Five questions in morphogenesis 1.How are tissues formed from populations of cells? 2.How are organs constructed from tissues? 3.How do organs form in particular locations, and how do migrating cells reach their destinations? 4.How do organs and their cells grow, and how is their growth coordinated throughout development? 5.How do organs achieve polarity?

Morphogenesis: Cell adhesion The rearrangement of cell layers bring about changes to the form of developing embryo, primarily during gastrulation. Plants undergo cell division and cell expansion only. Each type of cell has a different set of protein in cell membrane Some of these differences are responsible for forming the structures of the tissues and organs during development Cell layer rearrangement is driven by mechanical forces. Cell adhesiveness (to each other and the extracellular matrix) and cell motility are key to morphogenesis. Cell surface proteins determine the specificity and strength of the adhesiveness while cytosketetal (internal) proteins determine cell motility. These processes are controlled by spatio-temporal gene expression

Re-aggregation of cells from amphibian neurulae Jigannes Holtfreter (1901 – 1992)

Figure 3.2 Sorting out and reconstruction of spatial relationships in aggregates of embryonic amphibian cells

Differential adhesion hypothesis Cell sorting based on themodynamic principles. The cells rearrange themselves into the most thermodynalmcally stable pattern Strength A-A is greater than A-B, B-B, so A-A become central. Strong A-A (pigmented cells) interaction exclude B (retina) and locate inside. No adhesion activity between A and B, so they can for separate aggregates Thermodynamic difference could be caused by different types of adhesion molecules. Aggregates formed by mixing 7-day chick embryo neural retina cells with pigmented retina cells

Hierarchy of cell sorting in order of decreasing surface tensions

Cadherin-mediated cell adhesion Cadherins can provide adhesive specificity. Mixtures of cells transfected with different cadherins will sort into masses of same cadherin-type cells. Epithelial cells (L-cells) that do not adhere strongly nor express cadherins on their cell surface can be transfected with different cadherins (E-, P-, N-cadherin). E-Cad; expressed in all early embryo, in epithelial tissues after dev, P-Cad; on the plancental N-Cad: Neural Cell types, Nerve system R-Cad; retinal Difference in cell surface tension and the tendency of cells to bind together depend on the strength of Cad interaction. Ie: Strong interaction btwn Cad migrate internally. Cadherins bind through their extracellular domains but associate with cytoskeleton through intercellular domains

The importance of cadherins for maintaining cohesion between developing cells can be demonstrated by interfering with their production N-cadherin lacking the extracellular domain injected Anti-sense Oligo Against Cad mRNA Morpholino- N- Canderin injection: The neural plate cells fail to invaginate into embryos and to form neural tube

Figure 3.7 Importance of the types of cadherin for correct morphogenesis Changes in the pattern of expression of cell adhesion molecules accompany neural tube formation. The neural tube separates from the ectoderm after its formation through changes in cell adhesion. Neural plate cells (and the rest of the ectoderm) initially express L-CAM (surface adhesion molecule) but as the neural fold develops, the neural plate cells express N- cadherin and N-CAM but the adjacent ectodermal cells express E-cadherin. These changes (in adhesiveness) allow the neural tube to sink below the epidermis and may explain how the cells sort.

Ventral furrow formation during Drosophila gastrulation internalizes the cells that will become the mesoderm Mesoderm invagination in Drosophila is due to changes in cell shape, controlled by genes that pattern the D/V axis. Gastrulation begins in Drosophila when a strip of cells form the ventral furrow that then becomes the mesodermal tube. The cells individually spread out to form a layer of mesoderm on the inside of the ectoderm. This process requires the expression of twist and snail which indirectly control the expression of cytoskeletal cell components.

Figure 3.10 Getting mesodermal cells inside the embryo during Drosophila gastrulation by regulation of the cytoskeleton

Figure 3.11 Tracheal development in Drosophila

Figure 3.12 Cell Shape change

Figure 3.12 Cell migration Focal Adhesion Polarization Protrusion of the cell leading edges Adhesion of cells to Extracelluar substrates Release of Ad in the rear

Cell Signaling: Induction: Inductive induction: One cells send signals which lead to formation of organ/tissues (another cells) Ex) vertebrate eye development from UNC Chapel Hill embryo images E8 E9 neuroectoderm surface ectoderm Optic groove Optic vesicle Mouse eye development – morphogenesis guided by neighboring cues

Ectodermal competence and the ability to respond to the optic vesicle inducer in Xenopus Not all the interaction results in an induction. - Optic vesicle only can induce lens ectoderm at head, but not abdomen that is not competent.

Figure 3.14 Lens induction in amphibians: Cascade of induction: reciprocal and sequential iduction

Figure 3.14 Lens induction in amphibians (Part 3) Optic cup >>the pigment layer And neural retina Instructive induction: A signal from inducing cells is necessary for initiating new gene expression in the responding cells. Ex) optic vesicle only under a region of head ectoderm.>>> ectoderm forss a lens Permissive induction: the responding tissues has already been specified and needs only an evironment cues which allows expression of traits ex) Fibronectin, Laminin

Figure 3.15 Schematic diagram of induction of the mouse lens

Otx1-KOWT Fossat et al. (2007) Otx2 is an evolutionary conserved head determinant

Induction of optic and nasal structures by Pax6 in the rat embryo – as a potential competence factors

6.6 Feather induction in the chick – regional specification of induction regional specification of induction: “Feather induction” Skin: outer epidermis and dermis >>> Epidermis sends signals which allow dermis to become a condensed dermal mesenchyme Shh expression

6.7 Regional specificity of induction in the chick regional specification: The original source of the mesenchyme Determine the types of cutaneous structure made by the epidermal epithelium.

Identities of inductive signal Paracrine: short-ranged signaling – most secreted morphogens Endocrine: more dependent on the existence of certain types of receptors in responding cells – hormones Juxtacrine: contact-mediated signaling – Notch signaling

Mechanisms of inductive interaction

Paracrine and endocrine factors in development Growth factors (including fibroblast growth factor (Fgf)) Hedgehog (Hh) Wingless/Int1 (Wnt) Bone morphogenic factor (BMP) Hormones (ex. Prolactin, thyroid hormones)

Structure and function of a receptor tyrosine kinase

The widely used RTK signal transduction pathway

Fibroblast growth factor (Fgf) 24 isoforms in vertebrates. Activate Fgf receptors, which contain tyrosine kinase activity. Mediates MAPK pathway to activate transcription factors, which induce gene expression related with differentiation Ex) Fgf8 is important during limb development and lens induction

Fgf8 in the developing chick (Part 1)

Fgf8 in the developing chick (Part 2) L-Maf in situ hybridizationFgf8 in situ hybridization Neural retina of optic vesicle

Figure 3.22 Activation of MITF transcription factor through the binding of stem cell factor by the Kit RTK protein MITF and Kit are present in the migrating melanocyte precursor cells Like Mitf mutant mice, Steel (stem cell factor for Kit) and White (Kit protein) mutant mice are white because of their pigment cells failed to migrate

Figure 3.23 A STAT pathway: the casein gene activation pathway activated by prolactin

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 Its short rib can not support breath

Hedgehog pathway Drosophila segment-polarity gene of which mutation results in unusually short and stubby larva Three isotypes in vertebrate Sonic hedgehog Indian hedeghog Desert hedgehog Hedgehog ( 고슴도치 )

limb bud The sonic hedgehog gene is shown by in situ hybridization to be expressed in the chick nervous system (red arrow), gut (blue arrow), and limb bud (black arrow) of a 3-day chick embryo

Head of a cyclopic lamb born of a ewe who had eaten Veratrum californicum early in pregnancy Cyclopamine Veratrum californicum made the jervine and cyclopamine,which are teratogene, -inhibits cholesterol synthesis, which is needed for HS production. The cerebral hemispheres fused, resulting in the formation a single central eye and no Pituary gland

Figure 3.25 Hedgehog signal transduction pathway

Limb digit formation by Hh signaling

Hh signaling at ciliunm

The Wnt signal transduction pathways (Part 1) – Canonical Wnt pathway Wnts: family of Cystine-rich glycoproteins Fly: wingless, Mammals; Int -inducing the dorsal cells of the somites to becom muscle -Specification of the midbrain -Stem cell proliferation -Establishing the polarity of limb

Wnt proteins play several roles in the development of the urogenital organs Wnt4-CKO WT Wnt4: kidney development and female sex development

The Wnt signal transduction pathways (Part 2) – Planar Cell Polarity (PCP)

The Wnt signal transduction pathways (Part 3) – Calcium Signaling

TGF  /BMP pathway Homodimeric TGF  ligands bind to heterotetrameric receptors, which possess Ser/Thr kinase activity TGF  family: important for the extracellular remodeling and cell division, ductal formation of kidney, lungs, and salivary glands BMP family: Multifunctional – cell division, cell death, cell migration, and differentiation Decapentaplegic (Dpp; Drosophila homolog of vertebrates BMP4) controls axial development Antagonizing factors (chordin, noggin, follistatin) Diffuse through the binding to proteoglycan (Dally) Smad pathway Smad 2&3: mediate TGF  signaling pathway Smad 1&5: mediate BMP signaling pathway Smad 4: co-Smad cooperatively acting as a transcription modulator with effecter Smads

The Smad pathway activated by TGF-  superfamily ligands (Part 1)

The Smad pathway activated by TGF-  superfamily ligands (Part 2)

Figure 3.31 Apoptosis pathways in nematodes and mammals Apoptosis is necessary for the proper spacing and orientation of neurons, generation of middle ear space and space btwn fingers. Nematode: exactly 13 cells die during development -trance cell lineage -mutant isolation with less cell death or more cell death, Ced-9, Ced-4, Ced-3 Mammals; Bcl2 (CED-9): anti-cell death Apaf1(CED04): apoptotic protease activating factor 1 Caspase-9, Caspaee-3 (CED-3): protease

Figure 3.32 Disruption of normal brain development by blocking apoptosis Apaf1-null mice: severe craniofacial abnromalties Brain overgrowth, webbing btw their toes

Cell-to-cell direct interaction Notch : lateral inhibition (neural vs. glial fate determination) Eph receptors : axon guidance (cell migration) ECM-integrin signaling : cell-to-matrix interaction Cadherin signaling : cell-to-cell adhesion Gap-junction : direct connecting two neighboring cells’ cytoplasm Cell-to-cell direct interaction

Mechanism of Notch activity Notch signaling is involved in the formation of various organs(kidney, heart and pancreas) Delta, Jagged or serrate proteins activate Notch of the neighboring cells The binding of Delta to Notch tells the receiving cells not to become neural In eye development, their interaction seem to regulate which cells become optic neruron or glia cells

Figure 3.34 C. elegans vulval precursor cells and their descendants Anchor cell secretes LIN-3(EGF)-TRK activation. LIN-3 determine the P6.p cell and 1’ central cell, Lower LIN-3 cause P5 and P7 Cell to form a lateral Vulval lineage LIN-12 (Notch) in P5 and P7 can be activated by P6 cell

Lateral Inhibition 3o3o 3o3o 1/2 o 3o3o 3o3o Lin-12 (notch) Lag-2 (delta) 1/2 o 2o2o 2o2o 3o3o 3o3o 3o3o 3o3o 3o3o 3o3o 3o3o 3o3o Lin-3 1/2 o 1o1o

Two possible mode of Notch signaling in vertebrate neurogenesis Lateral inhibition Intra-lineage signaling Dong et al. (2012)

Asymmetric inheritance of mindbomb into two daughter cells par-3 MOControl

Cross-talk between Signaling Pathways No cell receives single inductive signal, but numerous signals act simultaneously. Each signaling pathways are not separated, but they cross-talk each other at many steps. Some events requires the cooperative signaling events. But, some signals block the other signaling pathway. How can cells filter the real inductive signal from many co- integrated signals?

6.38 Four ways of maintaining differentiation after the initial signal has been given (Part 1)

6.38 Four ways of maintaining differentiation after the initial signal has been given (Part 2)

6.38 Four ways of maintaining differentiation after the initial signal has been given (Part 3)

6.38 Four ways of maintaining differentiation after the initial signal has been given (Part 4)

Epithelia and mesenchyme Epithelia: sheets or tubes of connected cells, originated from all germ layers Mesenchyme: loosely packed and unconnected cells, originated from mesoderm or neural crest

Figure 3.37 Extracellular matrices in the developing embryo: source of Developmental signals

Figure 3.38 Simplified diagram of the fibronectin receptor complex

Figure 3.39 Role of the extracellular matrix in cell differentiation

Figure 3.40 Basement membrane-directed gene expression in mammary gland tissue

Figure 3.41 Epithelial-mesenchymal transition, or EMT