Morphogenesis

• Dr. Jonathan Bard, Computational Biology Research Group, Weatherall institute of Molecular Medicine, OxfordUK OX3 9DS, UK

Figure 1: A section through an early mouse kidney shows the mix of collecting ducts and nephron tubules in a mesenchyme matrix that have been generated by the morphogenetic processes of tube branching (collecting duct system) and mesenchyme-to-epithelial transformation (nephrons)

Morphogenesis means the generation of form, and usually in the context of developmental biology where it means the generation of tissue organization and shape in animal and plant embryos (it also covers the generation of internal organization in complex single-cell organisms such as Acetabularia - an area not discussed here). Morphogenesis therefore deals with apparently straightforward problems such as: how epithelial ducts branch in glands (Figure 1), how nerves migrate to and recognize their targets, how mesenchymal cells come together to form pre-muscle and pre-bone condensations, how tendons link to the appropriate bones, and how cells change their shapes.

Morphogenesis also covers more complicated questions which involve several coordinated morphogenetic processes such as: how bones are shaped, and how the early mammalian heart tube reorganizes itself and its associated blood vessels to produce the 4-chambered adult heart. Indeed, it covers anything and everything to do with biological shaping and developmental anatomy. While the questions are straightforward, they have proven difficult to answer.

Morphogenesis is one of the four key interrelated classes of event that characterize all of development:

1. Patterning: The setting up of the positions of future events across space (at a variety of scales)
2. Regulation of timing: The 'clock' mechanisms that regulate when events happen. Clocks can directly regulate morphogenesis of individual tissues such as somites, and changes of relative timing of events (heterochrony) can drive the evolution of new body plans.
3. Cell differentiation: Changes in a cell's set of expressed genes (its molecular phenotype)
4. Morphogenesis: The processes that generate tissue organization and shape and are usually the downstream response to the timing and patterning.

Each of these processes usually involves signaling from one tissue to another, the signals resulting in changes in protein activity and in gene expression that generate events (motors of change) that can be either cell-autonomous or can involve cell cooperation.

"Morphogenesis is important"

• It is responsible for tissue organization and hence for much of an organism's anatomy, physiology and behavior.
• Mutations that affect morphogenesis underpin many human congenital abnormalities.
• Mutations that alter shape alter the fitness of a species under selection pressure and so drive evolutionary change.

"Morphogenesis is difficult to study": Current knowledge about the morphogenesis of complex tissues is limited for three reasons:

1. Many of the key events take place during early development when organ rudiments are small and difficult to study, although genetic manipulation is now allowing morphogenesis to be investigated in organisms such as Drosophila [1]] with very small embryos.
2. Most tissues will not develop much of their form in vitro and so are inaccessible to standard experimental manipulation.
3. The intrinsic complexity of morphogenesis (see below) makes experimentation difficult.

The Basics

Tissue organization arises from cells exhibiting a set of well-defined morphogenetic behaviors (the morphogenetic toolkit - Table 1) that include movement, shape change, differential growth and apoptosis (programmed cell death). Differential growth is particularly important in plant morphogenesis, not considered here, where there is no cell movement (except by pollen tubes) and little apoptosis.

As to scale, morphogenetic events extend from the organization of subcellular structures (such as filopodia) through migration of single cells (e.g. a neural crest cell moving at a rate of about a micron a minute) to the coordinated activity of the thousands of cells that achieves the complex foldings that enable a simple heart tube to reorganize itself over several days (in the mouse) into a four-chambered organ. We know virtually nothing about how such coordination is achieved.

As the development of every tissue in the body involves morphogenesis, it has an enormous literature. This article makes no attempt at being comprehensive or to consider any example of developmental anatomy in detail, but only sets out to provide the basics (details of the molecular basis of morphogenetic mechanisms can be found in Davies 2005). Specific reviews can be found using Pubmed and Google, while textbooks that discuss morphogenesis include: Slack (2005) (a good introduction) and Gilbert (2006) (broad coverage). For a review of the pre-1990 material, see Bard (1990). References to some key examples are given in Table 1.

This article discusses the cellular processes of morphogenesis; the molecular basis of these processes is discussed in the article on the Cellular mechanisms of morphogenesis

The participating cells

Three classes of cell types in early embryos can be distinguished on the basis of geometry:

1D: This class covers single cells, and their most important morphogenetic process is movement. Examples include neural crest cells, primordial germ cells, and somite derivatives, and this area has attracted considerable interest. The direction of cell movement within an embryo is controlled by tracks (see contact guidance and haptotaxis Table 1), signaling gradients (chemotaxis) or boundary interactions. The key problems in analyzing cell migration in vivo are identifying which cells start to move, the signals for initiating movement, the nature of the migration pathways and the mode of stopping. This area of research has been strengthened by our ability to label specific cell populations with transgenic markers (β-galactosidase, green fluorescent protein etc.) to allow us to follow their migrations through development.

2D (actually: sheets of cells): Polarized, monolayer epithelial cells make strong side-to-side adhesions to their neighbors, secrete a basal lamina to which other cells can adhere, and maintain an apical surface to which other cells cannot make adhesions (which is why they remain a monolayer). Epithelia sheets form bounding surfaces (e.g. surface ectoderm and the mesothelial linings of body cavities) and tubes (e.g. gut) which may arborize (e.g. the kidney collecting duct system)(Figure 1)). Endothelia form the tubes of the vascular system, and are anatomically similar to epithelia but use different adhesion and matrix molecules. The most important mophogenetic processes of epithelia and endothelia are folding, movement (e.g. gastrulation and epiboly), controlled growth (e.g. the extension and branching of ducts) and convergent extension (the mechanism that, through changes in cell shape and neighbor relations, allows tubes and sheets to change their form - e.g. Drosophila limb and sea-urchin gut extension).

3D: These are groups of cells (usually mesenchymal) that can adhere directly or indirectly (e.g. via extracellular matrix molecules) with other similar cells over the whole of their surface and so are generally found in 3D associations. Many mesenchymal cells are primitive and will undergo one or more morphogenetic processes (e.g. movement) to set up a basic scaffold of tissue organization before condensing and differentiating into a range of cell types (dermis, cartilage, bone, muscle, tendon etc.). Later morphogenesis builds on this scaffold.

Figure 2: Confocal micrograph of an early (E13) mouse kidney with two mesnchymal aggregates (red) attached to a kidney duct: the upper one has already formed a lumen and made a basement lamina (green) on its way to becoming a nephron. This picture highlights the way in which epithelia form 2D structures within a 3D matrix of mesenchyme.

Epithelial and mesenchymal cells can occasionally turn into one another and the associated 3D <-> 2D transformations force mesenchymal masses to acquire lumens (e.g. blood vessel and nephron formation (Figure 1)) and epithelial cells to lose side-to-side adhesions and so may delaminate and migrate away from their sheets (e.g. neural crest cell migration, somite breakdown). Most functional tissues are of course complex 3D structures composed of both mesenchymal and epithelial cells and their derivatives, together with nervous and vascular tissue. The morphogenetic processes that lead to their final structures are rich and complex, and not well understood.

Experimental approaches

The first major approach to investigating morphogenesis was to look at the intrinsic morphogenetic properties of cells: Townes and Holtfreter (1955 - a classic) showed that randomized aggregates of cells from a mix of amphibian embryonic tissues would not only sort themselves out into their cell types but also generate some structure. The paper demonstrated that the cells themselves had morphogenetic properties that they could use, and stimulated a great deal of work in the '60 '70s and '80s on the morphogenetic abilities of cells.

A second approach was to analyze cell behaviour in tissues that that will develop in culture where they can be experimentally manipulated. As chick and amphibian embryos are relatively large and accessible, they have been the model species of choice for studying morphogenesis (e.g. neural crest and nerve migrations, corneal development, gastrulation and epithelial morphogenesis), although there has also been work on the transparent sea urchin (e.g. its gastrulation) and mouse embryos (particularly the ducted glands: kidney, salivary gland, lung etc.). Neither of these approaches has been of much use in studying small invertebrate embryos.

Figure 3: Some basic modes of morphogenetic movement. (From Slack (2005), permission being sought)

All this experimental work has culminated in the elucidation of a set of properties that cells can use in generating tissue organization (Figure 3) and that can be called The Morphogenetic Toolkit Table 1. This includes properties like cell movement and its constraints, epithelial reorganization and branching and the formation of spaces.

Most current work in the general area of morphogenesis focuses on

• The molecular basis of these tools.
• Which of these tools cells use for making a particular tissue, and how they use them.

Both approaches capitalize on the use of transgenic animals where gene manipulation has led to changes in tissue organization or to the marking of specific cells (e.g. with green fluorescent protein). Such molecular approaches can be used for all the main model organisms – the mouse Mus musculis, the zebrafish Brachidanio rerio, the fruitfly Drosophila melanogaster and the roundworm Caenorhabditis elegans.

The bigger picture

Any full investigation of the morphogenesis of a tissue always starts with a detailed understanding of its developmental anatomy. This is followed by experimentation to discover:

1. "The cellular organization that underpins morphogenesis". This covers the initial geometry and any surfaces or boundaries that will constrain subsequent cell behavior.
2. "The signals that initiate morphogenesis together with the initiating and recipient cells". Much is known about this (see Gilbert). An example of a signal is the growth factor GDNF, which initiates both mouse kidney morphogenesis and the colonization of the mouse gut by the neural crest cells that will form the enteric nervous system.
3. "The cell-based processes that drive tissue formation" This well-defined set (the morphogenetic toolkit Table 1) often involves cells behaving cooperatively, but we know little about how they do this.
4. "The molecular drivers of cell processes" Morphogenesis is a dynamic process driven by a limited number of molecular mechanisms involving the cell surface (e.g. adhesion molecules) and the cytoskeleton. The key drivers are:
   1. Actin contraction within the cytoskeleton This provides the molecular basis of cell movement, epithelial folding etc.
   2. CAM-mediated cell condensations: A first step in the development of bones, muscles, cartilage etc.
   3. Contextual growth The buckling of epithelia in the ciliary body of the chick eye and in the human brain are driven by growth constrained by fixed boundaries.
   4. Apoptosis The digits separate through the apoptotic loss of inter-digit mesenchyme.
   5. The hydration of glycosaminoglycans This can generate cavities (e.g. their swelling is responsible for the anterior and posterior chambers of the eye, as well as synovial cavities in joints and cardiac jelly in the early heart).
   6. Cell differentiation If mesenchymal cells become epithelial, they reorganize from a 3D mass to a 2D sheet (and vice versa; e.g. early nephron formation - figure).
   7. Other occasional forces Blood flow in the early heart is forced into two streams, and their separate pressures on the endocardial tube in the outflow tract distorts this soft tissue and leads to the formation of the spiral septum
5. How morphogenetic processes are terminated Little attention has been paid to this, but two examples illustrate the possibilities

• A key gene is down-regulated. This occurs in the salivary gland where branching morphogenesis is facilitated by a hyaluronidase. Once the enzyme is lost, branching stops.
• The new structure is intrinsically stable. An interesting example is boundary formation mediated by eph-ephrin interactions. Where an eph+ cell contacts an appropriate ephrin+ cell, migratory activity is blocked in both cells and mixing of the cell types is therefore inhibited. Such interactions set up stable boundaries between rhombomeres in the hindbrain (they also control the paths of spinal nerves and keep arteries and veins apart).

The current situation

Morphogenesis was an important area of research in the 70s and early '80s, but activity then declined as the focus of research in development moved to discovering and studying the genes involved in networks that regulate differentiation. Morphogenesis is now back on the agenda for three reasons.

1. The discovery of molecules (e.g. ephs and ephrins) that control tissue organization, so that molecular genetic techniques can be applied to the analysis of morphogenesis.
2. The development of transgenic mouse technology that allows the morphogenetic roles of molecules to be tested.
3. The development of tissue engineering, which involves applying knowledge of morphogenesis to make structures useful to clinical medicine.

The net result has been an enormous amount of work in the first decade of the 21st century that has explained much about the molecular basics of morphogenesis, albeit with less known about how these are integrated at the cellular level. There are thus major areas where our understanding is very limited, and problems that need solving include:

• How neurons organize themselves to make a functioning nervous system
• How epithelia rearrange themselves into the convoluted shapes seen in the heart, the ear and the gut.
• How mesenchyme cells in a mere condensation form muscles and bone, with all the complex shaping that this requires.
• How muscles, tendons, bones and ligament become organized and integrated.

The reader will note that some of these questions go beyond the definition of morphogenesis given earlier. So be it! Development is a difficult subject, its borders are fuzzy and molecular insights change our thinking. These are however exciting times for the subject and our ability to combine traditional and molecular experimental approaches with some clever thinking will revolutionize our approaches to investigating how specific tissues acquire their form. It seems likely that the next decade will bring real information about the details of complex tissue morphogenesis in all of the main model organisms.

References

These are some well-known books that discuss morphogenesis (the Townes article is still worth reading). Detailed research articles are cited in the Table 1 subpage, while reviews can be found via Pubmed.

• Bard, JBL (1990) Morphogenesis: the cellular and molecular processes of developmental anatomy Cambridge University Press.

• Davies JA (2005) Mechanisms of Morphogenesis. Academic Press

• Gilbert SF (2006) Developmental Biology (8th edn.). Sinauer Ass.

• Slack J (2005) Essential Developmental Biology (2nd edn) Blackwell Publishing.

• Townes and Holtfreter (1955) Directed movements and selective adhesion of embryonic amphibian cells. J. exp Zool. 128:53-120.

Internal references

• Valentino Braitenberg (2007) Brain. Scholarpedia, 2(11):2918.

• Jamie Davies (2008) Cellular mechanisms of morphogenesis. Scholarpedia, 3(2):3615.

• Olaf Sporns (2007) Complexity. Scholarpedia, 2(10):1623.

• John B. Furness (2007) Enteric nervous system. Scholarpedia, 2(10):4064.

• Hans Meinhardt (2006) Gierer-Meinhardt model. Scholarpedia, 1(12):1418.

• Hermann Haken (2007) Synergetics. Scholarpedia, 2(1):1400.

See Also

Cellular mechanisms of morphogenesis, Gierer-Meinhardt model, Pattern formation, Self-organization, Synergetics

Sponsored by: Eugene M. Izhikevich, Editor-in-Chief of Scholarpedia, the peer-reviewed open-access encyclopedia

Reviewed by: Anonymous

Accepted on: 2008-05-09 20:06:22 GMT