Sex Determination in Drosophila Malenogaster

One of the most important decisions in development is whether to be male or female. In Drosophila melanogaster, most cells make this choice independent of their neighbors such that diploid cells with one X chromosome (XY) are male and those with two X chromosomes (XX) are female. X-chromosome number is relayed through regulatory proteins that act together to activate Sex-lethal (Sxl) in XX animals. The resulting SXL female specific RNA binding protein modulates the expression of a set of downstream genes, ultimately leading to sexually dimorphic structures and behaviors. Despite the apparent simplicity of this mechanism, Sxl activity is controlled by a host of transcriptional and posttranscriptional mechanisms that tailor its function to specific developmental scenarios.

1.0 Genotypic Sex determination

In genotypic sex determination system,the sex chromosomes play the decisive role in the inheritance and determination of sex,and it may occur in one of the two ways: 

1. In the Y-chromosome mechanism of sex determination(e.g.,human) ,the Y chromosome of the heterogametic sex is active in determining the sex of an individual.Individuals carrying the Y-chromosome are genetically male,while individuals lacking the Y-chromosomes are genetically female.
2. In the X-chromosome autosome balance system(e.g.,Drosophila)the main factor in sex determination is the ratio between the number of X-chromosomes and the number of sets of autosomes.In this system the Y-chromosomes has no effect on sex determination,but is required for male fertility.

2.0Sex Determination in Drosophila by X/A Ratio

• The number of X chromosomes : sets of autosomes (X:A)ratio determines sex in Drosophila 
• First, the X:A ratio is read during development. For wild-type Drosophila, the ratio that sets the initial switch for development into females (XX) is 2X : 2 sets of autosomes=1.0 , and the ratio that sets the initial switch for development into males (XY) is 1X : 2 sets of autosomes .
• This information is transmitted to the sex determination genes, which make the choice between the alternative female and male developmental pathways, starting with the master regulatory gene Sex-lethal (Sxl).
• Loss-of-function mutants of Sxl are lethal for female embryo development (meaning that Sxl needs to be active in females), but they have no effect on male embryo development (meaning that Sxl expression is not necessary for male development). However, gain-of-function mutants are lethal for male embryo development, which means that Sxl needs to be inactive in males 
• Alternative splicing of the Sxl pre-mRNA in embryos destined to become females or males sets in motion the two different pathways.

3.0Genic balance theory

• In 1921, one of the founders of modern genetics Calvin Bridges found in Dr. Melanogaster several females with a triploid set of chromosomes (3X:3A). 
• After crossing these females with normal males (2X:2A), individuals with an unusual expression of sexual characteristics were found in their offspring along with normal females. 
• All offspring split into classes depending on the ratio of sex chromosomes (X) and autosomes (A):
• 3X:3A, triploid females.
• 2X:2A, normal female (ratio X:A is 1).
• (2X:2Y):2A, females.
• XY:2A, normal females (ratio X:A is 0.5).
• 2X:3A and (2X + Y):3A, intersexes (according to Bridges terminology) with varying ratio X:A 0.5 to 1. 
• These were individuals with a mixed expression of male and female characteristics. 
• These flies either completely lacked the sectors of the body determined by sex, or during development to a certain point formed organs of one sex, and then organs of the other sex.
• X:3A, supermales, i.e. individuals with hypertrophied male signs, being, however, sterile (ratio X:A is less than 0.5).
• 3X:2A, superfemales, i.e. individuals with an abnormal development of the ovaries and other disorders of sex characters (ratio X:A is greater than 1).
• In all the cases when females appear, the ratio of the number of X chromosomes to autosomes is one. The presence of the male Y chromosome does not affect the normal development of the female. 
• According to Bridges, the gender in Drosophila is determined by the balance of sex chromosomes and a set of autosomes, while the Y chromosome does not play in sex determination; hence, the genic balance theory of sex determination. 
• Indeed, in the Y chromosome there are genes of 11 fertility factors influencing the formation of the sperm that do not participate in the formation of the male sexual characteristics. Moreover, it is known that individuals XO in Drosophila are males.

4.0Genes for sex determination in Drosophila

• There are numerous genes in Drosophila that affect the proper sex differentiation including Sxl (sex lethal), da (daughterless), sis (sisterless), tra (transformer), and dsx (double sex). 
• In this regard, genetic interpretations of the genic balance theory of sex determination can be worked out.
• It was suggested that the ratio of the number of X chromosomes and autosomes might be “detected” by the Sxl gene at the early stages of embryonic development. This gene, in turn, controls simultaneously three aspects of differentiation: 
• The formation of sexual characteristics in somatic cells; 
• The formation of embryonic germ cells; and
• Implementation of dose compensation. 
• According to the Swiss scientist R. Niagara, at the initial stages of sex development in embryos of Drosophila the products of the following genes are crucial: sis-a and sis-b (known as XSEs, X Signal Elements, or numerator proteins) located in the X chromosome, and da, located in an autosome (known as ASEs, Autosomal Signal Elements, denominator proteins). 
• The da gene product enters the egg from the mother’s body. Its quantity always corresponds to two doses, as it is translated from the genes localized in the two maternal autosomes. 
• The quantity of products resulting from the genes sis-a and sis-b depends on how many X chromosomes, one or two, an individual possesses. 
• Therefore, the sis/da protein complex is characterized by the ratio of its components 1:2 in males or 2:2 in females.
• Observation of haploid and triploid larvae showed that gender in Drosophila is not determined by X:A, but rather by the number of X chromosomes. 
• Products of five X chromosome genes were found in Drosophila including sisA, sisB (scute), runt, unpaired (sisE), and dm (diminutive) called as numerators as well as several protein cofactors such as product of the autosomal gene deadpan. 
• One of the genes (obviously, da (daughterles)) encodes a protein that functions as autosomal control factor (ASE) and is referred to as the denominators.
• In three hours after fertilization and during the formation of the blastoderm, the products of the genes sisA, scute and runt stimulate the activity of the Sxl early promoter. 
• Its full activation is achieved by using the product of the X chromosome gene unpaired via the Janus kinase. 
• In diploid females (XX:2A), the activity level of UNPAIRED required to activate Sxl promoter is achieved by the 12th division of the blastoderm cells, in haploid females (XO:2A) by the 14th division, and in triploids (i.e. XXX:3A embryos) only in some cells (leading, as a result, to formation of partial gynandromorphs).
• The products of the above mentioned genes interact with a key regulatory region of the Sxl gene. The latter contains 8 regions that encode the amino acid sequence (i.e. exons) and are separated with noncoding regions (i.e. introns). 
• It also has two sites (i.e. promoters), “early” and “late” ones, that stimulate the transcription of RNA from this gene. Only in the case when the SIS/DA complex protein contains two doses of SIS, it can activate the beginning of transcription from the early promoter.
• In females the Sxl gene transcript does not contain exon3 with a stop codon. At the blastoderm stage, as a result of translation of that transcript, a complete SXL protein is formed, which activates transcription of tra gene that, further interacting with the protein of the tra2 gene, regulates the production of a specific RNA in females, dsxF (doublesex). 
• The presence of DSXF in females facilitates involvement in the cascade of the ix gene. Proteins of the dsxf and ix genes inactivate many genes that are specific to males, and eventually facilitate the development of a female. Under this scheme, the external (somatic) sexual characteristics are formed in females . 
• In males, activation of the late promoter (Pl) of the Sxl gene leads to the transcription of the third exon in which the stop codon UGA is located. Here the translation stops, resulting in a truncated protein. 
• In the absence of a normally functioning SXL protein, the tra gene forms a short non-functional protein molecule (because the translation is blocked by the UAG codon in the second exon).
• In males, disruption of the Sxl gene splicing results in inclusion of a specific exon into the transcript that contains the stop codon, and the protein is not synthesized. 
• In the absence of the SXL protein, mRNA of msl2 is not translated and the dose compensation occurs. In addition, in the absence of the normal TRA protein, male-specific genes dsx and fru begin to get transcribed . 
• The latter is translated into a zinc finger-type transcriptional factor, BTZ, which is responsible for all aspects related to the central nervous system (CNS) in males. Protein of the tra2 gene is present in both sexes. 
• In the absence of a functional product of the tra gene in males, there is no formation of a normal TRA/TRA2 multienzyme complex. 
• Moreover, in the absence of the normal products of the tra and tra2 genes, the DSXM protein is formed. It represses the development of female sex characteristics. 
• It was recently shown that the protein Nito (a product of the spenito gene) controls the alternative splicing of Sxl mRNA by interacting with the corresponding SXL protein and pre-RNA and thus engaging Sxl in self-regulation.
• Sex differentiation and the appearance of signs associated with sex, particularly behavioral ones, in Drosophila are related to primary sex determination. 
• For example, a complete protein of the Sxl gene leads to the activity of tra and tra2, and they, in turn, control the appearance of transcription regulators of the genes fru (fruitless), dsf (dissatisfaction), dsx, and fit (female-specific independent of transformer).
• The dsf gene product regulates the sex differentiation outside the nervous system and some aspects of sexual behavior (courtship), whereas fru influences the CNS development necessary for courtship, the development of muscles, etc. Other important sex-related genes control a number of characters. 
• For example, the tsx (turn on sex-specificity) gene encodes an odorant-binding protein, sxe1 (sex specificity enzyme), a phospholipase involved in signaling, and sxe2 that determines the cytochrome P450 involved in the metabolism of steroids in different organs.
• It should be noted that in Drosophila all 46 RNA types are described with different distributions between the sexes. It was also found that the activity of the Sxl gene in Drosophila is regulated by a long non-coding RNA. 
• This RNA activates the SxlPe promoter in females. For Drosophila, a “demasculinization” of the X chromosome is characteristic, which manifests itself in the transfer of some male genes from this chromosome to autosomes. 
• In humans and mice, on the contrary, genes expressed in spermatogonia are especially redundant in the X chromosome. The Y chromosome of Drosophila is believed to have originated from a specialized extra B chromosome, instead of a degenerating autosomal homologue of the X chromosome. 
• It contains only 16 genes, which together constitute about 0.5% of the total DNA of the Y chromosome. It is inherent in this chromosome that it has almost 11 times greater gain of genes than their loss.
• This is fundamentally different from the situation in mammals. In contrast to mammals, where sex hormones of the formed gonad affect the sexual identity of the whole organism, in Drosophila each cell is determined independently in terms of its sex.