DEVELOPMENT GENETICS

Course Outline:
A. Sex Determination
Sex determination is the process by which an organism develops as a male or a female. In many
species, including humans, sex determination is based on the presence or absence of certain sex
chromosomes. In humans, these sex chromosomes are known as X and Y chromosomes.

A1. Expression of the Sex Lethal Gene

The expression of the Sex lethal gene is involved in the sex determination process in fruit flies (Drosophila
melanogaster). In fruit flies, females have two X chromosomes (XX), while males have one X and one Y
chromosome (XY). The Sex lethal gene, abbreviated as Sxl, plays a crucial role in controlling the sexual
development of fruit flies.
The Sex lethal gene is located on the X chromosome, and its expression is regulated in a sex-specific
manner.
The sex-specific manner of gene expression means that the Sex lethal gene is regulated differently in
males and females, leading to distinct developmental outcomes.
In females, which have two X chromosomes (XX), both
copies of the Sex lethal gene on the X chromosomes are
active. This means that the gene is transcribed into RNA
and translated into functional protein molecules. The
production of these molecules is essential for the
development of female sexual characteristics.
On the other hand, in males, which have one X
and one Y chromosome (XY), only one copy of the Sex
lethal gene on the X chromosome is active. The gene on
the Y chromosome is different and not involved in
regulating sexual development. In males, the active copy
of the Sex lethal gene is responsible for controlling the
expression of other genes that are important for male
sexual development.
During early development in female fruit flies, the Sex
lethal gene produces functional RNA molecules that bind to
specific sites on the X chromosomes. This binding triggers a
cascade of molecular events that ultimately leads to the
expression of other genes involved in female sexual
development. As a result, the fruit fly develops as a female.
In contrast, in male fruit flies, the Sex lethal gene on the X
chromosome is inactivated. This inactivation prevents the
production of the functional RNA molecules. As a result, the
genes involved in female sexual development are not expressed,
leading to the development of male sexual characteristics.
It's important to note that the expression of the Sex lethal gene is just one example of how sex
determination can occur in certain organisms. Sex determination mechanisms can vary across different
species, and the specific genes involved can be different as well.

A2. The Action of the Transformer Gene

The transformer gene is located on one of the autosomes, which are non-sex chromosomes. Unlike
the Sex lethal gene that we discussed earlier; the transformer gene is not located on the X chromosome.
However, it is still involved in the regulation of sexual development.
During early development in fruit flies, the transformer gene is initially transcribed into RNA. This RNA
molecule then undergoes a process called splicing, where specific segments are removed, and the remaining
segments are joined together. The resulting spliced RNA molecule is then translated into a functional
Transformer protein.
 The Transformer protein plays a key role in determining the sexual fate of cells in fruit flies. In
females, the presence of two X chromosomes results in the production of sufficient levels of the
Transformer protein. This protein binds to specific target genes and promotes the development of female
sexual characteristics.
One of the key target genes that the Transformer protein interacts with is called Doublesex (dsx).
The Doublesex gene plays a crucial role in sexual development and is involved in the differentiation of
various male and female traits in fruit flies. The binding of the Transformer protein to the regulatory regions
of the Doublesex gene helps to activate its expression in female flies.
When the Doublesex gene is activated in females, it leads to the production of a functional Doublesex
protein. This protein, in turn, regulates the expression of other downstream genes involved in the
development of female-specific traits, such as the development of the ovaries and the external genitalia.
In males, which have only one X chromosome, the levels of the Transformer protein are significantly
reduced. As a result, the target genes are not activated properly, and male sexual characteristics develop
instead.

A3. The Msl-2 Protein

The Male-specific lethal-2 (MSL-2) protein is an essential component of the MSL complex, which
plays a crucial role in dosage compensation in fruit flies (Drosophila melanogaster). Dosage compensation is
a process that equalizes gene expression between males and females, as males have one X chromosome
while females have two.
The MSL complex is responsible for enhancing the transcription of genes on the single X
chromosome in males to achieve equal gene expression levels as in females. The MSL-2 protein is one of
the key components of this complex, along with other proteins like MSL-1, MSL-3, and MLE (Maleless).
MSL-2 is encoded by the msl-2 gene, which is located on the X chromosome. It is primarily
expressed in males, and its expression is regulated by the MSL-1 protein. MSL-2 plays a crucial role in
targeting the MSL complex to specific sites on the X chromosome, known as high-affinity sites (HAS).
These HAS regions are characterized by a specific DNA sequence motif, and MSL-2 binds to these sites,
facilitating the recruitment of the rest of the MSL complex.
Once the MSL complex is targeted to the X chromosome, it mediates a series of modifications,
including histone acetylation, to enhance transcriptional activity. This process leads to an increase in the
expression of X-linked genes in males, compensating for the dosage difference with females.
Understanding the function of MSL-2 and the MSL complex provides insights into the mechanisms
of dosage compensation in fruit flies and sheds light on the regulation of gene expression in different sexes.

A4. Results of the Splicing Cascade

Alternative splicing (AS) of pre-messenger (m)RNA is a pivotal mechanism in expanding proteomic
diversity, which determines the functions of mammalian cells. By conducting transcriptome analyses to
profile splicing events in human colorectal cancer (CRC) tissues compared to adjacent normal counterparts,
we noted differential splicing profiles of serine/arginine-rich splicing factor 3 (SRSF3) and mitogen-
activated protein 4 kinase 4 (MAP4K4) in cancerous tissues of CRC compared to adjacent normal tissues. In
addition to SRSF3-mediated autoregulation, RNA-binding motif protein 4 (RBM4) constituted another
mechanism in reprogramming the splicing profile of SRSF3. Upregulated expressions of SRSF3 in CRC
cells modulated utilization of MAP4K4 exon 16 in a sequence-dependent manner. Alternatively spliced
MAP4K4 variants exhibited differential effects on the phosphorylation of c-Jun N-terminal protein kinase 1
(JNK1) which subsequently modulated expression profiles of E-cadherin, N-cadherin, and vimentin, all of
which are involved in the migration and invasion of CRC cells. Collectively, RBM4-SRSF3-MAP4K4
constitutes a novel mechanism for manipulating the metastasis of CRC cells through the JNK1 signaling
pathway.
A5. Sex determination in mammals
Arguably the most defining moment in our lives is fertilization, the point at which we inherit either
an X or a Y chromosome from our father. The profoundly different journeys of male and female life are thus
decided by a genetic coin toss. These differences begin to unfold during fetal development, when the Y-
chromosomal Sry (“sex-determining region Y”) gene is activated in males and acts as a switch that diverts
the fate of the undifferentiated gonadal primordia, the genital ridges, towards testis development.
Mammalian sex is determined genetically by the presence of X and Y chromosomes. Individuals
homozygous for X (XX) are female, while heterozygous individuals (XY) are male. The presence of a Y
chromosome causes the development of male characteristics, while its absence results in female
characteristics. The XY system is also found in some insects and plants.

: Sex determination: The presence of X and Y chromosomes are one of the factors responsible for sex
determination in mammals, with males being the heterozygous sex. In birds, Z and W chromosomes
determine sex, with females being the heterozygous sex.
Avian sex determination is dependent on the presence of Z and W chromosomes. Homozygous for Z (ZZ)
results in a male, while heterozygous (ZW) results in a female. The W appears to be essential in determining
the sex of the individual, similar to the Y chromosome in mammals. Some fish, crustaceans, insects (such as
butterflies and moths), and reptiles use this system.

The sex of some species is not determined by genetics, but by some aspect of the environment. Sex
determination in some crocodiles and turtles, for example, is often dependent on the temperature during
critical periods of egg development. This is referred to as environmental sex determination or, more
specifically, as temperature-dependent sex determination. In many turtles, cooler temperatures during egg
incubation produce males, while warm temperatures produce females. In some crocodiles, moderate
temperatures produce males, while both warm and cool temperatures produce females. In some species, sex
is both genetic- and temperature-dependent.

Individuals of some species change their sex during their lives, alternating between male and female. If the
individual is female first, it is termed protogyny or “first female;” if it is male first, it is termed protandry or
“first male.” Oysters, for example, are born male, grow, become female, and lay eggs; some oyster species
change sex multiple times.

B. Program Cell Death

B1. The process of Apoptosis
Apoptosis is a process that occurs in multicellular when a cell intentionally “decides” to die. This often
occurs for the greater good of the whole organism, such as when the cell’s DNA has become damaged and it
may become cancerous.

Apoptosis is referred to as “programmed” cell death because it happens due to biochemical instructions in
the cell’s DNA; this is opposed to the process of “necrosis,” when a cell dies due to outside trauma or
deprivation.

Like many other complex cellular processes, apoptosis is triggered by signal molecules that tell the cell it’s
time to commit cellular “suicide.”

The two major types of apoptosis pathways are “intrinsic pathways,” where a cell receives a signal to
destroy itself from one of its own genes or proteins due to detection of DNA damage; and “extrinsic
pathways,” where a cell receives a signal to start apoptosis from other cells in the organism. The extrinsic
pathway may be triggered when the organism recognizes that a cell has outlived its usefulness or is no
longer a good investment for the organism to support.

Function of Apoptosis
Apoptosis is an important evolutionary adaptation because it allows organisms to destroy their own cells. At
first glance, that may sound like a terrible idea. Why would you destroy part of yourself?

Well, perhaps if that part of yourself had become dangerous to the rest, as in the case of cells with damaged
DNA that could become cancerous. Apoptosis is a major killer of pre-cancerous cells, and people with
mutations that prevent apoptosis from functioning correctly are much more likely to get cancer.
Multicellular organisms may also wish to lose cells that are no longer useful to the organism. We’ll share
some really spectacular examples of when cell death is a good thing below.

Examples of Apoptosis
From Tadpole to Frog
A spectacular example of this is found in frog tadpoles, which destroy and re-absorb entire body structures
as they undergo their transformation into frogs.

Cells from tadpole’s gills, fins, and tail are “told” to die by apoptosis signals as the tadpole matures. The raw
materials of these dissembled cells become building materials and food for their new growing limbs.

When Does Apoptosis Occur?

Apoptosis occurs when a cell’s existence is no longer useful to the organism. This can occur for a few
reasons.

If a cell has become badly stressed or damaged, it may commit apoptosis to prevent itself from becoming
dangerous to the organism as a whole. Cells with DNA damage, for example, may become cancerous, so it
is better for them to commit apoptosis before that can happen.

Other cellular stresses, such as oxygen deprivation, can also cause a cell to “decide” that it is dangerous or
costly to the host. Cells that can’t function properly may initiate apoptosis, just like cells that have
experienced DNA damage.

In a third scenario, cells may commit apoptosis because the organism doesn’t need them anymore due to its
natural development.

One famous example is that of the tadpole, whose gill, fin, and tail cells commit apoptosis as the tadpole
metamorphoses into a frog. These structures are needed when the tadpole lives in water – but become costly
and harmful when it moves onto dry land.

B2. Formation of Digits in the Tetrapod Limb

Snapshot Summary: The Tetrapod Limb
1.
The places where limbs emerge from the body axis depend upon Hox gene expression.

2.
The specification of the limb field into a hindlimb or forelimb bud is determined by Tbx4 and Tbx5
expression.

3.
The proximal-distal axis of the developing limb is determined by the induction of the ectoderm at the dorsal-
ventral boundary to form the apical ectodermal ridge (AER). This induction is caused by an FGF, probably
FGF10. The AER secretes FGF8, which keeps the underlying mesenchyme proliferative and
undifferentiated. This mesenchyme is called the progress zone.

4.
As the limb grows outward, the stylopod forms first, then the zeugopod, and the autopod is formed last.
Each of these phases involves the expression of Hox genes, and the formation of the autopod involves a
reversal of Hox gene expression that distinguishes fish fins from tetrapod limbs.

5.
The anterior-posterior axis is defined by the expression of Sonic hedgehog in the posterior mesoderm of the
limb bud. This region is called the zone of polarizing activity (ZPA). If the ZPA or Sonic hedgehog-
secreting cells or beads are placed in the anterior margin, they establish a second, mirror-image pattern of
Hox gene expression and a corresponding mirror-image duplication of the digits.
6.
The ZPA is established by the interaction of FGF8 from the AER and mesenchyme made competent to
express Sonic hedgehog by its expression of particular Hox genes. Sonic hedgehog acts, probably in an
indirect manner, to change the expression of the Hox genes in the limb bud.

7.
The dorsal-ventral axis is formed, in part, by the expression of Wnt7a in the dorsal portion of the limb
ectoderm. Wnt7a also maintains the expression of Sonic hedgehog in the ZPA and FGF4 in the posterior
AER. FGF4 and Sonic hedgehog reciprocally maintain each other's expression.

8.
Cell death in the limb is necessary for the formation of digits and joints. It is mediated by BMPs. The effects
of BMPs can be regulated by the Noggin protein, and the BMPs can be involved both in inducing apoptosis
and in differentiating the mesenchymal cells into cartilage.
References:

Meller, V. H., & Rattner, B. P. (2002). The roX genes encode redundant male-specific lethal transcripts
required for targeting of the MSL complex. EMBO Journal, 21(5), 1084–1091.
doi:10.1093/emboj/21.5.1084

Oh, H., Bone, J. R., & Kuroda, M. I. (2004). Multiple classes of MSL binding sites target dosage
compensation to the X chromosome of Drosophila. Current Biology, 14(5), 481–487.
doi:10.1016/j.cub.2004.03.006