DEVELOPMENTAL GENETICS

LESSON 1: SEX DETERMINATION

Expression of the ____________________(Using Drosophila Fly)

 Expression of the Sex lethal gene involves transcription from two different promoters.
 Two different promoters:
1) Establishment Promoter
 Only females transcribe the Sex lethal gene from the
_____________________________________ (PE), which generates a transcript that is
spliced and translated to generate functional protein.
2) Late Promoter
 After establishing the initial expression, sex lethal is transcribed from the
____________________________________ in both males and females. Transcription of
the Sex lethal gene from PE occurs only during the syncytial and cellular blastoderm stages.
Transcription from PE depends on the same proteins that determine the X/A ratio.

Female embryos Male embryos

The amount of X/A proteins is sufficiently high
that transcription from PE occurs. The PE-
derived Sex lethal transcript is spliced to
produce functional Sex lethal protein.
The initial Sex lethal protein that was generated
from the PE transcript in females functions to
prevent exon L3 from splicing to L2 in the PL
transcript. Instead, L2 splices to exon 4, which
can be translated to generate additional Sex
lethal protein.
The amount of X/A proteins is low, which
represses transcription from PE
The absence of Sex lethal protein in males
results in exon L2 splicing to L3 on the PL
transcript. This male transcript introduces a
translation termination codon into the ORF,
which again prevents the production of
functional Sex lethal protein. In this manner, Sex
lethal protein is initially produced only in
females, and it continues to be expressed only
in females.

 Later in development, and throughout the remaining life of the fly, the cells transcribe the Sex
lethal gene from PL in both males and females.
 The productive splicing of the PL Sex lethal transcript depends on the prior presence of Sex lethal
protein, which only occurs in females. This is typical of later steps of the sex-determination
pathway: The default pathway in Drosophila leads to male development, whereas regulated
activity leads to female- specific gene activation.

The exons are __________ and the introns are ______________ The gene contains two promoters
(red), PE that drives transcription early in female development and PL, which is transcribed in both
genders later in development. The location of the translation initiation codons (AUG) are shown in exons
E1 and L2, while the translation termination codons (Stop) are in exons L3 and 10. The

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__________________ designate the various splices that are used. In the early embryo, a single
mRNA is transcribed from the PE promoter and is only found in females. This mRNA encodes a functional
Sxl protein. Later in development, the Sxl gene is transcribed from the PL promoter. In females, the pre-
mRNA is spliced from exon L2 to 4 to generate the complete open reading frame. In males, the pre-
mRNA is spliced from exon L2 to L3, which produces a truncated open reading frame that encodes a
non-functional protein. The lower table summarizes the transcript that is produced from both promoters
in both genders. Only in the female is functional Sex lethal protein ever produced.

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Figure 1.9: Transcription and alternative splicing of the Drosophila Sex lethal (Sxl) gene.
(Image from Hyde introduction to Genetics p.745)

The Action of the transformer Gene

 The second key gene in the sex-specification cascade is transformer (tra). Like Sex lethal,
the transformer primary transcript is alternatively spliced.

Female Embryos Male Embryos

The transformer primary transcript is
spliced to one mRNA form in the presence
of the Sex lethal protein in females. This
female-specific tra mRNA can be
translated to yield a functional Tra protein.
In the absence of Sex lethal protein (in
males), the transformer primary transcript
is spliced into an alternative form.
Translation of this male tra mRNA is
terminated early due to the presence of a
translational termination codon in an exon
that is absent in the female-specific tra
mRNA. This yields a truncated and
nonfunctional Tra protein in males.

In females, the Tra protein interacts with
the Transformer-2 protein, which
regulates the splicing of the doublesex
(dsx) and fruitless (fru) primary
transcripts. The female-specific dsx and
fru mRNAs are translated to generate
female-specific Doublesex and Fruitless
proteins.
In males, the Transformer-2 protein, with
the truncated Tra protein, generates
different spliced male-specific dsx and fru
mRNAs that are translated to produce
male-specific and functional Doublesex
and Fruitless proteins.

The ______________________PROTEIN

 One final important control in sex determination is the production of the Male-specific
lethal-2 (Msl2) protein.
 In this case, the absence of the Sex lethal protein splices the msl-2 primary transcript into
an mRNA that is translated into a functional Msl2 protein.
 In females, the Sex lethal protein alters the splicing of the msl-2 primary transcript to
produce a female msl-2 mRNA that is translated into a truncated and nonfunctional Msl-2
protein.

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  The Msl-2 protein is essential for properly balancing expression of X-linked genes in the
two genders. Because females have two X chromosomes relative to one in males, they
have the possibility of expressing twice as much mRNA from each X-linked gene compared
to males. These differences in chromosome number can be lethal or deleterious. This is
resolved in Drosophila males by the Msl-2 protein functioning to double the transcription
of the X-linked genes. This increased transcription results in both genders exhibiting
similar levels of transcription of X-linked genes.

Results of the Splicing Cascade

 The end result of this cascade of alternative splicing is sex-specific control of gene
expression.
 Whereas:
1) The ___________________ is the overall control gene.
2) The _______________________, and
_______________________________________________ regulate
specific functions associated with the two sexes.
3) The _______________________ protein regulates the expression of genes
that control the development of the somatic and germ-line tissues in both sexes.
4) The _______________________ protein regulates the expression of genes
that allow differences in the central nervous system between the two sexes, which
results in sex specific behaviors. This difference is very important in the male- and
female-specific mating behaviors. The Msl-2 protein is critical for the
hyperactivation of Xlinked gene expression in males.
5) The doublesex and fruitless genes encode functional proteins in both sexes.
Aberrant splicing of either primary transcript may therefore result in the gender of
the individual switching. In contrast, the msl2 gene encodes a functional protein
only in males.
 If improper splicing of the msl-2 primary transcript occurs in males, hyperactivation of the
X-linked genes does not occur, and the fly dies due to genetic imbalance. In contrast, if
incorrect splicing of the gene occurs in females to produce functional Msl-2 protein, the
fly also dies due to hyperactivation of transcription of both X chromosomes and genetic
imbalance again.
 Mutants can be isolated in which the sex of the fly has been switched relative to its X/A
ratio, but mutations that affect the level of X chromosome transcription are lethal.

Sex determination in mammals

 Two issues must be resolved in sex determination.

1) First, differences must occur during the development of the two genders,
including both the somatic and germ tissues.

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 2) Second, differences in the number of sex chromosomes between the two
genders must be compensated for by altering the level of transcription of the
sex-linked genes in one of the genders. In Drosophila, this is achieved by
increasing the level of transcription of the X-linked genes in males (one X
chromosome) relative to females (two X chromosomes).
These two issues must also be resolved in mammalian sex determination.

 ___________, which is located on the Y chromosome. Evidence that the SRY gene is
sufficient to determine the gender of mammals was observed in mice and humans who
lack this gene on the Y chromosome. These XY(SRY deleted) individuals develop as
females. Similarly, when the SRY gene is translocated from the Y chromosome to an
autosome, XX individuals develop as males if they possess the SRY gene translocation.
The SRY gene encodes a DNA-binding protein with an HMG domain.
 ____________- is a 79-amino-acid DNA binding motif that is composed of three α-
helices. The HMG domain binds in the minor groove of DNA, which causes an
approximately 80-degree bend in the DNA. The result is that the DNA becomes
significantly unwound and even nucleosomes that are associated with some specific
genes are displaced. The end result of HMG binding is usually transcriptional
activation of specific genes.
 Many of the missense mutations in the SRY gene that cause human sex reversal are
located in the HMG domain, which suggests that this region of the protein is essential to
its function.
 Sex determination in mammals:
 Begins with the initial formation of an indeterminate gonad within the urogenital
system. If the SRY gene is present within these cells, then it is expressed, and
the resulting SRY protein performs two critical functions.
 First, it activates expression of the SOX9 gene, which also encodes a HMG-
containing protein. Unlike the Y-linked SRY gene, the SOX9 gene is an autosomal
gene. Transgenic mice that express SOX9 develop as males, even if they lack the
SRY gene. Thus, either SRY or SOX9 is sufficient to determine maleness in mice.
The SOX9 protein is involved in the activation of genes that encode testis-
determining factors and AMH (antiMüllerian hormone).
 Expression of AMH causes the Müllerian ducts to regress, which in combination
with the expression of the testis-determining factors, commits the indeterminate
gonad to accept the male fate. The second significant function of the SRY protein
is to block the expression of the WNT4 and DAX1 genes (see fig. 21.31). The
WNT4 protein activates the expression of genes that encode the ovary-
determining factors; the DAX1 protein blocks the expression of proteins that
activate expression of the testis-determining factors. In this manner WNT4 and
DAX1 combine to induce female gonad development, while also blocking male
gonad development. When SRY represses WNT4 and DAX1 expression, it is

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 preventing female gonad development in concert with the SOX9-induced
expression of AMH.

LESSON 2: PROGRAM CELL DEATH

 Most cells are determined to become a specific cell type, and in the absence of specific
cues, they adopt a different cell type. These homeotic transformations—one cell type
into another—are under genetic control. A different path that cells can undertake leads
to cell death.

__________________
- It is a mechanism of cell death that is controlled by the action of a specific
group of genes. Apoptosis involves the condensation of the cytoplasm,
fragmentation of the nuclear genome, and engulfment of the cell by
macrophages.
- It is a normal process in the development of an organism. It is involved in a
number of different developmental events, such as the patterning of
connections in the vertebrate neural system and establishing independent digits
during autopod development.
- The first genes involved in apoptosis were identified in
______________________________________. Studies of C. elegans
development revealed that 131 of 1090 cells die in the generation of the 959
somatic cells in the adult hermaphrodite, and 147 of 1178 cells die during the
generation of the 1031 cells in the male.
- Apoptosis is characterized by condensation of the cytoplasm and nucleus,
blebbing of the plasma membrane (a bubbled or blistered appearance),
fragmentation of the nuclear DNA, and, ultimately, engulfment of the cell by
macrophages.
- Whereas apoptosis is the result of a specific cascade of proteins that results in
the engulfment of a dying cell, necrosis results in cell injury and then death,
without a specific series of proteins mediating the process.

____________________
- It is a form of cell death that results from injury to the cell. Necrosis does not
require the action of a series of proteins.
- It exhibits cell swelling, fragmentation of organelles, and rupturing of the
plasma membrane.

The Process of Apoptosis

 The “decision” to enter apoptosis usually involves two groups of genes.

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 1) The first is ________________ and its related genes. These genes encode
proteins that associate with the mitochondrial membrane, which is disrupted
during apoptosis. Some of the Bcl-2 related proteins (Bcl-2 and Bcl-xi) suppress
apoptosis, and other members (Bax, Bad, and Bak) promote apoptosis.
2) The second group of genes involves ____________ and its related genes.
The p53 protein is a transcription factor that controls cell proliferation and DNA
repair. The role of these proteins in apoptosis is described in greater detail in
chapter 22. Once the cell is committed to enter apoptosis, a series of cysteine
proteases, called caspases, are activated.
 Some caspases act to cleave cytoskeletal proteins, which allow the cell to condense. Other
caspases cleave cell adhesion proteins, which allow the macrophage to move around and
engulf the dying cell. Still another caspase cleaves an inhibitor of a specific DNase, which
then allows the DNase to digest the genomic DNA in the cell.
 The outcome of apoptosis is to safely remove the cell without significant damage to the
surrounding cells. Many examples exist of the role of apoptosis during development. We
now look at two situations:
1. ________________________
2. The generation of digits

Formation of Digits in the Tetrapod Limb

 During embryogenesis, tetrapod vertebrate limbs begin by forming a limb bud that
develops into a paddle-like extension.
 Most tetrapod adults have individual digits, however, rather than paddle-shaped limbs.
 Two different mechanisms are likely to contribute to the formation of the adult autopod
(foot, hand, wing, and so on).
1. First, regions that will give rise to individual digits probably have a higher rate of
cell proliferation than the interdigital zones, leading to selective outgrowth of the
future digits.
2. Second, cells located in the interdigital zones are selectively removed by
apoptosis. Regulation of apoptotic cell death in specific regions during vertebrate
limb development leads to formation of different types of adult autopod
structures.

 Example:
1. In the chick hindlimb, bone morphogenetic proteins (BMPs) act as ligands to
stimulate a signaling cascade that initiates apoptosis in the interdigital zone.
2. In the duck hindlimb, an inhibitor of the BMP signaling cascade leads to lack of
apoptosis and formation of interdigital webbing in the adult.
 If the BMP signaling pathway is blocked in chick development, then interdigital webbing
occurs.

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