Molecular Regulation of Development
Overview
Life begins as a single cell; a fertilized egg or zygote. During cleavage, the zygote undergoes rapid, repeated mitotic divisions, producing a critical number of cells that become committed to follow specific developmental paths. The developmental process is guided at the molecular level by:
- genes
- gene products (proteins)
Differentiation is the systematic regulation of gene expression in unique populations of cells to produce the functional cell types that make up the tissues and organs. While all cells of an organism possess the same genome, it is the expression of particular genes and production of specific structural and functional proteins that distinguishes one cell from another after differentiation.
A muscle cell differs from a bone cell. While each possesses the identical genome, each expresses different protein profiles in accordance with their specialized functions. A muscle cell; contractile proteins, and myoglobin & a bone cell; the apparatus to synthesize collagen and enzymes to promote calcium deposition.
Human Genome
At one time, it was theorized that the human genome consists of over 100,000 genes; each gene coding a single protein. This is the "one gene, one protein" concept.
However, upon completion of the Human Genome Project, the human genome was determined to consist of about 23,000 genes. These genes, because of differential splicing, can be cut into multiple different mRNA's, sufficient to code for the large number of proteins that are known to exist.
The animation above illustrates how a single gene can be spliced to code for 3 different RNA's coding for 3 different proteins.
Totipotent vs Pluripotent
During development, cells become increasingly more specialized through the process of differentiation. In early cleavage, all cells of the zygote are totipotent; meaning that any single cell, on its own, is capable of forming a complete new individual. Splitting of the zygote during the two or four cell stage can result in formation of two, three or four new individuals from that single zygote. This is the basis for identical twins. Though identical triplets or quadruplets may also occur, they are exceedingly rare. During development, cells do not remain totipotent for very long.
At the blastocyst stage, the cells become committed to one of two diverse populations:
- outer cell mass (trophoblast) - that will form the supportive tissues like the amnion, chorion, placenta.
- inner cell mass (embryoblast), that will form the cells and tissues of the embryo proper
Differentiation is the final step in development of a tissue. Differentiated cells produce the proteins unique to a functional cell type.
Fig 2.
The illustration above shows that cells are totipotent during early cleavage, but within the blastocyst, they have become committed to one of two separate populations of pluripotent cells, those that form the embryo, embryoblast and those that form the embryonic membranes, trophoblast.
Cell Signaling
Communication between cells is critical to the development of an organism. In most aspects of development, one group of cells influences the fate of a second group of cells through an elaborate system of molecular signaling.
The first group of cells is called the inductor since it produces substances that will "induce" a second population of cells, the competent cells, to respond in a particular way. These interactions usually involve epithelia and mesenchymal cells.
Two major classes of proteins are considered to be integral to the developmental process.
- Transcription Factors (Gene Regulatory Proteins) - intracellular proteins that promote or inhibit expression of specific genes
- Morphogens - soluble substances or signaling molecules, that are secreted into the extracellular space and which influence the development of a specific population of cells.
Transcription Factors
Transcription factors or gene regulatory proteins are molecules that regulate specific genes by turning them on or off as needed during development. They are intracellular signals operating within the cells that produce them.
Some transcription factors are common to all cells while others are present in specific types of cells and at specific stages of development.
HOX genes and PAX genes are two important classes of genes that produce transcription factors guiding early development.
Morphogens
Morphogens are diffusible signaling molecules that direct the development of specific groups of cells in a concentration dependent manner. Morphogens are intercellular signals that act either by:
- juxtacrine signaling - cell to cell contact
- paracrine signaling. - secretion and diffusion
Cells that are competent to respond to the morphogens are generally exposed to different concentrations of the morphogen and respond accordingly.
In the illustration below, cells in an epithelial sheet, in this case the inductor, produce transcription factors that up regulate genes that produce a specific morphogen. The morphogen is secreted into the extracellular space and attaches to receptors on a different group of cells, the competent cells which in this case are mesenchymal cells.
Binding to the receptors on the cell surface initiates intracellular, signal transduction pathways that alter gene expression in the competent cell resulting in differentiation of that cell. The competent cell may be induced to form muscle proteins to become a muscle cell (as in the illustration), or maybe begin forming collagen bundles to become a fibroblast, a connective tissue cell to name just two.
Fig 3.
The illustration above shows transcription factors activate genes in one cell type to produce proteins (morphogens) that it secretes into the extracellular space. These signal molecules bind to receptors on competent cells activating intracellular transduction pathways that alter gene expression in the competent cell to produce new specific proteins and differentiation. In this case to become a myoblast, which fuses with others to make a skeletal muscle fiber.
Transcription Factors & Important Genes in Development
HOX Genes
HOX-genes encode a class of transcription factors that are sometimes referred to as “master switches” because they regulate the expression of large sets of downstream genes. They operate in a time and space sequence, and so guide the timing of the appearance of certain structures and the sequence in which body parts are formed.
In general, development proceeds in a cranial to caudal or proximal to distal direction.
HOX-genes are particularly important in the establishment of the cranio-caudal axis of the embryo through regionalization of the body (head, neck, thorax, abdomen, pelvis) and development of somites, limbs, vertebrae, and craniofacial structures.
Six Week Old Embryo
Fig 4.
HOX genes are important in establishing the regional divisions of the body such as head, neck, thorax, abdomen and pelvis. They also regulate the segmentation of the upper and lower limbs. In the image of a 6 week old embryo note regionalization of the embryo occurs in a cranial to caudal and proximal to distal sequence.
Expression of these genes usually occurs in a cranial to caudal or proximal to distal direction and as such, much of development proceeds with cranial parts of the embryo and proximal parts of the limbs being more advanced than caudal or distal parts.
In addition to controlling pattern formation in the body as a whole, they are also involved in the regionalization of specific organs such as the heart, GI tract, and urogenital system.
Fig 5.
Fig 6.
PAX Genes
In contrast to HOX genes, PAX genes appear to act singly rather than in a temporal or spatial combination. PAX genes are usually expressed in developmental processes involving epithelial to mesenchymal transformation and in the development of sensory organs and CNS.
Gastrulation and neural crest formation are both examples of processes that involve epithelial to mesenchymal transformation.
PAX3, expressed in epithelial cells at the edges of the neural folds, down regulates cadherin production, allowing cells to separate from the neural ectoderm and migrate into the body of the embryo as neural crest cells.
Neural crest cells respond to local concentrations of morphogens to differentiate into dorsal root ganglia, sympathetic ganglia, medulla of the adrenal (suprarenal) gland, melanocytes in the skin and a host of other tissues and organs.
Miscellaneous Genes
SRY-type HMG box (SOX) genes – the SRY gene (Sex-determining Region on the Y) located on the Y chromosomes of males codes for testis determining factor (TDF) that guides the development of the reproductive organs to become "male". In the absence of TDF, the default setting for sexual development is to become female.
T-box (TBX) genes – code for transcription factors that are involved in induction of mesoderm and notochord differentiation and coordination of limb bud outgrowth.
Signaling Molecules (Morphogens)
Transforming Growth Factor beta
The transforming growth factor beta family (TGF-beta) of morphogens consists of a large group of molecules that play a role in development. Among the members of the group are bone morphogenetic proteins (BMPs), signaling molecules important in differentiation of the neural tube and induction of skeletal differentiation.
Fibroblast Growth Factors
The approximately 22 members of the group of fibroblast growth factors (FGFs) play a key role in embryogenesis and fetal development by regulating cellular proliferation, differentiation, and migration.
They are important in promoting elongation of the upper and lower limbs, optic nerves and promoting proliferation of neural crest cellsHedgehog Family
Sonic hedgehog (Shh), the most ubiquitous member of the group, is involved in a whole host of developmental processes including patterning of the neural tube, establishment of left-right asymmetry, limb formation, development of the forebrain and head region of the embryo to name only a few.
Indian hedgehog and Desert hedgehog, are other, but less notable, members of the hedgehog family.One ring to rule them all. (Lord of the Rings; JRR Tolkien)Before the advent of molecular biology, embryologists hypothesized the existence of a master signal, a morphogen that directed all of embryologic development. Although it is now recognized that there are multiple signaling molecules at work during development, Sonic hedgehog (Shh) comes closest to fulfilling the role of "master signal" because of its role in the development of a large number of organs.Wnt Signaling
Wnt signaling is important in establishing polarity within epithelial sheets and plays a major role in convergent extension during gastrulation, notochord formation and neural tube development and in regulating limb patterning, somite development, midbrain development and urogenital tract differentiation.
Two Examples
Differentiation of the neural tube exemplifies how diffusion of a morphogen creates a concentration gradient that influences how cells in a specific organ are guided to develop into diverse cell types.
Fig 7. BMP secreted in the dorsal neural tube forms a concentration gradient that guides the development of specific neurons for processing sensory information in the dorsal horn of gray in the spinal cord, while sonic hedge hog (Shh) from the notochord and ventral neural tube guide the development of motor neurons in the ventral gray of the spinal cord
Bone morphogenetic protein (BMP) secreted by the midline ectoderm and dorsal neural tube, induces the dorsal part of the neural tube to develop neurons that process different sensory modalities, e.g. pain, touch, proprioception. The gradient of BMP determines which specific kinds of sensory processing neurons will be formed in each level.
Sonic hedge hog, Shh, secreted from the notochord and ventral part of the neural tube is most concentrated in the ventral part of the neural tube prompting the development of motor neurons.
The gradient established by the diffusion of Shh in the ventral part of the neural tube determines what kind of motor neurons will develop.
Cells closer to the source of the Shh, in the most ventral part of the neural tube become large alpha-motor neurons that innervate the skeletal muscles. Cells farther from the source of the Shh become presynaptic sympathetic neurons that are smaller and which activate smooth muscle cells of the viscera and blood vessels by way of the sympathetic nervous system.
Frequently, competent cells are exposed to morphogens from two or more signaling centers resulting in unique combinations of inductors prompting undifferentiated mesenchyme to differentiate into separate, but related tissues.
For example, some mesenchyme in the limb bud must be guided to develop into skeletal muscle, while neighboring mesenchyme must be guided to develop into fibroblasts for laying down bone matrix or connective tissue elements to form tendons. Microenvironments, comprised of unique combinations of morphogens guide the development of structures appropriate to their position and final location within the limb.
Fig 8. As a limb bud elongates, morphogens like, fibroblast growth factor (fgf8) (red), sonic hedgehog (Shh)(green) and retinoic acid (ra)(purple) released from specific signaling centers at different points along the limb bud, create gradients, such that mesenchyme cells in different regions of the limb are exposed to unique combinations of these morphogens guiding their development to form structures appropriate to their position within the limb bud.
Interplay Between Transcription Factors and Morphogens
Establishing left/right asymmetry, critical to proper positioning of the internal organs, is orchestrated by a cascade of events that occur early in development after the formation of the notochord and which involve a number of transcription factors and morphogens.
Development of Left/Right Asymmetry
Left/right asymmetry is established during gastrulation, by a gradient of fgf8 (fibroblast growth factor 8) secreted by cells of the primitive node. Cilia of these cells beating in a clockwise direction, form a gradient of fgf8 along the left side of the embryonic disc.
This activates NODAL (a gene coding for a transforming growth factor, Nodal) in cells on the left side disc. This up regulates a HOX gene for left sidedness PTX2.
An accumulation of serotonin (5-HT) on the left coupled with induction of MAD3, a transcription factor that inhibits NODAL on the right side of the embryonic disc, thus creating left and right developmental fields in the embryonic disc.
Sonic hedgehog (Shh)(hedgehog family) secreted by the notochord, in the midline of the embryonic disc prevents Nodal expression on the right side of the embryonic disc.
This coupled with a build up of monamine oxidase (MAO), a serotonin antagonist, creates separate right and left developmental fields thus establishing left-right asymmetry.
Cells that will give rise to left sided organs, such as left ventricle, stomach, spleen, and a lung with only two lobes, etc. and the mechanisms that lead to proper placement of these organs like folding of the heart tube and rotation of the gut, depend on these pathways.
Fig 9.
Morphogenesis
Transcription factors and morphogens regulate a relatively small number of cell behaviors that physically drive the developmental process. These include alterations in cell:
- adhesiveness - the ability of cells to 'stick together" to form sheets of epithelia or separate from one epithelium to move about within the body of the embryo to specific locations before they differentiate.
- number - control of cell division to produce a critical number of cells that make up the main mass of an organ.
- position - orientation to the body axes is important in alignment and placement of the organs.
- shape - shape must conform to function
- size - most differentiated cells exhibit size limits matched to their function.
Reconsider the process of convergent extension which is important in elongation of the neural tube and GI tract among others. Several of the above behaviors are important in the process which involves merging several rows of ectoderm cells into a single long tube. Convergent extension requires that these cells elongate and move from lateral to medial as they increase in numbers.
Morphogenesis is the process by which these cell behaviors convert a single cell zygote into a trilaminar embryonic disc that folds into a tubular body shape and in which the three primary germ layers are able to develop all the functional tissues, organs and organ systems to survive in the postpartum world.
Genetic defects or exposure to teratogenic agents can result in disturbances in normal development that are manifested as birth defects.
Disturbances in development can take one of two forms:
- Malformations - disturbance in the morphological events involved in the formation of a structure of body part (e.g. cleft palate)
- Deformations - disturbance due to mechanical trauma while in utero. (e.g. amputations due to constrictions by the umbilical cord)