Drew M. Noden Ph.D ~ Professor of Anatomy and Embryology
New York State College of Veterinary Medicine ~ Cornell University ~ Ithaca ~ New York

Dr. Noden is a professor of anatomy and embryology at the New York State College of Veterinary Medicine, where he has been on the faculty since 1979. He received his Ph.D from Washington University. His area of research is craniofacial development, and his primary interest is the control of cell movement and tissue assembly during the early stages of head and face formation.

DEFINITIONS

Morphogenesis ~ the formation and differentiation of tissues and organs
Polydactyly ~ an extremity having more than the normal number of digits
Syndactyly ~ union of two or more digits
Gastrulation ~ the process in which the embryo forms into a hollow, pouch-like structure
Neurulation ~ nervous tissue commences differentiation and the basic pattern of the vertebrate begins to emerge
Teratogen ~ an agent relating to, or causing, developmental malformations

Most of us consider the birth of a litter of puppies or kittens as demarcating the beginning of their lives and we anticipate the events to come, including the gradual, often comical, development of locomotory coordination. Birth, however, is but one event in a continuum that includes embryonic and fetal development. Fertilization marks the formation of a genetically unique animal with nearly equal genetic contributions from each parent. The events that take place between fertilization and birth are mysterious to many people and are usually only of concern when one or more animals in a litter exhibit undesirable features such as congenital malformations. How does the fertilized egg, which measures approximately 1/250 inch in diameter, become transformed in just two months to the neonatal kitten or puppy, and what kinds of disruptive processes lead to birth defects? These questions will be addressed in this article.

A MATTER OF SURVIVAL

The first problem the developing organism must face is survival. Unlike t he eggs of most other vertebrates, the mammalian egg does not contain significant quantities of yolk upon which to draw for nutrition. When released from the ovary, the unfertilized egg is surrounded by follicular cells that supplied its nutritional requirements while in the ovary, but these cells are sloughed in a couple of days following fertilization .

The fertilized egg divides slowly for the first few days, then very rapidly. During the first 12 to 14 days, the embryos move freely between the left and right uterine horns. Throughout this period, the embryos are dependent upon the fluids within the uterine cavity f or their nutrition; this fluid is secreted by specialized cells in the walls of the uterus and is sometimes called uterine milk.

Subsequent survival of the embryo depends upon the formation of a placenta. This organ, which is formed by both embryonic and maternal tissues, regulates the flow of metabolites, minerals, fluid, and dissolved gases (i.e., oxygen and carbon dioxide) between fetal an d maternal circulatory systems. The placenta ensures that the developing embryo is not rejected by the maternal immune system and acts as an endocrine organ to maintain and, at the appropriate time, terminate the pregnancy.

At the time of initial attachment of the embryo to the uterine wall, the developing embryo is a fluid-filled sphere about 1/8 inch in diameter that is called a blastocyst . There is a condensation of several hundred cells that appears as a small mass on one side of the blastocyst; these cells constitute the embryonic disc and subsequently will form the entire body of the embryo. All other cells on the surface of the blastocyst are part of the trophoblast, which will contribute to the formation of the placenta.

Trophoblast cells covering the embryo become specialized and release enzymes that cause the uterine epithelial cells and underlying tissues to degenerate. This process brings the developing embryonic placental tissues in contact with the wall of maternal uterine blood vessels. In the developing human, these blood vessel walls are eroded, but they remain intact in carnivores. Shortly after these contacts are made, blood vessels proliferate throughout the trophoblastic components of the placenta and join with those inside the embryo.

TRANSFORMATION: EMBRYONIC DISC TO EMBRYO TO FETUS

How does the embryonic disc, which is an aggregate of several hundred cells identical in appearance, become transformed into a fetus, a developing organism in which most of the body parts have formed? These changes occur during the embryonic period (Figure 2), which lasts approximately three weeks in dogs and cats (six weeks in humans). At the end of this period, the developing organism is called a fetus. There are four processes that facilitate the embryonic transformations:

• GROWTH: an increase in size as a result of the rapid proliferation of cells and the formation of materials that surround and separate cells (i.e., the matrix in bone and cartilage). Growth is essential for embryonic development although this process is not restricted to this period.
• DIFFERENTIATION: the process whereby cells acquire and then express those features that are characteristic of each different type of tissue in the body; often there are stages in the differentiation process in which cells express traits unique to developing tissues but quite distinct from mature tissues.
• MORPHOGENESIS: changes in the shape or location of cells and tissues. Morphogenetic processes include the migrations of individual cells or clusters from one part of an embryo to another, and the folding of flat sheets of cells to form a tube such as the spinal cord or the intestine, a duct, or an endocrine organ.
• PATTERNING: this provides the context within which the preceding pro cesses operate. Patterning is the establishment of precise locations and times at which changes in growth, pathways of differentiation, and morphogenetic movements will occur.

The essential information for embryonic development (i.e., for regulating and integrating the above four processes) lies within the set of genes inherited from both parents. Genetic information, however, is two – dimensional; much like the text of this article, it contains information that has meaning only when it is read and interpreted. In the developing embryo, the process of reading and interpreting this information (i.e., the process of controlling which genes are active in specific cells at appropriate times and
in particular locations) is effected by interactions between cells and tissues. The examples that follow illustrate how tissue interactions regulate growth and morphogenesis, and indicate how disruptions of this regulation can lead to some of the common birth defects seen in small domesticated animals.

CONTROL OF GROWTH

Throughout an animal’s life the rate of cell proliferation in each tissue is regulated precisely. Some tissues, such as bone marrow, the dermis, and the lining of the intestine contain populations of rapidly dividing cells that continuously replace dying or sloughed cells. In other tissues, especially in the brain and spinal cord, cell proliferation ceases after the fetal or infant period. For example, most neurons in the cerebellum are generated during the first 2 to 3 weeks after birth; this process ceases by 3 months of age (3 years in humans), after which more neurons can never be generated. Some norm ally quiescent tissues, such as the lining of blood vessels, can be reactivated to proliferate rapidly in response to injury, disease, or in the case of blood vessels serving muscles, following exercise.

Interactions between tissues important in the regulation of growth in the embryo can be demonstrated by examining two systems: the eye an d the limb. The embryonic eye (eye vesicle) looks very much like a fluid-filled jug with the lens acting as a stopper. Surrounding the eye are the precursors of muscles and, at later stages, of the skeleton that will form the orbit (socket) in which the eye sits. The fluid within the eye, called vitreous humor, also exerts a growth-enhancing pressure. If the embryonic eye is punctured or develops with an abnormal gap between the lens and the eye, the fluid leaks out and the eye fails to enlarge to its normal size.

Small eyes may occur together with malformations of the brain or face or as isolated defects. Depending upon the stage at which growth is arrested, the eye and orbit may be absent (anophthalmos), very small (microphthalmos) and dysfunctional, or slightly reduced. Some dog breeds (Collie, Doberman Pinscher, Akita) have been bred for proportionately small but functional eyes.

The nature of the interactions controlling embryonic growth has been elucidated by studies on the developing limb. It has been shown that each limb develops sequentially from the proximal area (shoulder, hip) to the distal area (paws); this process begins during the third week of development and within about 10 days all limb parts are formed. The epithelium (future skin) covering the distal margin of each limb bud has a thickened apical ridge, the significance of which has puzzled embryologists for a long time.

In the 1950s, a group of researchers opened fertile chick eggs after about 3 days of incubation (early limb bud stage) and gently cut off the apical ridge, leaving the rest of the limb intact. Immediately after the surgery, all cells that had been underlying this ridge stopped dividing, and growth of the limb ceased. The more proximal tissues (those closest to the body) that already had begun to form when the apical cap was removed continued to develop normally. Depending upon the exact stage of the removal, limbs with only a humerus (upper arm bone), or with a humerus, radius, and ulna (upper and lower arm bones) but no distal structures (wrist, digits) could be obtained. Subsequent research has revealed that this region of the epithelium releases growth-promoting factors that stimulate division of underlying cells.

While these results illustrate the critical role of the apical cap in limb development, they do not indicate what causes the cap to form. When researchers transplanted the apical cap to a different part of the embryo, it failed to promote limb growth in the new site and soon degenerated. In contrast, when non-limb epithelium was grafted in the place of the apical cap, it formed a new cap and limb development proceeded normally. Thus, there is something very special about the limb cells underlying the cap: they are necessary for the formation and maintenance of the limb. Similar reciprocal tissue interactions occur in many embryonic organ systems.

Alterations in the interactions between the apical cap and underlying limb cells can result in limb deformities. For example, if the cap-forming stimulus emanating from the limb cells is too great, the apical cap will be larger than normal and the limb will develop with extra digits (polydactyly). This anomaly is seen sporadically in all animals, but is most common as an autosomal dominant condition in cats. For unknown reasons, forelimbs are affected more frequently than hind limbs.

Conversely, when the apical cap is too small, the animal will be born with a reduction in the number of digits and often fusion of digits (syndactyly). The severity of the defects varies with the time at which the cap becomes abnormal.

Other domestic animals have fewer digits than dogs and cats. The horse has three, but two of those are greatly reduced; ruminants such as cattle, goats, and sheep normally have two digits. It is not known how the genes in these species cause a reduced number of digits. These species also occasionally show polydactyly and limb reduction; for example, mule foot calves have only a single digit on one or more of their limbs.

There are many aspects of growth that are not understood. For example, we do not know how the later growth of the limb skeleton is controlled so that all four limbs are virtually identical in length. When one compares this constancy of length in each animal with the enormous diversity in limb lengths among different breeds of dog, it is evident that growth is regulated precisely but at the same time subject to great genetically – based variation.