Stem Cells and Development:
Lecture 1: Principles of Development and Juxtracrine Signaling
Principles of Development:
Embryo development is progressive, and the fate of cells becomes determined at different times. As
development proceeds, the organizational complexity increases. The cell fate is usually stably
conferred. Fate describes what a group of cells would develop into. If a group of cells is isolated and
cultured a neutral environment and they develop according to their normal fate, then the cells are
specified. Cells are determined if they still develop according to their origin when they are transferred
to a different region of an embryo and so they are immune to the other influences of the new location.
Cells are determined as a result of induction.
Induction: synchronized changes that occur as a result of interaction between two or more cells with
distinct cell histories. One group act as inducers and the other as a responder. Cells need to be more
competent to respond to an inducing signal
Reciprocal Induction:
A feature of inductive events is the reciprocal nature of many inductive interaction e.g. Within the
eye, once the lens is induced, it can in turn induce other tissues such as the optic vesicle. It can
influence the optic vesicle to become the optic cup which differentiates into two layers, the pigmented
layer of the retina and the neural layer of the retina.
Epithelial-Mesenchymal interactions are the best examples of inductive event.
What mediates induction?
- Paracrine Signaling, Endocrine Signaling, Juxtracrine Signaling and Autocrine Signaling
Juxtracrine Signaling:
Proteins from inducing cells can interact with receptors from adjacent cells without the need of
diffusion.
Notch Mutation: Dominant Mutation. The loss of one copy leads to notching of the adult wings,
whilst the loss of two copies results in embryonic lethal. This is because the loss of notch leads to the
hypertrophy of the NS
In sensible development of the fruit fly:
The loss of notch in the larval stages triggers over-production of sensory bristles in the adult and
failure to maintain appropriate spacing. Notch interacts with either Delta or Serrate, which are also
membrane proteins. Notch contains 36 EGF-like repeats and binding to Notch requires EGF repeats
11 and 12.
The Notch Intracellular Domain (ICD) is liberated by cleavage and it traffics to the nucleus form the
cell membrane. Once in the nucleus, the ICD binds to the TR CSL (Cbf1, Su(H), Lag2). In the
absence of Notch signaling, CSL binds to the co-repressor proteins NCoR and represses transcription.
ICD binds to the CSL and displaces the co-repressors and recruits transcription co-activators to
activate transcription.
Targets of Notch-Signaling – CSL consensus recognition sequence 5’-GTGGGAA-3’. Principal
targets are enhanced of split (E(spl)) genes in the drosophila and in mammals, the related hairy
enhancer of the split family genes (HES).
HES genes are basic helix-loop-helix (bHLH) TF. They contain an N-terminal bHLH domain. It binds
DNA as homo or heterodimers with other HES proteins. They are recognized by their E-box sequence
(5’-CANNTG-3’). Each bHLH domain recognizes one half of the sequence. Basic AA at the amino
terminus of the first helix mediates DNA recognition. C-terminal WRPW motif recruits the co-
repressor Groucho. The HES genes are transcriptional repressors.
,The competence to form sensibly is conferred in the larval stages by the expression of the ‘pro-neural
genes’. Expression of pro-neural genes is initially broad, since many cells express it and form pro-
neural clusters. However, expression is progressively restricted to a single cell in each cluster, which
then generates the sensillum. Notch controls the restriction of pro-neural gene expression. This
restriction is restricted by a process called lateral inhibition. Delta expressed on one cell interacts with
Notch, which is expressed on the neighboring cell to activate the Notch Pathway and this eventually
repress Delta. Cells engage in competitive interaction and from a mutually repressive loop. Slight
differences in Delta expression are amplified. Once cell wins and maintains Delta therefore activates
Notch signaling in its neighbors. The Notch activation represses the pro-neural genes.
Inductive signaling by notch: Notch signaling between flanking cells is also used to establish
boundaries. The notch signaling between dorsal and ventral layers of the cells is required to form the
wing margin
Glycosylation of Notch regulates the interaction with ligands. During the Notch transit to the plasma
membrane, Notch passes through the ER. Notch can be glycosylated in the ER if the cells expresses
glycosyltransferase enzymes. The glycosylation of notch in the EGF domains affects interaction with
ligands (it blocks serrate interaction but enhances delta binding).
Fringe, a glycosyltransferase, modifies Notch signaling to enable boundary formation in the wing> ti
catalyzes the addition of the second of the four sugar groups to Notch. Fringe is expressed together
with Serrate in the dorsal compartment of the wing. Delta is expressed in the ventral compartment of
the wing. Fringe modifies Notch and blocks serrate binding but helps Delta to bind.
Fringe with Delta and Serrate without Fringe, restricts Notch activation to the boundary of the dorsal
and ventral surfaces of the wing, allowing the wing margin to form. Mammalian have 3 Fringe
Homologous: Radical Fringe, Lunatic Fringe and Manic Fringe
Somite Formation: Somites arise by boundary formation. The first phase of somites is a process of
periodization or segmentation. Segmented blocks of somites appear progressively from the anterior
(caudal) end of the animal. New somites appear around every 90 minutes and ultimately 42-44
somites are formed, Somites are specified in the PSM (pre-somitic mesoderm)/ Pre-patterned
mesoderm does not induce a boundary, but un-patterned paraxial mesoderm does produce an ectopic
boundary. Ectopic Notch activation in the PSM triggers boundary formation (inducing somite
boundaries). Ectopic Notch activation in the PSM triggers boundary formation (inducing somite
boundaries).
Notch Pathway and Segmentation clock:
Gene targets of the Notch Signaling Pathway: Hes-1, Hes-7 and Lunatic Fringe. They are expressed in
an oscillating pattern within the pre-somatic mesoderm. Waves of expression go anteriorly across the
PSM. Cells caudally first express hes-1/7 and then expression is lost form these cells and the more
anterior cells express them. The band of expression narrows, and it moves progressively across the
PSM until a small band is left that persists and marks the future posterior region of the somite.
The Clock and Wave Front Model:
The primitive node is a source of a morphogen Fgf8 that inhibits the somite formation in the PSM>
As the primitive node regresses the more anterior cells of the PSM can escape this inhibition. When
coupled with oscillation, activation of the Notch pathway and hes1/7 expression, the regression of the
primitive streak allows the regular progressive addition of pairs of somites
,Lecture 2: Paracrine Signaling in Development
Paracrine Signaling involves a secretory cell that release the signal/molecule adjacent to target cells.
This type of signaling explains the morphogen gradient models of development.
Morphogen Gradient Model:
A soluble substance (A morphogen) diffuses from its site of synthesis to its site of degradation (sink).
Therefore, a gradient is formed from the source to the sink and if the cells responds differently
according to the levels of the morphogen, then different cell fates can be achieved across the gradient.
‘French Flag’ Model: the analogy for operation of a gradient of positional information. Positional
information is delivered by a gradient of diffusible morphogen extending from a source to sink.
Thresholds on the left indicate the cellular properties that enable the gradient to be interpreted.
Example – cells become blue at one concentration but as the morphogen levels decline below a
certain threshold, they become white
Gradient Modelling:
Inductive signals trigger differentiation of the somites. Secreted signaling molecules generated by the
neural tube, the notochord and the epidermis trigger the differentiation of the somites. Wnt signaling
from the neural tube and the epidermis induces differentiation of the dermatome and myotome. The
dermatome is induced by neurotrophin (NT30) and Wnt1 is generated by the neural tube. The
myotome is induced by two distinct signals, epaxial myoblasts are induced by Wnt1 and Wnt3a,
whilst the hypaxial myoblasts are induced by Wnt from the epidermis and BMP4 produced by the
lateral plate mesoderm
Wnt (wireless) gene encodes secreted glycoprotein ligands that can acts as both shorting-range
signaling molecules and long-range morphogens. This gene is required to pattern the Drosophila
wings and other adult body structures. Initial allele is a partial loss of function or a hylomorphic
mutation and complete loss of function, or null, allele is lethal and shows defects in embryo cuticle
pattern. Wingless is a morphogen.
Drosophila larvae are segmented, and each abdominal segment develops specialised hook structures
called denticles. Wingless is transcribed and expressed in a stripe of cells at the posterior of each
segment. Wingless protein diffuses from the expressing cells; therefore, it establishes a protein
gradient. The presence of the Wingless protein prevents the epidermal cells from developing
denticles. It exhibits graded distribution of protein from a source, and a graded response in the
surrounding cells. The Drosophila wing is formed form an initial flat sheet of cells, that later
invaginates to from the two cell layers that adhere to generate the dorsal and the ventral surfaces of
the wing. High levels of Wingless triggers the expression of Achaete. Intermediate levels drive the
expression of Distal-Less. Low level drive vestigial expression. In the absence of wingless, the
margin is not formed, and the wing does not expand, hence the wingless mutant phenotype.
Wingless Signaling Pathway:
2 important Features:
- Activation of the wingless signaling pathway occurs by inhibition of an inhibitor
- DNA binding TF (TCF) is converted form a transcriptional repressor to an activator by
changes in the associated proteins. Activation is accompanied by a relief in repression.
Extracellular Wnt signal stimulates several intra-cellular signal transduction cascades. Wnt proteins
are secreted glycoproteins that bind to the N-terminal extra-cellular cysteine rich domain of the
Frizzled (Fz) receptor family of which there is ten Fz in humans and 4 in Drosophila. Fz protein is a
seven transmembrane – spanning protein that are probably coupled to G-proteins. LRP/Arrow is a
core receptor for Wingless, therefore the receptor of Wnt is made from Frizzled and LRP/Arrow. It is
a single transmembrane protein and it is related to a family of lipoprotein receptors. After binding of
Wnt to the receptor complex, the signal is transduced to cytoplasmic phosphoprotein, called
, Dishelleved (Dsh). The Wnt signal then branches in 3 major cascades. Canonical, Planar Cell Polarity
and Wnt/Ca2+. Dsh is an important downstream component of this transduction pathway and it is
involved in all three major branches of Wnt signaling.
Properties of the Signal: Wnt Proteins – secreted protein of 350-400 aa. They have to be lipid
modified since active Wnt requires lipid moiety. They adhere to the ECM and act on over1-5 cell
diameters. This has a gradient of action.
Properties of the Receptor: Receptor is composed of Frizzled and the co-receptor LRP/Arrow.
Frizzled is the core receptor and is a 7-pass transmembrane protein. It is most probably a G-protein
couple receptor and it has multiple Frizzled family members.
Properties of the Transducer: -catenin/Armadillo is a cytoplasmic nuclear signaling mediator. It is
also identified as a component of cell adherent junction. It contains tandemly repeated 40 aa motifs
(12 repeats) and the armadillo repeat that forms a superhelix with a positively charged groove,
provides an interaction surface with binding partners. Stability is controlled by phosphorylation by
GSK3/Shaggy. Phosphorylated B-catenin/Armadillo is targeted for proteasome degradation by
destruction complex including APC/Axin.
-catenin stability: Axin provides the scaffold for the destruction complex. -catenin is
phosphorylated by casein 1 at Ser45. GSK3 is phosphorylates Thr41, Ser37 and Ser33.
Phosphorylated -catenin is bound by the ubiquitin ligase -cTRCp. Ubiquitination of -catenin
targets it for proteasome degradation. APC regulates the transition from phosphorylation to
ubiquitination.
TCF (LEF-1) is a binding partner for -catenin. DNA binding proteins that contain HMG (high
mobility group) domain. In the absence of the Wnt signaling, TCF binds to a co-repressor (Groucho)
and it represses transcription. Binding of -catenin to TCF switches it into a transcriptional activator.
This means that the TCF switches from a repressor to an activator.
In the absence of nuclear -catenin, TCF acts as a transcriptional repressor by binding to the co-
repressor Groucho/TLE proteins. -catenin displaces Groucho/TLE by binding to an additional low
affinity site that overlaps the Groucho/ TLE binding site. -catenin/TCF activates transcription.
Lecture 1: Principles of Development and Juxtracrine Signaling
Principles of Development:
Embryo development is progressive, and the fate of cells becomes determined at different times. As
development proceeds, the organizational complexity increases. The cell fate is usually stably
conferred. Fate describes what a group of cells would develop into. If a group of cells is isolated and
cultured a neutral environment and they develop according to their normal fate, then the cells are
specified. Cells are determined if they still develop according to their origin when they are transferred
to a different region of an embryo and so they are immune to the other influences of the new location.
Cells are determined as a result of induction.
Induction: synchronized changes that occur as a result of interaction between two or more cells with
distinct cell histories. One group act as inducers and the other as a responder. Cells need to be more
competent to respond to an inducing signal
Reciprocal Induction:
A feature of inductive events is the reciprocal nature of many inductive interaction e.g. Within the
eye, once the lens is induced, it can in turn induce other tissues such as the optic vesicle. It can
influence the optic vesicle to become the optic cup which differentiates into two layers, the pigmented
layer of the retina and the neural layer of the retina.
Epithelial-Mesenchymal interactions are the best examples of inductive event.
What mediates induction?
- Paracrine Signaling, Endocrine Signaling, Juxtracrine Signaling and Autocrine Signaling
Juxtracrine Signaling:
Proteins from inducing cells can interact with receptors from adjacent cells without the need of
diffusion.
Notch Mutation: Dominant Mutation. The loss of one copy leads to notching of the adult wings,
whilst the loss of two copies results in embryonic lethal. This is because the loss of notch leads to the
hypertrophy of the NS
In sensible development of the fruit fly:
The loss of notch in the larval stages triggers over-production of sensory bristles in the adult and
failure to maintain appropriate spacing. Notch interacts with either Delta or Serrate, which are also
membrane proteins. Notch contains 36 EGF-like repeats and binding to Notch requires EGF repeats
11 and 12.
The Notch Intracellular Domain (ICD) is liberated by cleavage and it traffics to the nucleus form the
cell membrane. Once in the nucleus, the ICD binds to the TR CSL (Cbf1, Su(H), Lag2). In the
absence of Notch signaling, CSL binds to the co-repressor proteins NCoR and represses transcription.
ICD binds to the CSL and displaces the co-repressors and recruits transcription co-activators to
activate transcription.
Targets of Notch-Signaling – CSL consensus recognition sequence 5’-GTGGGAA-3’. Principal
targets are enhanced of split (E(spl)) genes in the drosophila and in mammals, the related hairy
enhancer of the split family genes (HES).
HES genes are basic helix-loop-helix (bHLH) TF. They contain an N-terminal bHLH domain. It binds
DNA as homo or heterodimers with other HES proteins. They are recognized by their E-box sequence
(5’-CANNTG-3’). Each bHLH domain recognizes one half of the sequence. Basic AA at the amino
terminus of the first helix mediates DNA recognition. C-terminal WRPW motif recruits the co-
repressor Groucho. The HES genes are transcriptional repressors.
,The competence to form sensibly is conferred in the larval stages by the expression of the ‘pro-neural
genes’. Expression of pro-neural genes is initially broad, since many cells express it and form pro-
neural clusters. However, expression is progressively restricted to a single cell in each cluster, which
then generates the sensillum. Notch controls the restriction of pro-neural gene expression. This
restriction is restricted by a process called lateral inhibition. Delta expressed on one cell interacts with
Notch, which is expressed on the neighboring cell to activate the Notch Pathway and this eventually
repress Delta. Cells engage in competitive interaction and from a mutually repressive loop. Slight
differences in Delta expression are amplified. Once cell wins and maintains Delta therefore activates
Notch signaling in its neighbors. The Notch activation represses the pro-neural genes.
Inductive signaling by notch: Notch signaling between flanking cells is also used to establish
boundaries. The notch signaling between dorsal and ventral layers of the cells is required to form the
wing margin
Glycosylation of Notch regulates the interaction with ligands. During the Notch transit to the plasma
membrane, Notch passes through the ER. Notch can be glycosylated in the ER if the cells expresses
glycosyltransferase enzymes. The glycosylation of notch in the EGF domains affects interaction with
ligands (it blocks serrate interaction but enhances delta binding).
Fringe, a glycosyltransferase, modifies Notch signaling to enable boundary formation in the wing> ti
catalyzes the addition of the second of the four sugar groups to Notch. Fringe is expressed together
with Serrate in the dorsal compartment of the wing. Delta is expressed in the ventral compartment of
the wing. Fringe modifies Notch and blocks serrate binding but helps Delta to bind.
Fringe with Delta and Serrate without Fringe, restricts Notch activation to the boundary of the dorsal
and ventral surfaces of the wing, allowing the wing margin to form. Mammalian have 3 Fringe
Homologous: Radical Fringe, Lunatic Fringe and Manic Fringe
Somite Formation: Somites arise by boundary formation. The first phase of somites is a process of
periodization or segmentation. Segmented blocks of somites appear progressively from the anterior
(caudal) end of the animal. New somites appear around every 90 minutes and ultimately 42-44
somites are formed, Somites are specified in the PSM (pre-somitic mesoderm)/ Pre-patterned
mesoderm does not induce a boundary, but un-patterned paraxial mesoderm does produce an ectopic
boundary. Ectopic Notch activation in the PSM triggers boundary formation (inducing somite
boundaries). Ectopic Notch activation in the PSM triggers boundary formation (inducing somite
boundaries).
Notch Pathway and Segmentation clock:
Gene targets of the Notch Signaling Pathway: Hes-1, Hes-7 and Lunatic Fringe. They are expressed in
an oscillating pattern within the pre-somatic mesoderm. Waves of expression go anteriorly across the
PSM. Cells caudally first express hes-1/7 and then expression is lost form these cells and the more
anterior cells express them. The band of expression narrows, and it moves progressively across the
PSM until a small band is left that persists and marks the future posterior region of the somite.
The Clock and Wave Front Model:
The primitive node is a source of a morphogen Fgf8 that inhibits the somite formation in the PSM>
As the primitive node regresses the more anterior cells of the PSM can escape this inhibition. When
coupled with oscillation, activation of the Notch pathway and hes1/7 expression, the regression of the
primitive streak allows the regular progressive addition of pairs of somites
,Lecture 2: Paracrine Signaling in Development
Paracrine Signaling involves a secretory cell that release the signal/molecule adjacent to target cells.
This type of signaling explains the morphogen gradient models of development.
Morphogen Gradient Model:
A soluble substance (A morphogen) diffuses from its site of synthesis to its site of degradation (sink).
Therefore, a gradient is formed from the source to the sink and if the cells responds differently
according to the levels of the morphogen, then different cell fates can be achieved across the gradient.
‘French Flag’ Model: the analogy for operation of a gradient of positional information. Positional
information is delivered by a gradient of diffusible morphogen extending from a source to sink.
Thresholds on the left indicate the cellular properties that enable the gradient to be interpreted.
Example – cells become blue at one concentration but as the morphogen levels decline below a
certain threshold, they become white
Gradient Modelling:
Inductive signals trigger differentiation of the somites. Secreted signaling molecules generated by the
neural tube, the notochord and the epidermis trigger the differentiation of the somites. Wnt signaling
from the neural tube and the epidermis induces differentiation of the dermatome and myotome. The
dermatome is induced by neurotrophin (NT30) and Wnt1 is generated by the neural tube. The
myotome is induced by two distinct signals, epaxial myoblasts are induced by Wnt1 and Wnt3a,
whilst the hypaxial myoblasts are induced by Wnt from the epidermis and BMP4 produced by the
lateral plate mesoderm
Wnt (wireless) gene encodes secreted glycoprotein ligands that can acts as both shorting-range
signaling molecules and long-range morphogens. This gene is required to pattern the Drosophila
wings and other adult body structures. Initial allele is a partial loss of function or a hylomorphic
mutation and complete loss of function, or null, allele is lethal and shows defects in embryo cuticle
pattern. Wingless is a morphogen.
Drosophila larvae are segmented, and each abdominal segment develops specialised hook structures
called denticles. Wingless is transcribed and expressed in a stripe of cells at the posterior of each
segment. Wingless protein diffuses from the expressing cells; therefore, it establishes a protein
gradient. The presence of the Wingless protein prevents the epidermal cells from developing
denticles. It exhibits graded distribution of protein from a source, and a graded response in the
surrounding cells. The Drosophila wing is formed form an initial flat sheet of cells, that later
invaginates to from the two cell layers that adhere to generate the dorsal and the ventral surfaces of
the wing. High levels of Wingless triggers the expression of Achaete. Intermediate levels drive the
expression of Distal-Less. Low level drive vestigial expression. In the absence of wingless, the
margin is not formed, and the wing does not expand, hence the wingless mutant phenotype.
Wingless Signaling Pathway:
2 important Features:
- Activation of the wingless signaling pathway occurs by inhibition of an inhibitor
- DNA binding TF (TCF) is converted form a transcriptional repressor to an activator by
changes in the associated proteins. Activation is accompanied by a relief in repression.
Extracellular Wnt signal stimulates several intra-cellular signal transduction cascades. Wnt proteins
are secreted glycoproteins that bind to the N-terminal extra-cellular cysteine rich domain of the
Frizzled (Fz) receptor family of which there is ten Fz in humans and 4 in Drosophila. Fz protein is a
seven transmembrane – spanning protein that are probably coupled to G-proteins. LRP/Arrow is a
core receptor for Wingless, therefore the receptor of Wnt is made from Frizzled and LRP/Arrow. It is
a single transmembrane protein and it is related to a family of lipoprotein receptors. After binding of
Wnt to the receptor complex, the signal is transduced to cytoplasmic phosphoprotein, called
, Dishelleved (Dsh). The Wnt signal then branches in 3 major cascades. Canonical, Planar Cell Polarity
and Wnt/Ca2+. Dsh is an important downstream component of this transduction pathway and it is
involved in all three major branches of Wnt signaling.
Properties of the Signal: Wnt Proteins – secreted protein of 350-400 aa. They have to be lipid
modified since active Wnt requires lipid moiety. They adhere to the ECM and act on over1-5 cell
diameters. This has a gradient of action.
Properties of the Receptor: Receptor is composed of Frizzled and the co-receptor LRP/Arrow.
Frizzled is the core receptor and is a 7-pass transmembrane protein. It is most probably a G-protein
couple receptor and it has multiple Frizzled family members.
Properties of the Transducer: -catenin/Armadillo is a cytoplasmic nuclear signaling mediator. It is
also identified as a component of cell adherent junction. It contains tandemly repeated 40 aa motifs
(12 repeats) and the armadillo repeat that forms a superhelix with a positively charged groove,
provides an interaction surface with binding partners. Stability is controlled by phosphorylation by
GSK3/Shaggy. Phosphorylated B-catenin/Armadillo is targeted for proteasome degradation by
destruction complex including APC/Axin.
-catenin stability: Axin provides the scaffold for the destruction complex. -catenin is
phosphorylated by casein 1 at Ser45. GSK3 is phosphorylates Thr41, Ser37 and Ser33.
Phosphorylated -catenin is bound by the ubiquitin ligase -cTRCp. Ubiquitination of -catenin
targets it for proteasome degradation. APC regulates the transition from phosphorylation to
ubiquitination.
TCF (LEF-1) is a binding partner for -catenin. DNA binding proteins that contain HMG (high
mobility group) domain. In the absence of the Wnt signaling, TCF binds to a co-repressor (Groucho)
and it represses transcription. Binding of -catenin to TCF switches it into a transcriptional activator.
This means that the TCF switches from a repressor to an activator.
In the absence of nuclear -catenin, TCF acts as a transcriptional repressor by binding to the co-
repressor Groucho/TLE proteins. -catenin displaces Groucho/TLE by binding to an additional low
affinity site that overlaps the Groucho/ TLE binding site. -catenin/TCF activates transcription.
