Chapter 4 Notes

Cell-Cell Communication
in Development
 All that you touch
You Change.
All that you Change
Changes You.
The only lasting truth
Is Change.

– Octavia Butler (1998), Woman African American Sci-fi writer

 In dealing with such a complex system as the
embryo, it is futile to inquire whether a certain
organ rudiment is “determined” and whether some
feature of its surroundings, to the exclusion of
others, “determines” it. A score of different factors
may be involved, and their effects most intricately
interwoven. In order to resolve this tangle, we have
to inquire into the manner in which the system
under consideration reacts with other parts of the
embryo at successive stages of development and
under as great a variety of experimental conditions
as it is possible to impose.
– R.G. Harrison (1933), Professor of Ecology and Evolutionary Biology @ Cornell
University
 Morphogenesis
• The construction of organized form

• A great mystery throughout history.

– Angels, Many miracles, “tiny sensitive bodies”…

• Issue Summarized in Five Modern Questions

– How are separate tissues formed from populations of cells?
– How are organs constructed from tissues?
– How do organs form in their correct locations, how do migrating cells find their correct destinations?
– How do cells and organs grow and coordinate their growth?
– How do structures achieve polarity / Asymmetry?
 Cellular Interactions
• Remember from Chapter 1:
– Epithelial cells – form sheets and tubes and adhere to one another
– Mesenchymal cells – typically migrate individually and will form Extracellular matrixes of larger tissues

– Three Behaviors that require Cell-to-Cell Communication via the Cell Surface
• Differential Cell Adhesion/Affinity
• Cell Shape
• Cell Signaling / Communications
 Signaling Distances
• Very Short
– Autocrine Signaling – A single cell produces both ligand and receptor
– Juxtacrine Signaling – Contact Dependent – Signals on the surface of one cell are bound by receptors
on adjacent cells
– Synaptic Signaling – Across synaptic cleft, Neural tissue specific

• Local Effects
– Paracrine Signaling – signals diffuse only a short distance from signaling cell to local “neighborhood”
cells

• Long Distance
– Endocrine Signaling – Hormones secreted into the bloodstream affect other parts of the body by
stimulating responses in distant cells
• Relatively slow - relies on circulation
• Act at very dilute concentrations
• Affect gene expression directly – very “powerful” effects
 Cell Communication Basics
• Signaling Molecules (ligands) bind to receptors
embedded in the cell membrane
• Binding of a receptor to its ligand alters the
receptors shape (conformational change)
– Can alter the Intracellular domain of the receptor allowing
the external signal to be relayed to the inside of the cell

• This signal can be “Transduced” through a series of

conformational changes / phosphorylation /
production of other small molecules (cAMP, Ca++)
that lead to cellular responses

• Culminates in:
– Enzymatic / Biochemical Changes / Cytoskeletal
alterations (Fast)
– Activating Gene Expression (slower)
 Differential Cell Adhesion
• Each type of cell expresses a definitive set of cell surface adhesion molecules that are partially
responsible for the ability of groups of cells to form structures, tissues, organs during development.

• Towns and Holtfreter (1955) – Re-aggregation experiments

– Took different cell types from embryos, disassociated the cells, and then mixed different types of cells
together. They found that:
– Reaggregated cells became spatially segregated again by type
– Final positions of the cell types reflected their “correct” positions within an intact embryo!
– Interpreted as Selective Affinity
 Selective Affinity
• The “automatic” cellular sorting observed was attributed
to each cell type’s differential adhesion to the other types of
cells.

– Inner surface of the ectoderm has a positive affinity for

mesodermal cells and a negative affinity for endoderm

– Mesoderm has a positive affinity for ectoderm on the outer

surface and a positive affinity for endoderm on the inner surface.

• Towns and Holtfreter’s Final conclusion suggested that

these affinities can change during development allowing
relationships between cells/tissue/structures to change and
be modified.
 Thermodynamic Model of Interaction
• 41 years later (1996-97), Towns and Holtfreter’s
hypothesis was confirmed through the work of
Steinberg, Foty and others.

• Changes in cell surface adhesion molecules alter

the strength of the affinity between individual cells.
– Changes in the number of receptors
– Changes in the the type of receptors
– Changes in cell morphology of the cell (changes
accessibility of receptors)

• Cells will arrange themselves into the most

thermodynamicallly stable configuration
– Changes in gene expression will result in
changes of cell interactions
 Cadherins and Cell Adhesion
• Most common cell-surface adhesion molecule

• Ca++-dependent Homophilic binders

– Only bind to similar cadherins on other cells

• Transmembrane proteins that create physical

linkages between cells and the actin cytoskeleton

• Form Adherens junctions with additional internal

proteins (catenins) that hold epithelial cells together

• Major functions:
– Anchor cells together
– Help assemble and link to the actin cytoskeleton
– Signaling function that can affect gene transcription
 Classes of Cadherins
• Different types of cells express different Cadherins
• Similar cells have similar cadherins – Very strong interaction
• Differences in # and in type change the strength of cell-cell interactions
• Cell with different cadherins Repel on another and form boundaries

– E-Cadherins – expressed on all early embryonic cells, later restricted to only epithelial cells
– P-Cadherins – Placental plays a role in connecting embryo to uterus
– N-Cadherins – restricted to neural tissues
– R-Cadherins – involved in retina formation
– Other classes exist, these are the major ones

– Protocadherins – lack the protein domain allowing attachment to the cytoskeleton through
catenins. Helps keep similar migrating epithelial cells together.
 Cadherins and Development
• Play numerous important roles during various developmental stages,
some examples:

• Neurulation:
– N-cadherin required for proper separation of neural tube ectodermal cells
from those that will become epidermis
– All ectoderm cells express E-cadherin but those that will become the neural
tube loose it and express N-cadherin
– Without this change, nervous system development fails

• Uterine Wall implantation

– Trophoblast cells of early mammalian embryo (those that surround the
inner cell mass that will from the embryo) express a number of adherence
molecules allowing it to stick to the endometrium of the Uterus
• E and P-cadherins
• Integrins that bind Collagen
• Glycosyltransferase enzymes
Cadherins and Adherens Junctions Video
 Extracellular Matrix and Development
• The Extracellular Matrix (ECM) is an insoluble
non-cellular matrix of macromolecules between
cells

• Critical for Development

– Cell adhesion
– Cell migration
– Formation of epithelial sheets/tubes
– Directions for cell movements
– Signals for developmental events

• Composed of:
– Collagen
– Proteoglycans
– Elastin
– Glycoproteins – fibronectin, laminin
 Connection to the ECM – Integrins
• Family of Extracellular matrix receptors identified in 1986.
• Also Calcium Dependent

• Create physical connections between the ECM and the

Cytoskeleton

• Bind to RGD sequences in ECM (Arg-Gly-Asp)

– Fibronectin
– Laminin
– Vitronectin
– Collagens

• Connect to Actin microfilaments through α-actinin and talin.

• Critical for cell movements, Focal adhesions, involved in

gene expression, prevention of apoptosis
– Anoikis - apoptosis due to loss of contact with ECM
 Epithelial → Mesenchymal Transition (EMT)
• When a polarized, adhered, stationary cell
is transformed in an orderly way into an
invasive and motile mesenchymal cell that
can move to form new structures
– Typically initiated by paracrine factors from
neighbors
– Cadherins are down-regulated and ECM
attachments are released
– Actin cytoskeleton is rearranged for
movement
– Cells begin to secrete mesenchymal ECM
molecules

• Examples:
– Formation of neural crest cells, somite
formation, mesoderm formation
– Wound healing in adults
– Metastatic tumors
 Induction and Competence
• During ALL stages of development cell differentiation and behavior are regulated by signals
sent from one cell and received by another

• Induction – A proximate (short range) interaction where one cell(s) changes the behavior of
another cell
– Inducer – cell producing the signal (often a paracrine factor)
– Responder – The cell/tissue being induced by the signal.
• Must have a receptor allowing it to receive the signal
• Must be able to respond to the signal with a change in biology (see below…)

• Competence – The ability to respond to an inducing signal

– Must be actively and specifically acquired, not all cells are capable of responding to a particular signal
– Have to have the right receiving equipment = receptors
Xenopus Optic Vesicle Induction Experiments
• Experimental Model used to investigate induction and competence
interactions
 Complex Series of Inductions…
• In Amphibians…
– The first inducer of the lens may be the foregut endoderm and heart-forming mesoderm
that underlie the lens-forming ectoderm during early and mid gastrula stages.
– The next inducer appears to be the neural plate, promoting the production of the Pax6
transcription factor in neural ectoderm
– Pax6 provides competence to respond to inducers from optic cup cells

– So even though the inducer appears to be the optic vesicle, the responding cells have already
been induced at least two previous time
 Reciprocal Inductions of the Mouse Lens
• The Optic vesicle (neural ectoderm) extends towards the surface ectoderm from the forebrain
– This induces the lens placode (thickening on surface ectoderm) to form
• As the lens placode thickens it induces the optic vesicle to reshape into the optic cup
• The Optic cup induces the center of the lens to invaginate.
• This invagination induces the optic cup to differentiate into two layers of the retina.
• Further invagination of the lens placode forms the Lens vesicle / Lens capsule.
• The lens placode induces the overlying ectoderm to form the Cornea.
 Instructive vs. Permissive
• Two major modes of induction:

• Instructive interactions –
– A signal from the inducing cell is Necessary for initiating new gene expression in the
responding cell.
– Without the inducing signal the responding cell is incapable of responding in the required
way

• Permissive interactions –
– In this type the responding cell already has been specified (is expressing or has the
ability to express the correct genes) it just needs the correct environment and signals to
allow the expression of those traits
 Epithelial-Mesenchymal Interactions
• All organs are composed of an epithelium and
an associated group of mesenchyme.

• Regional Specification
– Interactions between epithelium and mesenchyme
can create regionally specified structures =
location, location, location
• Dermis/Epidermis interactions induce Feather
formation; different types in different parts of
the body

• Genetic specificity of induction

– Mesenchyme may instruct epithelium as to which
genes to activate, but epithelium can only comply
so far as the genome permits
• Transplant experiments … next slide
 Interspecies Induction Experiments
• Sperman and Schotte (1932) transplanted flank ectoderm
from a frog gastrula onto the region of a newt gastrula
destined to become the mouth, and vise-versa.

• The chimeric larvae developed mouthparts in the correct

locations
– The mesenchyme instructed the ectoderm to make a mouth

• However the specific structures made were those from the

original ectoderm embryo
– The ectoderm responded by making the only mouth it “knew”
how to make
– The only structure “permitted” by the genome of the responding
cells
 Induction of Tracheal Development
• In Drosophila the respiratory system develops from epithelial sacs.

– Reorganization of about 80 cells in each sac produce primary,

secondary, and tertiary branches without cell division or apoptosis

– Initiated when nearby cells secrete Branchless (BNL) protein is a

chemoattractant which is bound by a cell membrane receptor on
epithelial cells.

– The cell receiving the most signal are attracted and migrate towards
the signal via changes in their cytoskeleton and changes in
Cadherin expression
• Express the Breathless protein (BTL)
• Leader cells guide the rest of the surrounding cells.
• The following cells receive a signal from the leading cells causing
them to become the tracheal tube

– Tertiary branch cells are also interacting with developing

musculature to ensure a close association
 Morphogen Gradients
• Gradients of paracrine factors that regulate gene expression

• Morphogen – A diffusible molecule that affects distant cell

fates by concentration
– Greek = “Form-giver”
– Transcription factors produced within a cell or secreted
paracrine factors
– Source – Cell(s) producing the Morphogen / Transmitting
cell
– Sink – Receiving cells affected by the morphogen

– Act in Analog – action on the receiving cell is dependent on

concentration
• Contrast to Morphogenic determinants that act Digitally (on/off)
• Cell fate is determined by the relative amounts of soluble
molecules present. Distance from source is important.
• A single morphogen might determine 3 or more cell fates
depending on concentration received
 Morphogen in Xenopus development

• Morphogen = Activin, a paracrine factor in the

TGF-B family was embedded into beads and
placed on unspecified cells
– High concentrations = induced expression of
googsecoid transcription factor activating dorsal-most
structures
– Medium concentrations = induced Xbra gene which
specifies cells to become muscles
– Low concentrations = Cells not activated but follow
default pathway and became blood vessels and heart
Signal Transduction Cascades – The Response to Inducers
• For a ligand to induce a cellular response, it
must bind to a receptor which starts a cascade
of events within the cell that ultimately
regulate a response.
– Molecular “Tag”

• Paracrine factors bind to surface receptors

and initiate a series of enzymatic reactions
within the receiving cell.

– End points:
• Regulation of transcription factors
• Remodeling of the cytoskeleton (change
shape / alter movement, migration)
Signal Transduction Cascades – The Response to Inducers
• Variations on a Theme. All following pathways share
a common overall structure:

• Receptor spans the membrane

• Paracrine factor binds to extracellular domain
• Induces a conformational change that is conveyed to
the cytoplasmic domain
• This activates cytoplasmic proteins, often kinases
• Sometimes the receptor itself has a cytoplasmic
domain with enzymatic function that becomes
activated (RTK = Receptor Tyrosine Kinase)
• Initiates a cascade of inductions ultimately activating
a transcription factor or altering the cytoskeleton of
the cell
 In Class Student Presentations
• The following Pathways will be presented by student teams
• Exam questions will be taken from a pool of student written questions distributed to
the class prior to the next exam.

• Pathways presented:
– FGF & RTK Signal Transduction Pathway (Figure 4.20)
– JAK-STAT Pathway (Figure 4.21)
– Hedgehog Pathway (Figure 4.24)
– Wnt Signal Transduction Pathway (Figure 4.28)
– Smad Pathway (Figure 4.30)
– Notch Signaling Mechanism = Juxtracrine Signaling pathway (Figure 4.37)
 Major Paracrine Pathways
• Fibroblast growth factor (FGF) and JAK-STAT pathways
– Involved in growth and differentiation. Breathless (BTL) is a FGF protein

• Hedgehog family
– Induce cell types and create boundaries between tissues

• Wnt family
– Establishment of polarity in limbs, proliferation of stem cells, urogenital development, induction of dorsal cells to become
somites, specification of midbrain

• TGF-β superfamily (Transforming Growth Factors)

– TGF-β family
– Activin family
– Bone morphogenic proteins (BMP) family
– Nodal proteins
– Vg1 family
– Epidermal growth factors, neurotrophils, stem cell factors, hepatocyte factors
– Others...
 Fibroblast Growth Factors – RTK
• Large family with about 2 dozen members known
• Bind to cell surface Receptor Tyrosine Kinases (RTK)
• Ligand binding causes Autophosphorylation of the cytoplasmic domain of the
receptor
• Phosphorylated domain recognized by adaptor protein (SOS) that binds and activates
Guanine nucleotide release factor (GNRP)
• GNRP binds to and activates the G-protein RAS.
– Assisted by GEF (Guanine Nucleotide Exchange Factor)

• Activated RAS(GTP-bound state) binds to and activates RAF (a kinase – adds

phosphate groups to other proteins)
– RAS is slowly INACTIVATED by GAP (GTPase Activating Protein) that promotes the GTPase activity of RAS to self-
inactivate (back to the GDP bound state) – In this way the signal is only transiently activated.

• RAF phosphorylates and activates MEK

• MEK phosphorylates and activates ERK
• ERK is a kinase that can enter the nucleus and activates a series of transcription
factors by phosphorylating them
Fibroblast Growth Factors – RTK
 JAK-STAT Pathway
• Another paracrine signal pathway using RTK
• Important in differentiation of blood cells, growth of limbs, activation of casein
gene during lactation.
 Hedgehog Family
• At least three vertebrate homologs
– Sonic hedgehog, desert hedgehog, indian hedgehog
• Active Hedgehog proteins require complexed
cholesterol for production and signal reception
• Normally the patched protein (receptor for
Hedgehog) is bound to and represses
Smoothened
• When activated smoothened acts to release Ci
from microtubules and prevents its cleavage and
phosphorylation by Slimb and PKA
• Intact Ci can then activate genes that its cleaved
version was just repressing
 Wnt Family
• A group of at least 15 Cysteine-rich Glycoproteins paracrine factors
• Bind to transmembrane receptors in the Frizzled family

• Several pathway variations

– Can activate transcription factors (β-catenin)
– Interact with cell cycle and Tumor Suppressor genes (APC-Adenomatous
polyposis coli)
– Affect Cytoskeletal remodeling enzymes
– Interact with Glycogen metabolism system (GSK3–Glycogen synthase kinase 3)
– Can increase intracellular Ca++ concentrations activating Ca++ responsive
pathways
Wnt Family
 TGF-β Superfamily
• TGF = Transforming Growth Factors

• Ligands for homodimers or heterodimers that are secreted

from producing cells

• These ligand dimers bind to paired dimerized receptors on

receiving cell’s surface.
– Cross-phosphorylation of receptors allows interaction with additional
intracellular components
Smad Pathway / TGF-β Ligands
 Juxtacrine Signaling
• Surface proteins on neighboring cells contact directly without any diffusion of signaling molecules.

• Notch Pathway:
– Notch is a transmembrane protein that can bind several other transmembrane proteins on other cells
(Delta, Jagged, Serrate)
– When bound, Notch undergoes a conformational change that allows a cellular protease to cleave of a
portion of the intracellular domain
– The cleaved-off portion acts as a transcription factor, entering the nucleus, recruiting histone
acetyltransferases (p300) and activating transcription.

– Involved in many processes:

• Many vertebrate organs: Kidneys, pancreas, heart, nervous system
• Help optic cells decide fates and maintain borders between cell types
• Patterning of the nematode vulva
Notch Pathway
END Chapter 4 Notes