Bio 127 - Section I Introduction to Developmental Biology Cell-Cell Communication in Development Gilbert 9e – Chapter 3

It has to be EXTREMELY well coordinated for the single-celled fertilized ovum to develop into the complex adult This coordination requires a systematic way for the cells to know what’s happening around them so that they can change their gene expression correctly They must also then change the signals they are sending out to let surrounding cells know what changes they are making

Developmental Activities Coordinated in this Way 1. The formation of tissues from a mix of individual cells 2. The formation of organs from a mix of tissue types 3. The formation of cells, tissues and organs in specific locations 4. The growth and death of cells, tissues and organs 5. The achievement of polarity in cells, tissue and organs

Membrane molecules sense: The plasma membranes of cells are designed to sense what is happening in their environment Membrane molecules sense: other cell membranes soluble signals sent by other cells the type of extracellular matrix that surrounds them A few signals can get past the plasma membrane

Most cells in the embryo have molecules on their surface that identify who they are DevBio9e-Fig-03-01-0.jpg These molecules also instruct them who they should be in contact with

Sorting out and reconstruction of spatial relationships in aggregates of embryonic amphibian cells All cell types can do it DevBio9e-Fig-03-02-0.jpg

Aggregates formed by mixing 7-day chick embryo neural retina cells with pigmented retina cells DevBio9e-Fig-03-03-0.jpg ....just to show that it’s more than an artist’s rendition....

Figure 3.4 Hierarchy of cell sorting in order of decreasing surface tensions The more adhesive the cell’s plasma membrane is, the more it migrates to the middle of a cell mixture. DevBio9e-Fig-03-04-0.jpg

The molecular biology of cell adhesion: Cadherins The calcium-dependent adhesion molecules (or cadherins) are the main source of adhesive activity on the cell surface DevBio9e-Fig-03-05-1R.jpg The more you express, the more central you become in a mixture

Importance of amount of cadherin for morphogenesis DevBio9e-Fig-03-06-0.jpg --- A nearly perfect linear relationship

Importance of type of cadherin for morphogenesis Early embryo cells all express E-cadherin DevBio9e-Fig-03-07-0.jpg Presumptive neural tube cells lose E-cadherin and gain N-cadherin. N-cadherin expression does something very similar in limb cartilage.

Cadherins can activate migration through actin DevBio9e-Fig-03-05-1R.jpg Cadherin binding outside of the cell can cause actin-based migration in some cells

Disruption of N-Cadherin in Frog Embryos failed migration failed actin assembly DevBio9e-Fig-03-08-0.jpg blocked normal

The cadherins activate migration through Rho GTPase Migratory cells have Rho in their cadherin-actin apparatus – cadherin activates Rho, Rho activates actin-myosin migration.

Drosophila gastrulation DevBio9e-Fig-03-09-0.jpg The cells that have Rho activated migrate to become the mesoderm.

Migration is started by expression of Twist and Snail which causes Rho and B-catenin to translocate in cells DevBio9e-Fig-03-10-0.jpg Rho build-up on E-cad causes actin polymerization and migration

Tracheal Development in Drosophila Rho can also be linked to cell surface receptors and cause chemotactic migration. The cadherin attachments remain strong and the cells migrate as a cohesive unit. DevBio9e-Fig-03-11-0.jpg

Figure 3.12 Cell migration Mesenchymal Cell Migration is also Rho-Dependent DevBio9e-Fig-03-12-0.jpg - not always cadherin-dependent however!

One-Way, Two-Way and Reciprocal Communications Strategies Ligands Receptors 2nd Messengers Target Mechanisms

Cell Signaling Terminology Paracrine Endocrine Synaptic Induction Inducer Responder Signal Competence Signal Transduction Permissive Signals Instructive Signals

Ectodermal competence and the ability to respond to the optic vesicle inducer in Xenopus DevBio9e-Fig-03-13-0.jpg

HOW? Optic vesicle secretes...... Head ectoderm expresses...... BMP 4 Fgf 8 Head ectoderm expresses...... Sox 2 L-Maf Pax 6 Lens genes turned on...... crystallin others

Induced Differentiation

Induction Cascades We know that tissues tend to aggregate through cell contact It’s common for tissues to play off each other to produce an organ Anything from two tissues signaling back and forth to many tissues coordinating each other’s actions The common theme is that a change in gene expression internally (TF’s, functional proteins) is often accompanied by a change in secreted proteins (paracrine, endocrine factors)

Eye formation is a classically studied cascade of induction DevBio9e-Fig-03-13-0.jpg Simple lens induction...

Reciprocal induction DevBio9e-Fig-03-15-1R.jpg

DevBio9e-Fig-03-15-2R.jpg

DevBio9e-Fig-03-15-3R.jpg

The reciprocal interaction between an epithelium and a closely associated mesenchyme is a very common means of organ development (organogenesis)

These are backwards DevBio9e-Table-03-01-0.jpg

Different mesenchyme induces different epithelial structures DevBio9e-Fig-03-17-0.jpg

Also, different epithelium can only become what they are competent to become DevBio9e-Fig-03-18-0.jpg

A little about some of the actual molecules..... Growth Factors carry most of the signals Hormones and neurotransmitters later on 4 big families: FGF, Hedgehog, Wnt, TGF-b Receptors and signaling cascades premade for them make you competent

Fibroblast growth factor (FGF) family are classic growth factors: FGF 1-8 lots of others: VEGF, HGF, PDGF, etc. can change transcription of genes 2 ways RTK Pathway: receptor tyrosine kinase JAK-STAT: JAK activates STAT TF’s

Optic vesicle secretes...... BMP 4 Fgf 8 Head ectoderm expresses...... Sox 2 L-Maf Pax 6 Lens genes turned on...... crystallin others

Figure 3.20 Fgf8 in the developing chick (Part 1) DevBio9e-Fig-03-20-1R.jpg

Figure 3.20 Fgf8 in the developing chick (Part 2) FGF8 in optic vesicle L-Maf expression in ectoderm DevBio9e-Fig-03-20-2R.jpg

Figure 3.24 A mutation in the gene for FgfR3 causes the premature constitutive activation of the STAT pathway and the production of phosphorylated Stat1 protein DevBio9e-Fig-03-24-0.jpg

Hedgehog family includes sonic (shh), desert (dhh) and indian (ihh) in vertebrates Change transcription through an interesting series of inhibitory activities the patched receptor inhibits the smoothened protein until hedgehog binds smoothened then moves to inhibit proteins that inhibit the Gli activator protein

Figure 3.26 (A) Sonic hedgehog expression is shown by in situ hybridization in the nervous system, gut, and limb bud of a chick embryo. (B) Head of a cyclopic lamb DevBio9e-Fig-03-26-0.jpg Both shh and patched proteins require cholesterol. Blocking its production can cause cyclopism.

Wnt family has 15 members in vertebrates Glycoproteins with lipid tails! Work through frizzled receptors and disheveled activators (fly guys!) Also activate by inhibition of an inhibitor

Interestingly, Wnt can do much of what cadherins can do Send catenins to the nucleus Activate rho and change the cytoskeleton This is called “crosstalk” and it is very important in cell signaling

Remember the structure of the cadherin system Implantation of the mammalian embryo in adhere to the uterine wall E- and P-cadherin Integrin and uterine ECM Proteins that bind sugars on uterine wall Rho proteins associate with catenins and actin system. They can change actin’s structure.

The receptors are also a large family of proteins The TGF-b superfamily is a very large family of very active peptide growth factors involved in the development of most tissues The receptors are also a large family of proteins They are serine-threonine kinases, not RTK They work through the activation of SMAD transcription factors

Figure 3.29 Relationships among members of the TGF-β superfamily We’ll hear a lot of these names again this semester! DevBio9e-Fig-03-29-0.jpg

Remember: The Big 4 are just part of the story We’ll talk about others as they come into play

The Delta-Notch family: “Juxtacrine” signals Transmembrane proteins on cells in contact Delta, Jagged or Serrate bind to Notch family Signals go both ways The Notch signal is interesting in that it’s internal domain is cleaved and enters nucleus This activates a dormant transcription factor

Figure 3.33 Mechanism of Notch activity DevBio9e-Fig-03-33-0.jpg

Apoptosis: genetically programmed cell death Absolutely essential to control cell numbers, cell quality and to create space The space between our fingers 2/3 of all neurons we make The middle ear The cerebral ventricles Frog tails Male mammary epithelium

Figure 3.32 Disruption of normal brain development by blocking apoptosis DevBio9e-Fig-03-32-0.jpg

Apoptosis Often cells are set to apoptose by default They require a signal to keep them alive The signal can be soluble or can be attachment, such as cadherins, integrins These are guarantees that cells remain where they should be in the body

Figure 3.31 Apoptosis pathways in nematodes and mammals Early work done mostly in worms Mammalian homologs A signal that turns on CED-9 (Bcl2) saves the cell from death DevBio9e-Fig-03-31-0.jpg

Other Related Strategies of Developmental Biology Maintaining the differentiated state B. The extracellular matrix as a source of developmental signals C. Epithelial-mesenchymal transition

Maintaining the Differentiated State Just changing gene expression is not enough Maintaining the new expression pattern is essential for differentiation So far, four ways to do this have been described

Four ways of maintaining differentiation after the initial signal has been given (Part 1) TF Positive feedback loop Trithorax opens promoter DevBio9e-Fig-03-36-1R.jpg

Four ways of maintaining differentiation after the initial signal has been given (Part 2) Autocrine loop Paracrine loop DevBio9e-Fig-03-36-2R.jpg

The Role of the Extracellular Matrix (ECM) As development proceeds, all cells secrete sugars and proteins to create solid substrate between the cells Nearly all cells require adhesion to survive Cell migration is also dependent on ECM

Figure 3.37 Extracellular matrices in the developing embryo A fibronectin tract allows mesoderm migration during gastrulation DevBio9e-Fig-03-37-0.jpg The epithelial cells secrete fibronectin into basal lamina and then can use it migrate upon.

Figure 3.38 Simplified diagram of the fibronectin receptor complex Integrins bind the ECM to the cytoskeleton DevBio9e-Fig-03-38-0.jpg

Figure 3.40 Basement membrane-directed gene expression in mammary gland tissue Plated on plastic (A) Plated on basal lamina (B,C,D) DevBio9e-Fig-03-40-0.jpg

Epithelial to Mesenchymal Transition A key type of differentiation in many tissue forming activities in embryos and adults formation of mesoderm from epiblast formation of neural crest cells from neural tube formation of coronary arteries from epicardium formation of vertebrae from somites wound healing in skin and vasculature metastasis of epithelial cancers

Figure 3.41 Epithelial-mesenchymal transition, or EMT (Part 1) If not accompanied by differentiation, loss of connections would lead to death DevBio9e-Fig-03-41-1R.jpg

Figure 3.41 Epithelial-mesenchymal transition, or EMT (Part 2) DevBio9e-Fig-03-41-2R.jpg