in embryogenesis and for the remainder of the life cycle, the Sxl gene is transcribed from the late promoter (PL) regardless of the X:A ratio or any other condition. This late promoter is active in every cell in the animal, beginning with midembryogenesis and persisting for the lifetime of the organism. The primary transcript produced by Sxl transcription from the late promoter is much larger than the primary transcript from the early promoter and is subject to alternative mRNA splicing, depending on the presence or absence of preexisting active SXL protein in the cell. The SXL protein is an RNA-binding protein that alters the splicing of the nascent Sxl transcript coming from this late promoter. When mRNA splicing occurs in the presence of bound SXL protein, splicing of Sxl produces an mRNA that encodes more active SXL RNA-binding protein. This SXL protein in turn binds to more Sxl primary transcript from the late promoter, creating the spliced form of the mRNA that encodes functional SXL protein, and so forth. Thus a feedback, or autoregulatory, loop, controlled at the level of RNA splicing, maintains SXL activity throughout development in flies with an X:A ratio of 1.0.

MESSAGE
The autoregulatory loop exemplifies how an early developmental decision can be "remembered" for the rest of development, even after the initial signals that established the decision have long disappeared.

In contrast, when the X:A ratio is 0.5, the Sxl switch is set in the "off" position. The early promoter is not activated early in embryogenesis and hence the early X:A = 0.5 embryo has no SXL protein. As a consequence, in the absence of any active SXL protein, the primary Sxl transcript of the late, constitutive Sxl promoter is processed in the default mRNA splicing pattern. This default Sxl mRNA is nonfunctional, in the sense that it encodes a stop codon shortly after the translation-initiation codon of its protein-coding region. The small protein produced from this male-specific spliced mRNA has no biological activity. Thus, in Drosophila with a low level of active NUMNUM transcription factor, the absence of active SXL protein early in development predestines that there will be no SXL activity throughout the remainder of development.

Propagating the decision.

Not only does SXL have to have an autoregulatory maintenance function, but it must be capable of activating the shunt pathway that will lead to female-specific gene expression. It accomplishes this through the same RNA-binding activity. Only in the presence of SXL protein is the primary tra (transformer) transcript spliced to produce an mRNA-encoding active TRA protein (Figure 16-5a on the following page). In turn, TRA protein is an RNA-binding protein that produces female-specific splicing of the dsx (doublesex) nascent RNA. The mRNA produced by this splicing pattern encodes a DSX-F protein, a transcription factor that globally represses male-specific gene expression (Figure 16-5b).

In the absence of active SXL protein, the splicing pattern of tra primary transcript produces an mRNA that does not encode functional TRA protein. In the absence of active TRA protein, splicing of the dsx primary transcript leads to the production of a DSX-M transcription factor that represses female-specific gene expression (Figure 16-5b).

The way that genetic analysis has contributed to this understanding of sex determination is described in Genetics in Process 16-1on the following page.

© 1999 by W. H. Freeman and Company. All rights reserved.