Gene Transcription: Embryology: The Field Investigations of The Molecular, Cellular, and Structural

Embryology: the field investigations of the molecular, cellular, and structural
factors contributing to formation of an organism.

The process of progressing from a single cell through the period of establishing
organ primordia (the first 8 weeks of human development) is called the period of
embryogenesis (sometimes called the period of Organogenesis); the period
from that point on until birth is called the fetal period, a time when
differentiation continues while the fetus grows and gains weight.
The study of the embryological origins and causes for these birth defects was
called teratology.
example employing developing limb buds, it was shown that if a piece of tissue
from the posterior axial border of one limb was grafted to the anterior border of a
second limb, then digits on the host limb would be duplicated as the mirror
image of each other. This posterior signaling region was called the Zone of
polarizing activity (ZPA), and it is now known that the signaling molecule is
sonic hedgehog.
Gene Transcription
Genes are contained in a complex of DNA and proteins (mostly histones)
called chromatin, whose basic unit of structure is the Nucleosome (see Fig.
1.3). Each nucleosome is composed of an octamer of histone proteins and
approximately 140 base pairs of DNA. Nucleosomes themselves are joined
into clusters by binding of DNA existing between nucleosomes (linker
DNA) with other histone proteins (H1 histones; Fig. 1.3). Nucleosomes
keep the DNA tightly coiled, such that it cannot be transcribed. In this
inactive state, chromatin appears as beads of nucleosomes on a string of
DNA and is referred to as heterochromatin. For transcription to occur, this
DNA must be uncoiled from the beads. In this uncoiled state, chromatin is
referred to as euchromatin.
Genes reside within the DNA strand and contain regions called exons,
which can be translated into proteins, and introns, which are interspersed
between exons and which are not transcribed into proteins (see Fig. 1.4).
In addition to exons and introns, a typical gene includes the following: a
Promoter region that binds RNA polymerase for the initiation of
transcription; a transcription initiation site; a translation initiation site to
designate the first amino acid in the protein; a translation termination
codon; and a 3 untranslated region that includes a sequence (the poly A
addition site) that assists with stabilizing the mRNA, allows it to exit the
nucleus, and permits it to be translated into protein (Fig. 1.4). By
convention the 5 and 3 regions of a gene are specified in relation to the
RNA transcribed from the gene. Thus, DNA is transcribed from the 5 to
the 3 end, and the promoter region is upstream from the transcription
initiation site (Fig. 1.4). The promoter region, where the RNA polymerase
binds, usually contains the sequence TATA, and this site is called the TATA
box (Fig. 1.4). However, in order to bind to this site the polymerase
requires additional proteins called Transcription factors (see Fig. 1.5).
Transcription factors also have a specific DNA binding domain plus a
transactivating domain that activates or inhibits transcription of the gene
whose promoter or enhancer it has bound. In combination with other

proteins, transcription factors activate gene expression by causing the
DNA nucleosome complex to unwind, by releasing the polymerase so that
it can transcribe the DNA template, and by preventing new nucleosomes
from forming.
Enhancers are regulatory elements of DNA that activate utilization of promoters

to control their efficiency and the rate of transcription from the promoter.
Enhancers can reside anywhere along the DNA strand and do not have to reside
close to a promoter. Sometimes enhancers can inhibit transcription and are
called silencers. This phenomenon allows a transcription factor to activate one
gene while silencing another by binding to different enhancers.
The initial transcript of a gene is called nuclear RNA (nRNA) or sometimes

premessenger RNA. nRNA is longer than mRNA because it contains introns
that are removed (spliced out) as the nRNA moves from the nucleus to the
cytoplasm. In fact, this splicing process provides a means for cells to
produce different proteins from a single gene. For example, by removing
different introns, exons are spliced in different patterns, a process called
Alternative splicing (see Fig. 1.6). The process is carried out by
spliceosomes, which are complexes of small nuclear RNAs (snRNA) and
proteins that recognize specific splice sites at the 5 or 3 ends of the
nRNA. Proteins derived from the same gene are called splicing isoforms
(also called splice variants or alternative splice forms), and these afford
the opportunity for different cells to use the same gene to make proteins
specific for that cell type. For example, isoforms of the WT1 gene have
different functions in gonadal versus kidney development.
Even after a protein is made (translated), there may be posttranslational
modifications that affect its function. For example, some proteins have to
be cleaved to become active, or they might have to be phosphorylated.
Others need to combine with other proteins or be released from
sequestered sites or be targeted to specific cell regions. Thus, there are
many regulatory levels for synthesizing and activating proteins, such that
even though only 35,000 genes exist, the potential number of proteins
that can be synthesized is probably closer to three times the number of
genes.
Cell Signaling
Cell-to-cell signaling is essential for induction, for conference of
competency to respond, and for cross talk between inducing and
responding cells. These lines of communication are established by
paracrine interactions, whereby proteins synthesized by one cell diffuse
over short distances to interact with other cells, or by juxtacrine
interactions, which do not involve diffusable proteins. The diffusable
proteins responsible for paracrine signaling are called paracrine factors or
growth and differentiation factors (GDFs). There are a large number of
GDFs, but most are grouped into four families, and members of these
same families are used repeatedly to regulate development and
differentiation of organ systems. Furthermore, the same GDFs regulate
organ development throughout the animal kingdom from Drosophila to
humans. The four groups of GDFs include the fibroblast growth factor
(FGF), WNT, hedgehog, and transforming growth factor families.
Fgfs
Approximately two dozen FGF genes have been identified, and they can
produce hundreds of protein isoforms by altering their RNA splicing or
their initiation codons. FGF proteins produced by these genes activate a
collection of tyrosine receptor kinases called fibroblast growth factor
receptors (FGFRs). In turn, these receptors activate various signaling
pathways. FGFs are particularly important for angiogenesis, axon growth,
and mesoderm differentiation. Although there is redundancy in the family,
such that FGFs can sometimes substitute for one another, individual FGFs
may be responsible for specific developmental events. For example, FGF8
is important for development of the limbs and parts of the brain.
Hedgehog Proteins
There are three hedgehog genes, Desert, Indian, and sonic hedgehog.
Sonic hedgehog is involved in a number of developmental events
including limb patterning, neural tube induction and patterning, somite
differentiation, gut regionalization, and others. The receptor for the
hedgehog family is Patched, which binds to a protein called Smoothened.
The Smoothened protein transduces the hedgehog signal, but it is
inhibited by Patched until the hedgehog protein binds to this receptor.
Thus the role of the paracrine factor hedgehog in this example is to bind
to its receptor to remove the inhibition of a transducer that would
normally be active, not to activate the transducer directly.
WNT Proteins
There are at least 15 different WNT proteins that are involved in
developmental pathways. Their receptors are members of the frizzled
family of proteins. WNT proteins are involved in regulating limb
patterning, midbrain development, and some aspects of somite and
urogenital differentiation among other actions.
The TGF Superfamily
The TGF superfamily has over 30 members and includes the
transforming growth factor s, the bone morphogenetic proteins, the
activin family, the Mllerian inhibiting factor (MIF, anti-Mllerian
hormone), and others. The TGF members are important for extracellular
matrix formation and epithelial branching that occurs in lung, kidney, and
salivary gland development. The BMP family induces bone formation and
is involved in regulating cell division, cell death (apoptosis), and cell
migration among other functions.
Signal Transduction Pathways
Paracrine Factors
Paracrine factors act by signal transduction pathways either by activating
a pathway directly
P.9
or by blocking the activity of an inhibitor of a pathway (inhibiting an

inhibitor, as is the case with hedgehog signaling). Signal transduction
pathways include a signaling molecule (the ligand) and a receptor (see
Fig. 1.8). The receptor spans the cell membrane and has an extracellular
domain (the ligand binding region), a transmembrane domain, and a
cytoplasmic domain. When a ligand binds its receptor, it induces a
conformational change in the receptor that activates its cytoplasmic
domain. Usually, the result of this activation is to confer enzymatic activity
to the receptor, and most often this activity is a kinase that can
phosphorylate other proteins using ATP as a substrate. In turn,
phosphorylation activates these proteins to phosphorylate additional
proteins, and thus a cascade of protein interactions is established that
ultimately activates a transcription factor. This transcription factor then
activates or inhibits gene expression. The pathways are numerous and
complex and in some cases are characterized by one protein inhibiting
another that in turn activates another protein (much like the situation with
hedgehog signaling).
Juxtacrine Signaling
Juxtacrine signaling is mediated through signal transduction pathways as
well but does not involve diffusable factors. Instead, there are three ways
juxtacrine signaling occurs: (1) A protein on one cell surface interacts with
a receptor on an adjacent cell in a process analogous to paracrine
signaling (Fig. 1.8). The Notch pathway represents an example of this type
of signaling. The Notch receptor protein extends across the cell membrane
and binds to cells that have Delta, Serrate, or Jagged proteins in their cell
membranes. Binding of one of these proteins to Notch causes a
conformational change in the Notch protein such that part of it on the
cytoplasmic side of the membrane is cleaved. The cleaved portion then
binds to a transcription factor to activate gene expression. Notch signaling
is especially important in neuronal differentiation, blood vessel
specification, and somite segmentation. (2) Ligands in the extracellular
matrix secreted by one cell interact with their receptors on neighboring
cells. The extracellular matrix is the milieu in which cells reside. This
milieu consists of large molecules secreted by cells including collagen,
proteoglycans (chondroitin sulfates, hyaluronic acid, etc.), and
glycoproteins, such as fibronectin and laminin. These molecules provide a
substrate for cells on which they can anchor or migrate. For example,
laminin and type IV collagen are components of the basal lamina for
epithelial cell attachment, and fibronectin molecules form scaffolds for cell
migration. Receptors that link extracellular molecules such as fibronectin
and laminin to cells are called integrins. These receptors integrate
matrix molecules with a cell's cytoskeletal machinery, e.g., actin
microfilaments, thereby creating the ability to migrate along matrix
scaffolding by using contractile proteins, such as actin. Also, integrins can
induce gene expression and regulate differentiation as in the case of
chondrocytes that must be linked to the matrix to form cartilage. (3) There
is direct transmission of signals from one cell to another by gap junctions.
These junctions occur as channels between cells through which small
molecules and ions can pass. Such communication is important in tightly

connected cells like epithelia of the gut and neural tube because they
allow these cells to act in concert. The junctions themselves are made of
connexin proteins that form a channel, and these channels are
connected between adjacent cells.

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Embryology: the field investigations of the molecular, cellular, and structural

factors contributing to formation of an organism.

whose promoter or enhancer it has bound. In combination with other

Enhancers are regulatory elements of DNA that activate utilization of promoters

The initial transcript of a gene is called nuclear RNA (nRNA) or sometimes

or by blocking the activity of an inhibitor of a pathway (inhibiting an

molecules and ions can pass. Such communication is important in tightly

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