Molecular & Developmental Biology (BIOL3530)

With Dr. Brian E. Staveley
Department of Biology
Memorial University of Newfoundland

Morphogenesis: Change in Form in the Early Embryo

Morphogenesis
The rearrangement of cell layers bring about changes to the form of the developing embryo, primarily during gastrulation.
(Plants undergo cell division and cell expansion only.)
Rearrangement of cell layers 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) determine cell motility.
These processes are controlled by spatio-temporal gene expression.

Sorting out of dissociated cells demonstrates differences in cell adhesiveness in different tissues
Mixtures of early endoderm and early ectoderm of an amphibian blastula will separate.
Mixtures of presumptive epidermis and presumptive neural plate cells will sort so that epidermal cells move to the outside and neural cells move to the interior.

Cadherins can provide adhesive specificity
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).
Mixtures of cells transfected with different cadherins will sort into masses of same cadherin-type cells.
Cadherin molecules bind through their extracellular domains but associate with the cytoskeleton through their intercellular domains.

Cleavage and formation of the blastula
The first step in embryonic development is cleavage in short cell cycles.
Questions:
How are the positions of the cleavage planes determined?
How does cleavage lead to a hollow blastula with a clear inside-outside polarity?
 

Blastulation

The asters of the mitotic apparatus determine the plane of cleavage at cell division
The plane of cleavage directs the distribution of the cells contents.
The asters (at the cells poles) are microtubules (MT) which radiate from a centrosome, the organizing centre of MT growth).
Before mitosis, the centrosome duplicates, each one moves to opposite sides of the nucleus and form asters.
Usually the new plane of division is set up at right angles to the last.

Cells become polarized in early mouse and sea urchin blastulas
If planes of division are at right angles to the surface, then the cells remain as a single layer and the inside volume increase at every division (i.e. sea urchin).
Both in sea urchin blastula and mammalian morula, the cells become radially polarized (apical and basal faces differ.)
The sea urchin blastula, a hollow sphere (1 cell thick) has the outer surface covered in microvilli which develops the hyaline layer of extracellular matrix (ECM).
 Specialized junctions (including desmosomes) form between adjacent cells.
Golgi body orients towards apical surface.
Basal lamina (another ECM) is made.
Volume of blastocoel increases.

As mouse embryos undergo compaction, cell-cell contact is increased and microvilli are restricted to apical surface to result in polarization.
Some outer cell layer cleavages produce 1 polarized and 1 non-polarized cell.
The former become trophectoderm and the non-polarized cell contribute to the inner cell mass to become the embryo.
Changes in E-cadherin probably drive compaction.

Ion transport is involved in fluid accumulation in the blastocoel
Fluid pressure inside the blastocoel is a major force in maintaining a spherical blastula (hydrostatic pressure).
Tight junctions, which appear at the 8 cell stage, act to seal the epithelial cell layer by the 32 cell stage.
Sodium pumps become active on the blastocoel side on the cells (inward flux).
Water then flows in by osmosis
The epiblast is initially a solid mass of cells but apoptosis (programmed cell death) removes the internal cells to produce a fluid-filled cavity surrounded by an epithelial sheet.
 

Gastrulation

Gastrulation in the sea urchin:  cell migration and invagination
The most vegetal cells undergo an epithelial to mesenchymal transition.
This transition is associated with both changes in cell shape and cell movement.
The primary mesenchyme detach from each other and the surrounding hyaline layer and migrate as single cells into the blastocoel.
Next the endoderm begin to invaginate as a sheet of cells to form the archenteron which is probably started by apical cytoskeleton contraction.
The gut forms first as a short cylinder extended halfway across the blastocoel.
Convergent extension of the tip of the gut followed by extension of filopodia from the cylinder to contact the blastocoel wall follows.
With contact, the filopodia pull the elongating gut to eventually fuse with the mouth region.

Mesoderm invagination in Drosophila is due to changes in cell shape, controlled by genes that pattern the D/V axis
Gastrulation begins in flies when a strip of cells form the ventral furrow then 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.

Xenopus gastrulation involves several different types of tissue movement
The large yolk drives a much more complex series of tissue movements than seen in the sea urchin such as
1) involution, the migration (the roll in) of endoderm and mesoderm at the blastopore;
2) convergent extension, mesoderm, endoderm (on the inside) and future neural tissue (on the outside) movements and
3) epiboly the spreading of the ectoderm to cover the embryo.
Note that presumptive mesoderm (marked by expression of Brachyury) undergoes convergent extension to form the notochord.
Intercalation of adjacent layers of cells drive both convergent extension and epiboly.
Notochord elongation is caused by cell intercalation.
 

Neural tube formation

The neural tube forms from neural plate, a region of thickened ectoderm induced by the mesoderm.
In amphibians, the neural folds arise via convergent extension of the edges of the neural plate which fuse to form the neural tube then separate from the adjacent ectoderm (which becomes epidermis).
The neural groove is between the neural folds.
In birds and mammals, only the posterior neural tube forms as a solid rod which later develops a lumen.
In fish, the entire neural tube first develops as a solid rod.

Neural tube formation is driven by both internal and external forces
During Xenopus neuralation, cells at the edge of the neural plate undergo apical constriction (become wedge-shaped) which draws the edges into folds.
Possibly, the cells crawl down the inner surface of the adjacent ectoderm.
In birds and mammals, neuralation starts at the anterior end (mid-brain region) and proceeds in both directions.
In the middle of the chick neural furrow and later on the furrow sides (the hinge points), the cells become wedge-shaped through the action of cytoskeleton.
Isolated newt neural plate rolls in the wrong direction therefore forces outside of the neural plate are required for correct folding.

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.
 

Cell migration

In the sea urchin, after the primary mesenchyme enters the blastocoel, they form a ring in the vegetal region around the gut.
Later, two extensions of mesenchymal cells are formed towards the oral region.
The primary mesenchyme later secrete the sea urchins endoskeleton.
The directed migration of sea urchin primary mesenchyme cells is determined by the contacts of their filapodia to the blastocoel wall.
The primary mesenchyme cells move over the inner surface by extending up to six fine filopodia (40 micrometres long).
The filopodia make contact with the blastocoel wall, adhere and the cell moves toward the point of contact.  Since several contacts can be made, the point of strongest contact wins out and movement of the primary mesenchyme is a contest to find the most stable attachment.
The inner surface is covered with basal lamina which probably governs the strength of filopodia attachments.

Neural crest migration is controlled by environmental cues and adhesive differences
The neural crest cells of vertebrates originate at the edges of the neural folds, undergo an epithelial to mesenchymal transition as the tube closes and migrate away from the mid-line.
Epithelial to mesenchymal transition is controlled by the gene slug (similar to the snail gene of Drosophila) which is expressed in all migratory NC cells.  slug inhibition inhibits migration.
Neural crest cells migrate from the neural tube to give rise to cartilage (in the head), dermis pigment cells, medullary cells of the adrenal gland, glial Schwann cells and peripheral and autonomic system neurons.
In the trunk of the chick embryo, most neural crest cells migrate either ...
1) dorso-laterally under the ectoderm & over the somites to give rise to the pigment cells of the skin and feathers or
2) ventrally to give rise to the sympathetic and sensory ganglia.
In addition, a minority of the neural crest cells move through the somites to form:
1) dorsal root ganglia and others produce
2) sympathetic ganglia and
3) adrenal medulla.
Differences in cell surface molecules must dictate the various cell migration behaviours.

Slime mold aggregation involves chemotaxis and signal propagation
The myxamebae of Dictyostelium exhaust their supply of bacteria, they enter the multicellular stage of the slime mold life cycle.
Aggregation (the 1st stage) is the streaming of cells to a focus of aggregation which is followed by the cells adhering to each other at their posterior and anterior ends.
The cells move in pulses of inward movement driven by cAMP as a chemoattractant.
The cells extend Pseudopods towards the source of cAMP.
The ameba receives a pulse of cAMP which binds to membrane receptors which induces the ameba to move toward the signal and to produce a pulse of cAMP as well.
This chain reaction produces a wave of cAMP.
The cells are momentarily insensitive to the signal which generates a unidirectional (outward) pulse.
Phosphodiesterase (which cleaves cAMP) prevents an over accumulation of the cAMP signal.
 

Directed dilation

Hydrostatic pressure can provide the force to drive morphogenesis (i.e. in the mouse and amphibian blastula).
Directed dilation can result from an increase in hydrostatic pressure inside of a structure that has relatively stable constraints governing its circumference but weak constraints on its length.
Directed dilation causes an asymmetric shape change.
The rod of cells inside a sheath lengthens, as seen in the Xenopus notochord.
The Xenopus notochord becomes surrounded with extracellular material, to restrict its circumference.
The notochord cells develop fluid-filled vacuoles and expand.
This leads to hydrostatic pressure which results in the directed dilation and expansion of the notochord along the A/P axis.
If the sheath is removed (digested) the notochord buckles and fold and the cells become round instead of flat.

Circumferential contraction of hypodermal cell elongates the nematode embryo
The nematode embryo begins to elongate along A/P axis five hours after fertilization.
Within two hours the embryo decreases in circumference but increases four-fold in length.
Hypodermal (epidermal) cells change from being long circumferentially to be A/P elongated.
The hypodermal cells remain attached by desmosomes linked to circumferentially running actin filaments which contract to cause the cell elongation.

The direction of cell enlargement can determine the form of a plant leaf
Cell enlargement as a result of turgor pressure drives plant morphogenesis with as much as 50 fold increase in tissue volume.
New cell wall material must be synthesized and deposited.
The direction of cell growth is at a right angle to the orientation of cellulose fibrils in the cell wall which is determined by how the microtubules of the cytoskeleton position the enzymatic machinery that lay down the fibrils.
Leaf development depends upon cell division and cell elongation.
Mutations in Arabidopsis that affect cell elongation can produce thin or fat leaves.

email me at bestave@mun.ca