A Lorelei Verthandi

[Note: This was originally published in three parts in August 2007, and represents my attempt to represent the state of the research at the time as informed by my coursework at Dartmouth. It has been very lightly edited for style and formatting, but the content is otherwise unchanged.]

You’re probably aware that a person’s sex is typically determined by their combination of sex chromosomes. In humans, females have two X chromosomes, whereas males have an X and a Y. But how do you go from X and Y to boy and girl? And what does the cell have to do to compensate for the chromosome differences between the sexes?

Sex chromosomes pose two interesting questions in the study of development:

1. Sex determination: How does the cell interpret the data from the sex chromosomes to result in phenotypic sex?
2. Dosage compensation: The sex chromosomes carry many genes that aren’t sex-specific; that is, both male and female cells need the products of those genes in approximately equal amounts. Without dosage compensation, a cell with two X chromosomes will produce twice as much of a given X-linked gene product as a cell with one X chromosome. How does the cell regulate sex chromosome expression so that cells with unequal sex chromosomes express sex-linked genes equally?

The animal kingdom employs a number of different mechanisms for dealing with these two questions. Let’s take a brief look at sex determination and dosage compensation in three model organisms: the nematode worm (Caenorabditis elegans), the fruit fly (Drosophila melanogaster), and the common mouse (Mus musculus).

Part I: C. elegans

C. elegans is a tiny invertebrate worm with just one kind of sex chromosome: X. A typical worm with two X chromosomes (XX) is a hermaphrodite, producing both sperm and eggs and capable of self-fertilization. A typical worm with one X chromosome (XO) is a male; they’re smaller, produce only sperm, and are capable of mating with hermaphrodites.

The pathway leading to sex determination and dosage compensation is initiated by “reading” the X-to-autosome (X:A) ratio. (Autosomes are any chromosomes that aren’t sex chromosomes.) In C. elegans, the first main gene in the signal transduction pathway is xol-1 (XO lethal 1, so named because mutations of the gene are lethal to animals with XO genotype). The autosomes express a number of genes, such as sea-1, that promote xol-1 expression; these are called denominator factors, since they show up on the bottom of the X:A ratio. Each X chromosome carries genes like sex-1 and fox-1 that inhibit xol-1 expression; these are called numerator factors. An XO cell doesn’t produce enough numerator factors to “cancel out” the denominator factors, so xol-1 is “turned on.” An XX cell has twice as much of each numerator factor, enough to “turn off” xol-1.

Xol-1 is the first in a series of several regulatory genes. Since the genes regulate each other, activity alternates down the chain. If xol-1 is on, then it turns off sdc-2, which means her-1 gets turned on, etc. Alternatively, if xol-1 is off, then sdc-2 gets to turn on, and that turns off her-1, etc. This pathway ultimately leads to expression of transcription factors (gene-regulating proteins) specific for either hermaphrodite or male differentiation.

Loss-of-function mutations of some of the genes in this pathway can cause “transformation” to the wrong sexual phenotype. For example, in XO animals her-1 is normally turned on, and we expect to get a male. But if we mutate her-1 so it can’t perform its function, then the rest of the pathway downstream acts as if her-1 is off and we get a hermaphrodite phenotype. (Genes are often named according to the phenotype of the mutation that led to their discovery; thus, her-1 got its name for turning XO animals into hermaphrodites.)

So that’s sex determination, but what about dosage compensation? It turns out that dosage compensation is activated by sdc-2. Dosage compensation in C. elegans acts by cutting expression of both X chromosomes in half in XX animals. That way, two X chromosomes at half-expression result in the same amount of product as one X chromosome at full expression. That’s also why xol-1 mutations are lethal for XO animals; without xol-1 to regulate it, sdc-2 gets turned on when it shouldn’t be. That in turn activates dosage compensation, and with only one X-chromosome at half its normal expression, the cell doesn’t have enough X-linked gene product to survive.

C. elegans summary:
One sex chromosome — XO males, XX hermaphrodites. X:A ratio read by X-linked inhibition of xol-1, affecting downstream chain of regulatory genes. Dosage compensation reduces expression from each X chromosome by half in XX animals.

Part II: Drosophila

Drosophila’s sex chromosomes are perhaps more familiar-looking, in that it has an X and a Y chromosome, with XX animals being phenotypically female and XY animals being phenotypically male. As we’ll see, however, the mechanisms are vastly different from what we see in humans and other mammals.

Sex determination and dosage compensation begin as they did in C. elegans with reading the X:A ratio. This time, however, the X-linked numerator factors (like daughterless and sisterless) drive Sxl (sex lethal) expression from a promoter that results in the code for a fully-active SXL protein (nomenclature note: italics are typically used for the name of the gene, capital letters are used for the corresponding protein). The A-linked denominator factors (like deadpan) bind to the numerator factors to inhibit their activity. Thus, if the fly has only one X chromosome, the denominator factors will titrate out the numerator factors, so Sxl only gets up and running in XX animals.

SXL is a protein that alters RNA splicing. Most mRNA, when it is first transcribed from DNA, must first be processed within the nucleus before it is shipped out for protein translation. This processing includes removing undesired non-coding regions of RNA, called introns, and splicing back together the remaining regions, called exons. Normal Sxl mRNA has a premature stop codon in its third exon; if the cell tries to make protein from this mRNA, it will stop translation short, resulting in a non-functioning protein. SXL protein alters splicing to exclude this exon so the cell can make additional fully functional SXL. Thus, SXL drives its own positive feedback loop.

Both XX and XY animals express Sxl mRNA from the normal promoter. In XY animals, there isn’t ever any SXL around to splice this mRNA properly, so no SXL is made. In XX animals, the numerator factors drive SXL expression at the beginning of development from a special promoter that doesn’t require splicing, so there’s enough SXL to kick-start splicing of the normal mRNA and keep generating more SXL long after the numerator factors stop working.

SXL is then responsible for proper splicing of tra to its active form, which in turn (in conjunction with tra-2) alters splicing of the transcription factor dsx, which causes differentiation to the female phenotype.

SXL also inhibits msls, a gene which otherwise would initiate dosage compensation by increasing expression from the male X. (Note that in C. elegans, dosage compensation meant scaling down expression in the hermaphrodite, but in Drosophila, dosage compensation means scaling up expression in the male.)

Drosophila summary:
Two sex chromosomes — XX female, XY male. Numerator factors drive expression of fully-functional SXL in XX animal, kicking off SXL positive feedback loop. SXL activates pathway leading to female development. SXL inhibits msls pathway to dosage compensation. In the absence of SXL (i.e. in XY animals), msls drives increased expression from the single X chromosome.

Part III: Mus (mouse)

Of our three models, the mouse (being a mammal) is most closely analogous to humans. Here, as with the flies, XX animals are female and XY animals are male. This time, however, sex determination depends upon the Y-linked gene Sry instead of the X:A ratio. If you have Sry, you typically become a male. Note the implications of this for individuals with an atypical number of sex chromosomes. An XO fly would become a (pseudo)male due to the X:A ratio, whereas an XO human would become a female (with Turner syndrome) due to lack of Sry. An XXY fly would be female, whereas an XXY human would be male (with Klinefelter’s syndrome).

Overall sex determination in mammals relies primarily on differentiation of the gonad. The embryonic gonad is bipotential (neither male nor female but capable of differentiating into either) and contains both somatic cells and germ cells (the cells that will give rise to the germ line, i.e. sperm or eggs). The somatic cells of the gonad will differentiate into either Sertoli cells (for testes) or Follicle cells (for ovaries); the hormone production of the gonad will then affect development of the rest of the body.

So we’re looking for a cellular mechanism affecting gonad differentiation. As it turns out, there are two primary exogenous growth factors (proteins excreted from a cell that affect growth and development) expressed by the somatic cells: FGF9 and WNT4. FGF9 causes the somatic cells to differentiate into Sertoli cells, and inhibits Wnt4 expression. WNT4 causes the somatic cells to differentiate into Follicle cells, and inhibits Fgf9 expression. Initially, the somatic cells express both factors, and they balance each other out. But as development progresses, the balance gets tipped one way or the other.

In mammals, SRY promotes expression of FGF9, thus tipping the balance toward male development. However, a similar mechanism in other vertebrates might not require a single gene like Sry. For instance, for many animals (such as crocodiles) sex determination is affected by environmental factors like temperature. It’s entirely possible that these environmental factors could be affecting a balance such as that between FGF9 and WNT4.

This sex determination mechanism takes place primarily in the gonad; a different mechanism entirely is needed for dosage compensation in all the cells of the body. Unlike the worm mechanism of reducing each X by half in hermaphrodites or the fly mechanism of doubling expression of the male X, dosage compensation in mice and humans works by (almost) completely silencing one of the two female X chromosomes.

In both males and female cells, the autosomes produce enough of a certain blocking factor (BF) to bind to one X chromosome and block expression of the gene Xist. In a cell with only one X chromosome (i.e. a normal male), that’s the end of the story. In a cell with two X chromosomes (i.e. a normal female), Xist is expressed on the chromosome that did not receive BF. Xist is a gene that does not code for protein; its end product is untranslated RNA. Xist RNA aggregates to form a region in the nucleus that excludes RNA ploymerase II and transcription factors. The X chromosome migrates into this region, thus silencing it.

The paternal X chromosome carried by the sperm is imprinted so it will always be chosen for inactivation in the zygote. Once the embryo reaches the blastocyst stage of development, X-inactivation is temporarily turned off. As the cells then differentiate, X-inactivation is reinitiated. This time, the decision of which X chromosome to deactivate is random. Thus, the adult animal will be mosaic for X-inactivtion; some of the animal’s cells will have the paternal X deactivated, and some cells will have the maternal X deactivated.

The location where BF binds to the X chromosome, since the region runs opposite to Xist, is called Tsix. If Tsix is deleted from one X chromosome, then that chromosome will always be chosen for X-inactivation, since BF cannot bind it. A Tsix deletion in an XY cell will result in ectopic (out-of-place) X-inactivation, which is lethal.

Mouse summary:
Two sex chromosomes — XY male, XX female. FGF9 and WNT4, mutually inhibitory growth factors, are expressed in the gonad somatic cells. Y chromosome carries Sry, which promotes FGF9 and tips the balance to testis development; otherwise, WNT4 tips balance to ovary development. X-inactivation occurs by transcription of Xist RNA, which forms a nuclear domain excluding transcription factors. Autosomes produce enough blocking factor (BF) to rescue one X chromosome from inactivation, by binding Tsix and thereby blocking Xist. The paternal X is imprinted to be always chosen for inactivation in the zygote; at the blastocyst stage, X-inactivation is reset and randomized.