Alternative mechanisms of

signal transduction

The Cellular and Molecular

Basis of Development
Stem cells
• Stem cells in the embryo can give rise to all cells and tissues in the
developing organism.
• Adult stem cells maintain tissues in the mature organism.
• These types of stem cells and induced pluripotent stem cells (iPS) are
potential sources of cells for regeneration and/or repair of injured or
degenerating cells and organs.
 Morphogens
• These are diffusible molecules that specify which cell type will be generated at a
specific anatomical location.
• Morphogens also direct the migration of cells and their processes to their final
destination.
• These include retinoic acid, transforming growth factor-β (TGF-β), bone
morphogenetic proteins (BMPs), and the hedgehog and Wnt protein families
• Many morphogens are found in concentration gradients in the embryo.
• Different morphogens can be expressed in opposing gradients in the dorsoventral,
anteroposterior (AP), and mediolateral axes.
• The fate of a specific cell can be determined by its location along these different
gradients.
 Retinoic Acid
• The bioactive form of vitamin A is retinoic acid, which is formed by enzymatic oxidation
by retinol aldehyde dehydrogenase and subsequently retinal aldehyde dehydrogenase.
• Retinoic acid can also be actively degraded into inactive metabolites by enzymes such as
CYP26
• Normally, retinoic acid acts to “posteriorize” the body plan, and either excessive retinoic
acid or inhibition of its degradation leads to a truncated body axis where structures have
a more posterior nature.
• In contrast, insufficient retinoic acid or defects in the enzymes (e.g., retinal aldehyde
dehydrogenase) will lead to a more “anteriorized” structure.
• At the molecular level, retinoic acid binds to its receptors (transcription factors) inside
the cell, and their activation will regulate the expression of downstream genes.
• Hox genes are crucial targets of retinoic acid receptors in development.
• Because of their profound influence on early development, retinoids are powerful
teratogens, especially during the first trimester.
 Transforming Growth Factor-β/Bone Morphogenetic
Protein
• Members of the TGF-β superfamily include TGF-β, BMPs, and activin.
• These molecules contribute to the establishment of dorsoventral patterning,
cell fate decisions, and formation of specific organs and systems, including
the kidneys, nervous system, skeleton, and blood.
• In humans, there are three different forms of TGF-β (isoforms TGF-β1, TGF-
β2, and TGF-β3).
• Binding of these ligands to transmembrane kinase receptors results in
phosphorylation of intracellular receptor-associated Smad proteins (R-
Smads)
• The Smad proteins are a large family of
intercellular proteins that are divided into three
classes:
• Receptor activated (R-Smads), common-partner
Smads (co-Smads [e.g., Smad4]), and inhibitory
Smads (I-Smads).
• R-Smad/Smad4 complexes regulate target gene
transcription by interacting with other proteins
or as transcription factors by directly binding to
DNA.
• The diversity of TGF-β ligand, receptor, and R-
Smad combinations contributes to particular
developmental and cell-specific processes, often
in combination with other signaling pathways
 Sonic Hedgehog

• Sonic hedgehog (Shh) was the first mammalian ortholog of the

Drosophila gene hedgehog to be identified.
• Shh and other related proteins, such as desert hedgehog and Indian
hedgehog, are secreted morphogens critical for early patterning, cell
migration, and differentiation of many cell types and organ systems.
• The primary receptor for Shh is Patched
(PTCH in human, PTC family in mouse), a
transmembrane domain protein.
• In the absence of Shh, Patched inhibits
transmembrane domain, G-protein–linked
protein (Smoothened [Smo]).
• This results in inhibitions of downstream
signaling to the nucleus.
• However, in the presence of Shh, Ptc
inhibition is blocked and downstream events
follow, including transcriptional activation of
target genes, such as Ptc-1, Engrailed, and
others
• The role of Shh in patterning of the vertebrate ventral neural tube is one of its
best-studied activities.
• Shh is secreted at high levels by the notochord, and therefore the concentration
of Shh is highest in the floor plate of the neural tube and lowest in the roof
plate, where members of the TGF-β family are highly expressed.
• The cell fates of ventral interneuron classes and motor neurons are determined
by the relative Shh concentrations in the tissue and other factors.
• Mutations of SHH and PTCH have been associated with
holoprosencephaly in humans, a common congenital brain defect
resulting in the fusion of the two cerebral hemispheres, dorsalization
of forebrain structures, and anophthalmia or cyclopia.
 Gorlin syndrome
• Gorlin syndrome, often due to germline PTCH mutations, is a constellation
of congenital malformations mostly affecting the epidermis, craniofacial
structures, and nervous system.
• also known as nevoid basal cell carcinoma syndrome, is a condition that
affects many areas of the body and increases the risk of developing various
cancerous and noncancerous tumors.
• Rib and vertebrae anomalies
• Intracranial calcification
• Skeletal abnormalities: bifid ribs, kyphoscoliosis, early calcification of falx
cerebri (diagnosed with AP radiograph)
• Distinct faces: frontal and temporoparietal bossing, hypertelorism, and
mandibular prognathism
• Bilateral ovarian fibromas
• 10% develop cardiac fibromas
• Most people with Gorlin syndrome also develop noncancerous (benign)
tumors of the jaw, called keratocystic odontogenic tumors. Keratocystic
odontogenic tumors rarely develop later in adulthood. If untreated, these
tumors may cause painful facial swelling and tooth displacement.
• Mutations of the GLI3 gene, encoding a zinc finger that mediates Shh
signaling, are associated with autosomal dominant polydactyly
syndromes.
• Polydactyly is associated with different mutations, either mutations in a
gene itself or in a cis-regulatory element responsible for the expression of
a specific gene. Mutations in Hoxa- or Hoxd clusters are reported leading
to polydactyly. Interactions of Hoxd13 and GLI3 induce synpolydactyly, a
combination of extra and consolidated digits. Other signal transduction
pathways in this context are the Wnt signaling pathway or Notch
 Wnt/β-Catenin Pathway

• The Wnt-secreted glycoproteins are vertebrate orthologs of the

Drosophila gene Wingless.
• Similar to the other morphogens, the 19 Wnt family members control
several processes during development, including establishment of cell
polarity, proliferation, apoptosis, cell fate specification, and migration.
• Specific Wnts bind to 1 of 10 Frizzled (Fzd) seven-transmembrane domain
cell surface receptors, and with low-density, lipoprotein receptor–related
proteins 5 and 6 (LRP5/LRP6) coreceptors, thereby activating downstream
intracellular signaling events.
• In the absence of Wnt binding,
cytoplasmic β-catenin is phosphorylated
by glycogen synthase kinase 3 (GSK-3) and
targeted for degradation.
• In the presence of Wnts, GSK-3 is
inactivated, and β-catenin is not
phosphorylated and accumulates in the
cytoplasm.
• The β-catenin translocates to the nucleus,
where it activates target gene
transcription in a complex with T-cell
factor (TCF) transcription factors.
• β-Catenin/TCF target genes include
vascular endothelial growth factor (VEGF)
and matrix metalloproteinases.
• Dysregulated Wnt signaling is a prominent feature in many
developmental disorders, such as Williams-Beuren syndrome
(heart, neurodevelopmental, and facial defects), and in cancer.
• Williams syndrome is a developmental disorder that affects
many parts of the body. This condition is characterized by mild
to moderate intellectual disability or learning problems,
unique personality characteristics, distinctive facial features,
and heart and blood vessel (cardiovascular) problems.
• People with Williams syndrome typically have difficulty with
visual-spatial tasks such as drawing and assembling puzzles,
but they tend to do well on tasks that involve spoken
language, music, and learning by repetition
• Young children with Williams syndrome have distinctive facial
features including a broadforehead, a short nose, with a broad
tip, full cheeks, and a wide mouth with full lips. Many affected
people have dental problems such as teeth that are small,
widely spaced, crooked, or missing. In older children and
adults, the face appears longer and more gaunt.
• LRP5 mutations are found in the osteoporosis-pseudoglioma
syndrome (congenital blindness and juvenile osteoporosis).
• Similar to the Shh pathway, canonical Wnt pathway mutations have
been described in children with medulloblastoma, a common
malignant brain tumor.
Intercellular communication
• Development involves the interaction of a cell with its neighboring
cell either directly (gap junctions) or indirectly (cell adhesion
molecules).
 Gap Junctions
• Gap junctions are channels that permit ions and small molecules
(<1 kD) to directly pass from one cell to another, known as gap
junctional intercellular communication (GJIC).
• Large proteins and nucleic acids cannot transfer through gap
junctions.
• Gap junctions are made from hemi-channels present on the
surface of each cell known as connexons. Each connexon is made
up of six connexin molecules that form hexamers.
• In early development, gap junctions are usually open, permitting
exchange of small molecules in relatively large regions.
• As development proceeds, GJIC is more restricted, with
establishment of boundaries, such as in the rhombomeres of the
developing hindbrain.
• Gap junctions are particularly important for electrical coupling in
the heart and brain. Mutations of specific connexin molecules
are associated with human diseases (e.g., mutation of Cx43 is
associated with atherosclerosis).
 Cell Adhesion Molecules
• Cell adhesion molecules have large extracellular domains that interact with extracellular matrix
components or adhesion molecules on neighboring cells.
• These molecules often contain a transmembrane segment and a short cytoplasmic domain,
which regulates intracellular signalling cascades.
• An example of cell adhesion molecules is the cadherins, which have important roles during
embryonic development.
• Cadherins are critical for embryonic morphogenesis as they regulate the
• separation of cell layers (endothelial and epidermal),
• cell migration,
• cell sorting,
• establishment of well-defined boundaries,
• These properties result from cadherins mediating the interaction between the cell and its
extracellular milieu (both neighboring cells and extracellular matrix).
• Cadherins were originally classified by their site of expression; for example, E-cadherin is highly
expressed in epithelial cells, whereas N-cadherin is highly expressed in neural cells.
• The cadherin extracellular domain contains four
calcium-binding sites and five repeated
domains called extracellular cadherin domains.
• Each cadherin molecule forms a homodimer.
• In the intracellular domain, cadherin binds
directly to p120-catenin and to β-catenin,
which binds to α-catenin.
• This complex links the cadherin molecules to
the actin cytoskeleton.
• E-cadherin expression is lost as epithelial cells
transition to mesenchymal cells
• this is known as the epithelial-to-mesenchymal
transition [EMT].
• EMT is required for the formation of neural
crest cells during development, and the same
process also occurs during tumor development.
 Receptor tyrosine kinases (RTKs)
• Many growth factors signal by binding to and activating membranebound
RTKs.
• These kinases are essential for the regulation of cellular proliferation,
apoptosis, and migration.
 RECEPTOR TYROSINE KINASES
• Growth factors, such as insulin, epidermal growth factor, nerve growth factor, and other
neurotrophins, and members of the platelet-derived growth factor family, bind to cell surface
transmembrane receptors found on target cells.
• These receptors, members of the RTK superfamily, have three domains: (1) an extracellular
ligandbinding domain, (2) a transmembrane domain, and (3) an intracellular kinase domain.
• They arefound as monomers in the unbound state but dimerize upon ligand binding. This process
of dimerization brings the two intracellular kinase domains into close proximity such that one
kinase domain can phosphorylate and activate the other receptor (transphosphorylation), which
is required to fully activate the receptors.
• In turn, this initiates a series of intracellular signaling cascades.
• An inactivating mutation of one receptor subunit kinase domainresults in abolishment of
signaling; such a mutation in the kinase domain of the VEGF receptor 3 (VEGFR-3)results in the
autosomal dominantly inherited lymphatic disorder called Milroy disease.
Regulation of Angiogenesis by Receptor Tyrosine Kinases

• Endothelial cells are derived from a progenitor cell (the hemangioblast) that can give rise to both
the hematopoietic cell lineage and endothelial cells.
• The early endothelial cells proliferate and eventually coalesce to form the first primitive blood
vessels. This process is termed vasculogenesis.
• After the first blood vessels are formed, they undergo intensive remodeling and maturation into
the mature blood vessels in a process called angiogenesis.
• This maturation process involves the recruitment of vascular smooth muscle cells to the vessels to
stabilize them.
• Vasculogenesis and angiogenesis are both dependent on the function of two distinct RTK classes,
members of the VEGF and Tie receptor families.
• VEGF-A was shown to be essential for endothelial and blood cell development;
• VEGF-A knockout mice fail to develop blood or endothelial cells and die at early embryonic stages.
• A related molecule, VEGF-C, was shown to be crucial for the development of lymphatic endothelial
cells
 NOTCH-DELTA
PATHWAY
• The Notch signaling pathway is integral for cell fate determination, including
maintenance of stem cell niches, proliferation, apoptosis, and differentiation.
• These processes are essential for all aspects of organ development through
regulation of lateral and inductive cell-cell signaling.
• Notch proteins are single transmembrane receptors that interact with
membranebound Notch ligands (e.g., Delta-like ligands and serrate-like ligands
[Jagged]) on adjacent cells
• Ligand-receptor binding triggers proteolytic events leading to the release of the
Notch intracellular domain (NICD).
• When the NICD translocates to the nucleus, a series of intranuclear events culminates
in the induction of expression of a transcription factor that maintains the progenitor
state of the cell.
• In the initial cell-cell interaction, Notch receptor signaling maintains one cell as
an uncommitted progenitor.
• The adjacent cell maintains reduced Notch signaling and undergoes
differentiation.
• Inductive signaling with other surrounding cells expressing morphogens may
overcome a cell’s commitment to a default fate and lead to an alternative cell
fate.
• The findings that mutations in Jagged1 are associated with Alagille syndrome
(arteriohepatic dysplasia), with liver, kidney, cardiovascular, ocular, and skeletal
malformations, and that NOTCH-3 gene mutations are found in CADASIL
(cerebral autosomal dominant arteriopathy with subcortical infarcts and
leukoencephalopathy), an adult vascular degenerative disease with a tendency
to early-age onset of stroke-like events, support the importance of the Notch
signaling pathway in embryonic and postnatal development, respectively.
Transcription factors
• This set of evolutionarily conserved proteins activates or represses
downstream genes that are essential for a number of cellular
processes.
• Many transcription factors are members of the homeobox or helix-
loop-helix families.
Hox/Homeobox Proteins
• The Hox genes were first discovered in the fruit fly, Drosophila melanogaster.
• The order of the Hox genes along the AP axis is faithfully reproduced in their
organization at the level of the chromosome.
• Mutations in these genes of the HOM-C complex lead to dramatic
phenotypes (homeotic transformation) such as the Antennapedia gene, in
which legs instead of antennae sprout from the head of the fruit fly.
• In humans, the order of the Hox genes along the AP axis and chromosomal
location is conserved as well.
• Defects in HOXA1 have been shown to impair human neural development,
and mutations in HOXA13 and HOXD13 result in limb malformations
• All the Hox genes contain a 180-base-pair sequence, the homeobox, which
encodes a 60-amino-acid homeodomain composed of three α-helices.
• The third (recognition) helix binds to DNA sites that contain one or more
binding motifs in the promoters of their target genes.
• The homeodomain is highly conserved across evolution, whereas other
regions of the protein are not as well conserved.
• Mutations in the DNA-binding region of the homeobox gene NKX2.5 are
associated with cardiac atrial septal defects, and mutations in ARX are
associated with the central nervous system malformation syndrome
known as lissencephaly.
 Pax Genes
• The Pax genes all contain conserved bipartite DNAbinding motifs called the Pax (or paired) domain, and
most Pax family members also contain a homeodomain.
• PAX proteins have been shown to both activate and repress transcription of target genes.
• The Drosophila melanogaster ortholog of Pax6, eyeless, was shown to be essential for eye development
because homozygous mutant flies had no eyes. Eyeless shares a high degree of sequence conservation
with its human ortholog PAX6 and is associated with ocular malformations such as aniridia (absence of
the iris) and Peters anomaly.
• In human eye diseases, the level of PAX6 expression seems to be crucial because patients with only one
functional copy (haploinsufficiency) have ocular defects and patients without PAX6 function are
anophthalmic.
• PAX3 and PAX7 encode both homeodomain and Pax DNA-binding
domains.
• The human childhood cancer alveolar rhabdomyosarcoma results from a
translocation that results in the formation of a chimeric protein wherein
PAX3 or PAX7 (including both DNA domains) is fused to the strong
activating domains of the Forkhead family transcription factor FOXO1.
• The autosomal dominant human disease Waardenburg syndrome type I
results from mutations in the PAX3 gene. Patients with this syndrome
have hearing deficits, ocular defects (dystopia canthorum), and
pigmentation abnormalities best typified by a white forelock.
• There are marked differences among the various signaling pathways, but they
share many common features: ligands, membrane-bound receptors and
coreceptors, intracellular signaling domains, adapters, and effector molecules.
• Signaling pathways are co-opted at various times during development for stem
cell renewal, cell proliferation, migration, apoptosis, and differentiation.
• Knowledge of gene function has been acquired by reverse genetics using model
systems with loss- or gainof- function transgenic approaches. As well, much
insight has been gained by forward genetics that begins with the description of
abnormal phenotypes arising spontaneously in mice and humans and then
subsequent identification of the mutant gene.
• There is evidence of cross-talk among pathways. This communication among
various signaling pathways facilitates our understanding of the far-reaching
consequences of single gene mutations that result in malformation syndromes
affecting the development of multiple organ systems, or in cancers.