Morphogenesis

Morphogenesis is the process that leads to the appearance of form in specific patterns through the differential reproduction, growth, and movement of cells and tissues and interactions between tissues. Such movements are controlled by a variety of factors, including cell adhesion molecules (CAMs), the nature of the extracellular matrix (ECM), and the size of the cells themselves.

Most multicellular organisms begin their lives as fertilized eggs. Although this seemingly simple cell is highly organized, it is still relatively uncomplicated compared to the structural complexity of the parent. As each egg cell divides, the daughter cells become structurally and functionally distinct. This is the beginning of the process of cell differentiation, which can lead to complex multicellular organisms. As new cells appear, grow, mature, and divide, there is a point at which the eventual fate of these cells is determined. That is, at some point, the eventual location, structure, and function of the descendants of such cells are fixed. This fate determination frequently takes place very early in the development of an organism. After a particular fate is determined, it is the process of morphogenesis that allows the potential fate to be realized. Also, through the process of morphogenesis, patterns of form develop that increase the structural and functional complexity of an organism.

The Rules of Morphogenesis

Fate determination, morphogenesis, and pattern formation are not haphazard. In fact, these events are highly constrained, extremely stereotyped, and very predictable—so much so that models of morphogenesis have been formulated that rely on rules of development to establish the probabilities of particular forms developing. These rules consider two types of conditions—initial and boundary conditions—that work together to produce pattern formation in a cascading process. Initial conditions are simply the cellular conditions that prevail in a certain part of an organism, such as cell size and number and the chemical makeup of cell membranes. Boundary conditions are the conditions that exist at the boundaries of different types of tissue.

Only a few phenomena are responsible for setting the initial and boundary conditions within which a morphogenetic system operates. A variety of cellular phenomena are involved, including cell migration, cell division (producing new cell lineages), the rate of cell division, cell number, cell density, the orientation of daughter cells relative to parent cells, and even the places in which cell death occurs. Tissue-level phenomena also have an important impact on morphogenesis. In these cases, specific tissues possess certain properties and a limited ability to respond differently within the constraints allowed by those properties. Fluid mechanics, particularly hydrostatic pressure, has shown potential as a morphogenetic force that acts on biological tissues by generating internal pressure. For example, sheets of epithelial cells can become folded, but they seldom form a solid mass of cells. Finally, there are interactions that take place between different tissues. Among these interactions is induction, wherein one tissue will induce a specific response in an adjacent tissue. Examples of such induction include the mesenchymal induction of dental epithelium during the formation of teeth.

Limb Morphogenesis

Although far from completely understood, one of the most thoroughly studied morphogenetic systems is the development of the tetrapod limb. (A tetrapod is a vertebrate with two pairs of limbs.) Because amphibians (such as frogs and salamanders) and chickens have large, easily obtainable eggs, they have been studied most. It will be useful to summarize the course of limb development since it exemplifies many of the common features of morphogenesis and pattern formation.

Early in the formation of the limbs, a ridge forms along the flank of the embryo. Along this ridge, the apical ectodermal ridge (AER) develops as a thickened layer of epithelial cells. Undifferentiated connective tissue cells, called mesenchyme cells, accumulate underneath the AER and form the limb bud. These mesenchymal cells are derived from mesoderm that migrates into the limb bud following pathways of the ECM that are laced with CAMs, especially fibronectin. The AER is responsible for the formation of pattern in the proximodistal axis, which is a line from the base of a limb (the proximal end) to the outer end of the limb (the distal end).

Another focus of pattern formation also appears quite early in limb development; it is called the zone of polarizing activity (ZPA) and has influence over the anteroposterior axis of limb development. Together, the AER and the ZPA regulate a pattern of form that is shared by all tetrapods.

Under the influence of these pattern-generating centers, three types of mesenchymal chondrogenic foci will form de novo condensations (which are unconnected), bifurcations, and segmentations. Segmentations and bifurcations both show recognizable connections, and they appear in a consistent and stereotyped fashion. A linear series of mesenchymal condensations showing connections with one another are called segmentations; a Y-shaped condensation with a single proximal focus connected to two distal foci is called a bifurcation.

In all tetrapods, a bifurcation leads to the formation of two skeletal elements in the region between the proximal bone in the arm (or leg) and the hand (or foot). In frogs and amniotes, the condensations at the base of the putative fourth digit, showing connection to the bone in the forearm (or lower leg), are the first to appear as the result of a bifurcation event. Each subsequent proximal digit condensation appears as a bifurcation. All distal condensations, after the proximal digital element forms, appear as the result of segmentation events. Thus, the development of limb elements is asymmetric and always emanates from the axis of the first digit to form chondrogenic foci.

The morphogenetic control of this pattern, which has been conserved throughout the long evolutionary history of tetrapods, is both simple and complex. It is simple in that only a few morphogenetic processes are responsible for generating the complex pattern of limb structure, so different in all the various tetrapods. It is complex in that so many phenomena can influence the ultimate structure of any particular limb. For example, the number of somites that contribute mesoderm to the limb bud mesenchyme can dictate the number of limb elements that will eventually form.

Studying Morphogenesis

There are two general methods for studying morphogenesis. In the older—but still widely used—approach, tissues are removed from an organism in order to be examined under a microscope. Tissues are often stained (a variety of staining materials may be used) to make them more readily visible, and they are usually cut into thin slices called sections. The purpose is to determine the position of individual tissues. If carefully staged materials have been used, it is possible to get some ideas about whether cells have moved and, if so, approximately how far they have moved. There are problems with studying morphogenesis in this way: Details can be missed, or, more worrisome, preconceived notions can bias observations. For example, a small set of sections may support a theory, but because thin sections of tissue can vary greatly, many sections of the material under study may contravene a pet theory of development. One could simply reject the many conflicting sections, claiming them to be poorly prepared or somehow damaged material. Indeed, many sections are rejected. With the advent of transmission electron microscopy (TEM) and Scanning electron microscopy (SEM), more detailed observations could be made. New problems, however, accompany these methods. In TEM, the thinner sections that must be used increase the occurrence of variation, also increasing the chances of biased observations. SEM allows for the magnification of surfaces and a great increase in the depth of field, but it requires that materials be very carefully staged. In none of these methods is it possible to see individual cells move. Another method of studying the developmental processes involved in morphogenesis is using CRISPR/Cas9-mediated gene technology. This allows scientists to better examine the cellular changes in an expanded range of animals, like reptiles. However, this technology garnered some criticism because of its ability to alter embryo genomes and germline cells.

A second approach seeks to document cellular movements. Understanding such movements requires that individual cells be “marked.” Marked cells are then grafted onto an unmarked host organism and followed. Both natural and artificial markers can be used. Pigment granules such as melanin and stained glycogen are examples of natural markers. These markers, however, can be affected by cellular activities, making it hard to follow the cells, but there are other natural markers not so affected. For example, chick and quail cells stain differently, so the cells of one can be followed when they are transplanted into the other. One of the more frequently used artificial markers is tritiated thymidine. When it is introduced, this radioactive substance is permanently incorporated into the deoxyribonucleic acid (DNA) of the selected cells. Tritiated thymidine has several advantages: It works in any cell, it follows along with the cells, and it will be in all the offspring of the cell to which it was originally introduced. As cells continue to divide, the concentrations of tritiated thymidine eventually approach undetectable levels.

Many systems have been studied using these methods, including the development of the tetrapod limb and vertebrate head, somite differentiation, and the appearance of the primary germ layers that eventually yield all other tissues.

Morphogenesis is the reflection of all the interactions that take place during the formation of a living organism and of the patterns of structure that characterize this organism. The study of morphogenesis is motivated by the desire to understand the appearance of patterns of structure. Such patterns are often conserved through evolutionary history. It is hoped that a clear picture of how these patterns are formed and regulated will show how the origin of novel morphologies is constrained. Thus, it will be possible to understand both the origin of novel forms and the maintenance of unchanging forms.

Principal Terms

Bifurcation: The division of a Y-shaped and connected mesenchymal structure into a single proximal chondrogenic focus and two distal chondrogenic foci; this can lead to the formation of separate chondrification centers in a developing digit

Chondrification: The process by which undifferentiated connective cells transform into chondrocytes (cells that make cartilage) and begin forming extracellular matrix

Epithelium: The tissue that covers and lines all exposed surfaces of an organism, including internal body cavities such as the viscera and blood vessels

Limb Bud: Thickened epithelial cells along the lateral body fold that are underlain by mesoderm, creating a paddle-shaped extension from the trunk

Segmentation: The division of a structure into linearly arranged segments; it can lead to the formation of somites, or it can lead to the formation of separate chondrification centers in a developing digit

Zone of Polarizing Activity (ZPA): A region at the posterior base of the limb bud that seems to influence the distal development of pattern in a developing limb

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