By:Tim Fedak*, Tamara Franz-Odendaal, Brian Hall and Matt Vickaryous
During the last six columns we have attempted to demonstrate why palaeontologists need developmental biologists, and vice versa. In this column we discuss epigenetics and why this concept should be of interest to those who study traces of organisms long since deceased.
Epigenetics is often confused with epigenesis. Epigenesis historically refers to the process of development by which a complex structure (embryo, organ or tissue) is produced from a single cell homogeneous starting point. In contrast to epigenesis is preformation, long debunked in its extreme form, which was that a tiny version of the adult was present in the gamete(s), and which grew only in size to produce the adult organism. In a less extreme form, we know that eggs inherit preformed structures such as mitochondria, ribosomes, nuclei etc. Indeed, the egg is inherited as a fully formed structure.
Waddington coined the term “epigenetics” to merge epigenesis with genetics, producing the notion that embryos, organs, tissues and cells develop under genetic control. Importantly, for Waddington the genetic control was not autonomous, but rather responsive to environmental signals, where the environment might be factors internal to the organism or external. However, epigenetics as used today has a second meaning that can be characterized as the ”phenotype of the gene,” a concept that includes such processes as genetic imprinting and DNA methylation, which result in selection expression/repression of genes. Waddington’s definition and the new approach both highlight factors necessary for successful and selective transcription of DNA products and the interactions of these products in space and time. The environment of a cell provides the conditions necessary for the successful initiation of DNA transcription, whereas cell-cell interactions or physical factors such as temperature, pH and nutrition provide other conditions required for cell, tissue and organ differentiation (Robert 2004). Epigenetics then, recognizes that context is everything.
So why should palaeontologists be concerned with examples and details of epigenetics? The fossil record is depauperate (or completely lacking) in evidence of cell-cell interactions, let alone the ability to characterize the genetic code of the various study organisms, so epigenetics might initially seem to be of little interest to those who study hard tissue, or trace remains of long dead organisms. However, palaeontology in its most general sense is the study of ancient morphology and its evolution through time. Epigenetics reminds us that there is rarely a one-to-one coupling of gene mutation and morphological change. The evolution of morphology is not solely the product of heritable genetic mutations, but rather driven by a dynamic interplay of cellular components and tissues with their environments.
Internal Factors
The environment of a cell, its location and surroundings, are important epigenetic factors that influence the cell’s identity and activities. For example, osteoblasts remain as such until the bony matrix they secrete surrounds them and they become entombed as osteocytes. Along with position there are associated changes in cell shape and gene expression patterns.
As far as we know particular osteoblasts are not predetermined to become embedded and ultimately form osteocytes, the differentiation is apparently a consequence of position in relation to osteoid matrix.
Epithelial-mesenchymal tissue interactions are important epigenetic factors necessary for chondrogenesis (cartilage formation) and osteogenesis (bone formation) of the mandible of chicks and mice. While three types of epithelial-mesenchymal interactions are possible (cell-cell contact, diffusible factors from the epithelium, or matrix-mediated factors located in the basal lamina), factors located in the basal lamina have been demonstrated to be necessary for mouse mandibular skeletogenesis (Macdonald and Hall 2001). Due to differences in the timing and nature of neural crest migration, avian mandibular mesenchymal cells are chondrogenic prior to neural crest migration due to an interaction with cranial epithelium during neurulation; however mammalian mandibular mesenchymal cells also depend upon a later interaction with mandibular epithelium, perhaps because migration occurs prior to completion of neural tube folding and interaction with cranial epithelium. The ability for the matrix-mediated factors to be provided in spatially and temporally different patterns provides a means for heterochronic change. An awareness of epigenetics provides the context with which to interpret such features as: associations of the dermal skeleton with the epidermis; the functioning of odontodes, and the conditions underlying increase or decrease in skeletal elements, morphological changes in tooth patterns and tooth shapes, and so forth.
Embryonic induction and the competence of cells/tissues to respond to different inductive signals are also epigenetic interactions. They can be explained at both the cell and the tissue level (e.g., at the level of the cell—development of the vulva in the nematode—precursor cells need to be competent to respond to the signal from the anchor cell; e.g. at tissue level —eye development—ectoderm overlying the optic vesicle needs to be competent to respond to the inductive signal from the vesicle in order to form a lens placode). Take a cell/tissue out of its usual context/environment and its fate can change (within limits). But first it must be competent to respond to the new environment (Dawid, 2004). The evolution of external cheek pouches in pocket gophers and kangaroo rats or the origin of the turtle shell are classic examples.
When inductive interactions between tissues are disrupted, “upstream” phenotypes can be altered or fail completely. The Mexican tetra Astyanax mexicanus has two forms—a surface form that is pigmented and a cave form that is eyeless and unpigmented. Cave fish may have exchanged sight—unneeded in underground rivers—for more teeth and taste buds (see Vogel 2000; Jeffery et al. 2003). In normal eye development, the lens induces the optic cup to form the optic vesicle, composed of the inner and outer layers of the retina. The blind tetras initially form an optic primordium with a small lens but the eye subsequently degenerates. Jeffery and colleagues (2003) discovered differences in the expression of key eye genes (Pax6, shh) between the two morphs, with reduced Pax6 expression in the lens placodes. The degenerated lens is incapable of setting up later inductions involved in further eye development, and the eye degenerates after subsequent programmed cell death of the lens. Current hypotheses suggest that adaptation to cave environments may have occurred either once or multiple times during the evolutionary history of this species.
External Factors
Whether maternal characteristics (extra-embryonic egg shell and contents or in utero) or characteristics of the physical environment, the external environment around an embryo can greatly influence the process of development and the resulting morphology. One can develop chicken embryos in shell-less culture (up to about 19 days out of 21 in ovo developmental period). In response to the reduction of the calcium reservoir, much of the cartilaginous skeleton of these chicks fails to ossify. Interestingly, in the American alligator, a similar semi-shell-less culturing technique has proven to be much less detrimental to the embryo (see Ferguson, 1981). Unlike birds (and turtles), alligators are more dependent on contents within the egg (and not the egg shell) as a calcium source. Indeed, semi-shell-less alligator egg cultures appear to develop normally, even to hatching (Ferguson, 1981). An adequate interpretation of palaeohistology requires recognizing the nuances of metabolic regulation of skeletal development.
Experimental work on mice and other rodents highlights the importance of hormonal exposure during prenatal development (see Vandenbergh 2003). Testosterone levels increase within prenatal male mice, leading to sexually dimorphic characteristics such as increases in the size of the hypothalamus and anogenital distance; removal of the testosterone signal, either by surgery or pharmaceutical blockage, results in a pup exhibiting anatomical and behavioural female traits. Female pups located adjacent in the uterus to males are exposed to increased levels of testosterone from their brothers, and exhibit increased amounts of male sexual morphology and behaviour depending upon the amount of exposure (one adjacent male or two). The uterine environment has a measured affect on the adult morphology and variation within the litter.
Mechanical strain plays an important role in the development of bone morphology, processes, and remodelling. Strain is detected by osteocytes and is communicated via connections (cell processes) between osteocytes and osteoblasts on the bone surface, thereby modulating remodelling (Hall 2004).
The role of muscular activity on the growth of bones has been investigated in paralysed chick embryos (Hall and Herring 1990). Individual bones, or the elements of a single bone, vary in their dependence on muscle contraction for normal growth. As an example of a single bone, the development of the anterior portion of the mammalian dentary bone is influenced by the developing dentition but not by muscle action. Development of the posterior portion of the mammalian dentary bone is influenced by the muscle action but unaffected by the dentition. As an example comparing bones, the furcula is more dependent and the mandible less dependent on muscular activity for normal bone growth than the long bones, which showed a decrease in growth rate of about 52–63% of normal rate (this is comparable to overall reduction in embryonic growth). Not only does paralysis affect the growth rates but it can also stop some developmental processes. For example, in paralysed chick embryos, developmental arrest of fusion of the sternum, followed by its collapse and the displacement of the entire shoulder girdle was observed (also in Hall and Herring 1990). It was suggested that perhaps the sternum is incapable of resisting loads imposed on it because of the effects of paralysis on adjoining musculoskeletal elements (rib cage, muscle attachments to the sternum, etc).
Temperature can affect pigment pattern and/or sex ratios (temperature-dependent sex determination) in some reptiles. The American alligator expresses an F:M:F ratio—below 31.5ºC or above 35ºC and all embryos are female (Western, 1999). The temperature sensitivity period of gonadogenesis occurs during days 32 to 42 (of a 70 day average) at an ambient temperature of 33ºC.
The three-spined stickleback, Gasterosteus aculeatus, is distinguished by three individual spines on the back, close to the dorsal fin. This species shows great variation in the development of the series of bony plates along the sides of its body. In the past these variations were considered to constitute distinct and named forms or varieties of the one species. While variation in the development of these side plates has been considered a result of local habitat differences such as variation in temperature and particularly in salinity, as well as the sex and age of the fish (see Ahn and Gibson 2004, and Pelchel et al. 2001), increasing strides are being made on demonstrating how the morphological differences could be linked to gene regulation (Pennisi 2004). Hubbs (1922) and Lindsey and Arnason (1981) noted that modulation in the number of somites and hence number of vertebrae as a result of temperature most likely occurs in the posterior part of the body.
Newman and Müller (2000) propose that plasticity, extreme morphological variation that results from variations in the environment during development, may have been much more prevalent in the early evolution of multicellular life. For example, the fungal pathogen Candida albicans switches between very different forms such as budding cells, to thread-like hyphae, or strings of yeast-like cells with long septated filaments, with no apparent default morphology. They suggest that genetic integration, which limits the effect of environmental variability, is a highly derived condition and that epigenetic plasticity permitted rapid accommodation to highly variable conditions as well as a wider range of morphologies on which selection could act.
Considering the Fossil Record
Some of the cave fish forms of A. mexicanus have been described as belonging to a separate genus Anoptichthys (Wilkens and Strecker 2003). The eyed and eyeless form of A. mexicanus show morphological differences in their craniofacial skeletons, particularly in the circumorbital bones (Yamamoto et al. 2003). If these were found in the fossil record, the morphological differences may have led to misidentification (splitting into separate species) —but with our knowledge of developmental processes, we know they are the same species. The Mexican tetra example also demonstrates plasticity inherent in development, where loss of eyes occurred as well as increase in tooth and taste bud number. That is, there are different changes occurring in different tissues in the head region.
Examples initially known from the fossil record, but where our understanding has been expanded by incorporating studies on the epigenetics of development, include the transition from fins to tetrapod limbs, the origin of the mammalian middle ear ossicles, and the transformation of forelimbs to flippers in cetaceans.
For fins to limbs we have: homology between the endoskeletal elements; knowledge of the expansion of domains of expression of Hox genes associated with the transition; models of altered timing (heterochrony) to explain the loss of the fin rays (Hall, 2004).
For the middle ear ossicles we have: the evolutionary transition preserved in the ontogeny of marsupial embryos; an understanding of how the pharyngeal arches are patterned by Hox genes; knowledge of the major genes that initiate skeletogenesis; information on how groups of cells are specified and migrate within the embryo (Hall, 1999).
For flippers we have: mutants that display various degrees of loss of paired appendages; knowledge of the contribution of somitic cells that maintain the specialized apical ectodermal ridge (AER) in limbed tetrapods, and whose premature death deprives the AER of the factors required to maintain it; knowledge of the specialized interactions between the AER and underlying mesenchyme required for outgrowth and patterning of limb rudiments (Fedak and Hall, 2004).
Epigenetic relationships identify factors and interactions important for hypotheses and studies of morphological evolution. Also, growing attention to plasticity in developmental systems, which produce extreme polymorphisms depending upon external environmental factors, suggest the fossil record may include an (irreconcilable) bias producing taxa-splitting in systematic studies, because the exact environmental variables during development for fossil specimens will always be unavailable. Furthermore, the hypothesis that genetic integration is a highly derived condition and that plasticity was the norm early in multi-cellular evolution provides important context for interpreting the significance of the earliest fossil record of multi-cellular life.
Some might suggest that epigenetics is a catch-all term to account for all variation ‘noise’ not accounted for by heritable changes of the genetic code that some still consider the primary, if not only, factor driving evolution. However, the ‘code’ is context-dependant, and there is much information available from considering the details of this non-genetic variation. Epigenetics and developmental plasticity remain fundamental developmental concepts of great relevance to the study of morphological evolution in the fossil record.
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Created by Mark D Sutton on the 2006-02-25. (Version 2.0)