I hypothesize that the First Principles of Physiology (FPPs) were co-opted during the vertebrate transition from water to land, beginning with the acquisition of cholesterol by eukaryotes, facilitating unicellular evolution over the course of the first 4.5 billion years of the Earth's history, in service to the reduction in intracellular entropy, far from equilibrium. That mechanism was perpetuated by the advent of cholesterol in the cell membrane of unicellular eukaryotes, ultimately giving rise to the metazoan homologs of the gut, lung, kidney, skin, bone, and brain. Parathyroid hormone-related protein (PTHrP), whose cognate receptor underwent a gene duplication during the transition from fish to amphibians, facilitated gas exchange for the water-to-land transition, since PTHrP is necessary for the formation of lung alveoli: deletion of the PTHrP gene in mice causes the offspring to die within a few minutes of birth due to the absence of alveoli. Moreover, PTHrP is central to the development and homeostasis of the kidney, skin, gut, bone, and brain. Therefore, duplication of the PTHrP receptor gene is predicted to have facilitated the molecular evolution of all the necessary traits for land habitation through a common cellular and molecular motif. Subsequent duplication of the β-adrenergic receptor gene permitted blood pressure control within the lung microvasculature, allowing further evolution of the lung by increasing its surface area. I propose that such gene duplications were the result of shear stress on the microvasculature, locally generating radical oxygen species that caused DNA mutations, giving rise to duplication of the PTHrP and β-adrenergic receptor genes. I propose that one can determine the FPPs by systematically tracing the molecular homologies between the lung, skin, kidney, gut, bone, and brain across development, phylogeny, and pathophysiology as a type of “reverse evolution.” By tracing such relationships back to unicellular organisms, one can use the underlying principles to predict homeostatic failure as disease, thereby also potentially forming the basis for maneuvers that can treat or even prevent such failure.
- PTHrP receptor
- β-adrenergic receptor
- gene duplication
- First Principles of Physiology
“We dance round in a ring and suppose,
But the Secret sits in the middle and knows.”
The Secret Sits, Robert Frost
the premise of this review is that the First Principles of Physiology (FPPs) are knowable. We just need to be clever enough to identify, define, and validate them. My hypothesis is that such FPPs were co-opted during the transition of vertebrates from water to land, beginning with the acquisition of cholesterol by eukaryotes (62), facilitating unicellular evolution over the course of the first 4.5 billion years of the Earth's history in service to the reduction in intracellular entropy, far from equilibrium. That mechanism was initiated, supported, and perpetuated by the introduction of cholesterol into the cell membrane of unicellular eukaryotes (42, 62), ultimately giving rise to the metazoan homologs of the gut, lung, kidney, skin, bone, and brain (62). Central to this working hypothesis is that homeostatic control is “plastic,” allowing for inheritance of a range of set-points, rather than just one genetically fixed state (20). It is important to note that this perspective is one-hundred-and-eighty degrees out of synch with traditional genetic determinism (50, 76), in which evolution is considered to result from random mutation and natural selection. Yet such plasticity is totally in keeping with ideas such as the Barker Hypothesis for the fetal origins of adult disease (3), the role of epigenetics (74), and what we know of the variation in growth factor determination of morphogenesis and homeostasis (24). Table 1 defines several terms for readers who are not familiar with ideas that I will mention in this article.
Tiktaalik, the fish-to-tetrapod transitional fossil discovered by Neil Shubin in 2004 (13) provides a heuristic for the vertebrate water-to-land transition. To make that transition, Tiktaalik had to have been “preadapted” for respiration (as the primary selection pressure), kidney, skin, gut, bone, and brain traits amenable to land life. When thought of in the context of fish physiology as the antecedent for such a critical transition, importantly, the swim bladder has been definitively shown to be structurally, functionally (as a gas exchanger), and genomically homologous to the tetrapod lung (80). Both organs are outpouchings of the gut and mediate the uptake and release of atmospheric oxygen and carbon dioxide. Furthermore, among the most highly expressed genes in the zebrafish swim bladder is parathyroid hormone-related protein (PTHrP) (80), whose signaling receptor underwent a gene duplication event during the transition from fish to amphibians (47). That event made atmospheric gas exchange for the water-to-land transition possible, since PTHrP is necessary for the formation of lung alveoli: if one deletes the PTHrP gene in mice, the offspring die within a few minutes of birth due to the absence of alveoli (53). PTHrP is expressed in the epithelial cells that line the swim bladder of fish and the alveoli of land vertebrates. In alveoli, PTHrP stimulates the production of surfactant, which maintains the structure and function of the alveoli by reducing surface tension; in the absence of surfactant, the alveoli will collapse, rendering them dysfunctional.
PTHrP and Lung Cell-Molecular Evolutionary Homeostasis
PTHrP is a peptide secreted by alveolar type II cells in response to stretch (54, 71). PTHrP acts locally (i.e., in a paracrine manner) via cell surface receptors to induce specialized connective tissue fibroblasts to become lipofibroblasts (54, 59–70, 75) (Fig. 1). The lipofibroblasts appear to be critical in the evolution of the lung for two reasons: first, they protect the alveolus against oxidant injury (70) by actively recruiting and storing neutral lipids from the alveolar microcirculation (59), acting as antioxidants (70); secondly, the stored neutral lipids are actively “trafficked” from the lipofibroblasts to the alveolar type II cells for surfactant synthesis (59) through the mechanically coordinated effects of PTHrP (54, 66), leptin (66, 68), and prostaglandin E2 (69), which act via their cognate receptors that reside on the apposing surfaces of neighboring epithelial type II cells and lipofibroblasts. Ultimately, PTHrP regulates alveolar epithelial calcium uptake, suggesting its evolutionary history: calcium concentration in the alveolar lining aqueous, protein-containing hypophase regulates the formation of tubular myelin, which determines its effect on surface tension (4); tubular myelin is a lipid-β-defensin complex homolog of the lipid-β-defensin barrier formed by the stratum corneum in the skin (44) to prevent fluid leak and protect against microbial infection. Since the skin is the most primitive organ of land vertebrate gas exchange, it may have provided a molecular homeostatic co-option for further evolution of the lung.
It can be calculated that such a mechanism would have taken approximately 9 × 1016 years to have occurred by chance, which is orders of magnitude longer than the estimated 5 × 109 year existence of the Earth (62). An alternative mechanism is “descent with modification” (14): phylogenetically, PTHrP is a gene involved in fish adaptation for buoyancy, which was co-opted by land vertebrates for stretch-regulated (the biologic homolog of gravity) surfactant production (62, 66).
ADRP as a Deep Homology that Interconnects Evolved Functional Homologies
“Oh, the Places You'll Go!”
The key molecule that mediates neutral lipid trafficking (59) between the alveolar microcirculation, lipofibroblast, and epithelial type II cell is adipocyte differentiation-related protein (ADRP) (55). It is a member of the Perilipin-ADRP-TIP47, or PAT, family of intracellular lipid cargo proteins that mediate lipid uptake, storage, and secretion in a wide variety of cells, tissues, and organs, ranging from fat cells to endothelium, liver, and steroidogenic endocrine organs (39). PAT proteins are expressed in many organisms, ranging from mammals to slime molds and fungi (8). ADRP was first discovered to be involved in early adipocyte differentiation (25) and subsequently shown to be necessary for the uptake and storage of intracellular lipid droplets when overexpressed in Chinese hamster ovary cells, which do not naturally express ADRP (18).
In the lung, ADRP (Fig. 1) is physiologically upregulated by the stretching of the alveolar type II cells, which produce PTHrP; PTHrP then binds to its cognate receptor on the epithelial lipofibroblasts, stimulating PPARγ (61, 75), which then upregulates ADRP. This mechanism may have evolved initially to protect the alveolar wall against hyperoxia, since the rising atmospheric oxygen tension causes the differentiation of myofibroblasts into lipofibroblasts (12). This mechanism may subsequently have been co-opted to regulate surfactant synthesis during the vertebrate water-to-land transition (60), consistent with the phylogenetic adaptation of the alveolus from the swim bladder of fish to the highly adapted lungs of mammals and birds. This phenomenon is of particular interest in the context of exploiting such functional molecular homologies when one considers the homologies between the alveolar lipofibroblast and endocrine steroidogenesis. For example, oxygen in the atmosphere has not increased linearly from zero to 21%; rather, it has gone up and down episodically, ranging between 15 and 35% over the past 500 million years (5). Bearing in mind that hypoxia is the most stressful of all physiologic agonists, it would have put huge constraints on both the evolving lung and endocrine systems. Perhaps fortuitously the vertebrate pulmonary and endocrine systems were preadapted for such an adaptation through PAT genes; thus, the well-recognized effects of the adrenocortical system on lung development and homeostasis (38) can be seen as part of a logical evolutionary sequence of external and internal selection mechanisms (60).
This is not a tautology, or a “Just So Story,” since, for example, the same morphogenetic mechanisms occur during both ontogeny and phylogeny (67), and we observe the reversal of this evolutionary process in chronic lung diseases, in which there is “simplification” of the alveolar bed, resulting in a froglike structure in mammals. Experimentally, Besnard et al. (6) found that when they deleted a gene necessary for the synthesis of cholesterol, the most primitive of lung surfactants (45), specifically from mouse lung alveolar type II cells, the lung developmentally “compensated” by overexpressing the lipofibroblast population in the alveoli, suggesting that these cells have an evolutionary capacity to facilitate surfactant production, both ontogenetically and phylogenetically.
This rational cell/molecular approach to understanding how and why the lung evolved can be carried one step further, since catecholamine/β-adrenergic receptor signaling was essential for regulation of blood pressure in the lung independent of the systemic circulation, facilitating a further increase in the surface area of the evolving lung (62, 67). Our ancestors were organisms able to survive the whipsawing physiologic effects of alternating hyperoxia and hypoxia (5) by structurally and functionally adapting their pulmonary and endocrine systems (see below). Here again, the β-adrenergic receptor underwent a gene duplication during the fish-amphibian transition that allowed for a further increase in lung surface area to support metabolic demand (2). At this phase in vertebrate evolution the glucocorticoid receptor is documented to have evolved from the mineralocorticoid receptor, perhaps as a counterbalancing selection for the blood pressure-elevating effect of mineralocorticoids. The emergence of the physiologic glucocorticoid mechanism may have been further facilitated by the presence of pentacyclic triterpenoids, which are unique to the land environment, produced by rancidifying land vegetation. These compounds inhibit 11β-hydroxysteroid dehydrogenase type II (11βHSD2), which inactivates cortisol's blood pressure-stimulating activity, causing positive selection pressure for the tissue-specific expression of 11βHSD1,2 in a wide variety of glucocorticoid target organs, including the lung (81), thereby permitting local activation and inactivation of cortisol.
Reinforcing this hypothesis, when pituitary adrenocorticotropic hormone (ACTH) stimulates glucocorticoid production by the adrenal cortex, the hormone passes through the intra-adrenal portal vascular system of the adrenal medulla, providing it with uniquely high local concentrations of glucocorticoids (79). These high concentrations are needed to induce the medullary enzyme phenylethanolamine-N-methyltransferase (PNMT) (79), which controls the synthesis of catecholamine, thus coordinately upregulating both of the primary adrenal stress hormones for a maximally adapted “fight or flight” response.
PTHrP and Kidney Cell-Molecular Evolutionary Homeostasis
Akin to its role as a stretch-regulated gene product that maintains alveolar homeostasis, PTHrP is also integral to renal physiology (Fig. 2). In the glomerulus, PTHrP is produced by the epithelially derived podocytes that line them (32), maintaining the function of the mesangium (57), a stretch-sensitive fibroblast structure that determines systemic fluid volume and electrolyte homeostasis. Parenthetically, this molecular homology between the lung and kidney is not surprising, since ontogenetically both structures produce amniotic fluid during development (41). It should be borne in mind that the glomerulus also makes its appearance during the phylogenetic transition from fish to amphibians (7), and subsequently to reptiles, mammals, and birds.
PTHrP and Skin Cell-Molecular Evolutionary Homeostasis
PTHrP is essential for development of skin through its paracrine interaction between melanocytes and keratinocytes (49), generating the stratum corneum as a dual water- and bacterial-barrier essential for preventing desiccation in terrestrial vertebrates (43). It is noteworthy that the alveolar type II epithelial cells and the skin epithelium of the stratum corneum exhibit a functional homology at the cell/molecular level, packaging lipids together with host defense peptides, and secreting them in the form of lamellar bodies to generate lipid-based barriers against water loss (from the inside out), and host invasion (from the outside in) in both structures.
The evolutionary significance of the homology between lung and skin as barriers is further exemplified by the pathophysiology of asthma. Patients with asthma often have the skin disease atopic dermatitis. Both these phenotypes are common to humans and dogs and have been mechanistically linked through a common molecular defect in β-defensins, which mediate innate host defense in both skin and lung (44). In dogs, β-defensins determine coat color, which serves a multitude of adaptive advantages, ranging from protective coat coloration to reproductive strategies. The β-defensin CD103 has also been shown to cause atopic dermatitis in dogs, and possibly asthma, since it is also found in dog airway epithelial cells (11, 62, 78). Therefore, hierarchically, host defense and reproduction take evolutionarily adaptive precedence over wheezing due to asthma.
The Goodpasture Syndrome and Barrier Formation and Function: Exception That Proves the Rule?
Vertebrates transitioned from water to land approximately 300 million years ago, causing selection pressure for type IV collagen (34), which acts to physically maintain the integrity of the walls of the alveoli. Since the extracellular matrix forms during the process of cellular differentiation, it is highly likely that modification of the basement membrane occurred early in the evolutionary adaptation to land. Molecular evolutionary studies of Goodpasture's syndrome (34) have shown that the 3α isoform of type IV collagen evolved during the phylogenetic transition from fish to amphibians due to selection pressure for specific amino acid substitutions that rendered the molecule more hydrophobic and negatively charged. Goodpasture's syndrome is an autoimmune disease caused by catastrophic failure of both the kidney and lung epithelial barriers, caused by pathogenic circulating autoantibodies targeted to a set of discontinuous epitope sequences within the noncollagenous domain 1 (NC1) of the α3 chain of type IV collagen [α3(IV)NC1], referred to as the “Goodpasture autoantigen.” Basement membrane extracted NC1 domain preparations from Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio do not bind Goodpasture autoantibodies, while frog, chicken, mouse, and human α3(IV)NC1 domains bind autoantibodies. The α3(IV) chain is not present in worms (C. elegans) or flies (D. melanogaster), and is first detected in fish (D. rerio). Interestingly, native D. rerio α3(IV)NC1 does not bind Goodpasture autoantibodies. In contrast to the recombinant human α3(IV)NC1 domain, there is complete absence of autoantibody binding to recombinant D. rerio α3(IV)NC1. Three-dimensional molecular modeling of the human NC1 domain suggests that evolutionary alteration of electrostatic charge and polarity due to the emergence of critical serine, aspartic acid, and lysine amino acid residues, accompanied by the loss of asparagine and glutamine, contributes to the emergence of the two major Goodpasture epitopes on the human α3(IV)NC1 domain, as it evolved from fish over the ensuing 450 million years. The evolved α3(IV)NC1 domain forms a natural physicochemical barrier against the exudation of serum and proteins from the circulation into alveoli and glomeruli, due to its hydrophobic and electrostatic properties, respectively, which likely provided the molecular selection pressure for the evolution of this protein, given the rising oncotic and physical pressures on the evolving barriers of both the lung and kidney during the water-to-land transition. Taken together, the lung, kidney, and skin evolved critical physiologic barriers against desiccation in land-dwelling animals.
Internal and External Selection, PTHrP, and the Water-to-Land Transition
Another way to think about this co-option of cell/molecular mechanisms of evolution is as serial interactions between internal and external selection pressures. Such external environmental constraints to the transition from water-to-land as air breathing, gravitational orthostatic forces, and desiccation were all hypothetically adapted to through a common internal cell/molecular pathway for development and homeostasis—PTHrP and its cognate G protein-coupled receptor (61, 80). This model is also predictive, since PTHrP is a potent vasodilator (36), and an angiogenic factor (promotes capillary formation) (46), potentially explaining why glomeruli, as microvascular derivatives of the renal artery, may have evolved in the transition from fish to amphibians (7).
The significance of PTHrP in the vertebrate transition from water to land may be as follows: Such organisms must have spontaneously overexpressed their PTHrP signaling, initially for lung evolution from the swim bladder, particularly in physostomous fish like zebrafish, which possess a trachea-like pneumatic duct that connects the esophagus and swim bladder for gas filling and emptying. At the cell/molecular level, the smooth muscle that forms both the pneumatic duct and trachea are determined by FGF10 expression (31). The PTHrP-mediated mechanisms in the kidney and skin must have followed suit, since they would have protected against desiccation in the terrestrial adaptation. Calcification of bone in response to increased gravitational force on land would have further facilitated adaptation to land living (10); Wolff's Law states that bone will adapt to the load under which it is placed (17). PTHrP is a gravity-sensitive hormone (62, 65, 71) that is integral to bone development and homeostasis (27), determining bone calcium uptake and incorporation into cartilaginous structures, and facilitates the adaptation of terrestrial organisms to environmental gravitational forces (62, 65, 66, 71).
This scenario of a reiterative process for the acquisition of traits that facilitated water-to-land transition is consistent with data showing that vertebrates attempted the water-to-land transition several times (76). Based on parsimony, one can propose that these processes were all realized as a result of the PTHrP receptor gene duplication event, beginning with the lung, by necessity (52), and that organisms that could upregulate their PTHrP/PTHrP receptor signaling through ligand-receptor-mediated paracrine mechanisms evolved as the forebears of contemporary land vertebrates. In contrast, individuals who were unable to accomplish this feat became extinct. This perspective is supported by our demonstration of the correlation between the cell/molecular genetic motifs common to ontogeny and phylogeny of the lung and major environmental epochs (Fig. 3). Note the apparently seamless alternation between internal and external selection mechanisms in association with major ecologic stresses; we postulate that there are no gaps between these genetic adaptations because the data are derived from contemporary land vertebrates; conversely, those members of the species who failed to adapt died off, and thus are not represented in this analysis.
In 2006, Neil Shubin (56) announced the discovery of Tiktaalik, the fossilized remains of the organism that transitioned from water to land. But in the era of quantum mechanics, we have come to expect more than just a descriptive biologic “Big Bang.” The ability to live on land was very physiologically demanding, but according to the Romer Hypothesis (52), it was necessitated by the increase in carbon dioxide in the primordial atmosphere, causing the Earth's lakes, ponds and rivers to dry up, forcing our vertebrate ancestors to seek refuge on land or face extinction.
So from a physiologic perspective, how might a fish have evolved into a tetrapod? The biggest constraint was the ability to breathe air. It has long been thought, though controversial, that the swim bladders of fish evolved into the lungs of land vertebrates, since both are gas exchangers that are derived from the gut tube. The notion that evolution co-opted an organ for buoyancy into one that mediated oxygenation for metabolism is attractive, though there are certain anatomic constraints (48). That controversy has been put to rest by a recent study showing that at the molecular level, the swim bladder expresses all the homologous genes of the lung (80), including PTHrP, which, as discussed above, is a gravity-sensing gene that is necessary for the formation of alveoli in mammals (53). Thus, there is a functional genomic link between the water-to-land transition and PTHrP signaling, which underwent a gene duplication (47) sometime during the fish-amphibian transition, thereby helping to provide an explanation for the evolution of the lung from the swim bladder.
As also discussed above, equally important is that the organs necessary for barrier function against desiccation are also PTHrP dependent, both developmentally (16) and physiologically (37). The skin (22), kidney (9), and gut (19) all express both PTHrP and its receptor, and the signaling between the mesoderm and epithelium of these organs is mediated by the PTHrP/PTHrP receptor (9, 19, 22), which determines the structural and functional development of these organs to form homeostatically regulated physiologic barriers against water loss. Moreover, PTHrP is necessary for the calcification of cartilage (33), so it would have facilitated the evolution of the boney tetrapod limbs of Tiktaalik to accommodate the increased gravitational force on its skeleton on land, exhibiting the plasticity of the lung, skin, and bone. As an added note in support of this interrelationship, when the PTHrP gene is deleted in mice, they exhibit morphogenetic defects exclusively in lung, skin, and bone (26).
The angiogenic properties of PTHrP are another feature relevant to its utility in the water-to-land transition and organ adaptation. PTHrP promotes vascularization of bone (23) and skin (15), particularly when the vascular endothelium is cyclically distended, as in conditions of increased physiologic stress such as those involved in the water-to-land transition. PTHrP receptors exist in the lymphatic microcirculation as well (40). Additionally, PTHrP is a vasodilator, ultimately epistatically relieving tension on the remodeled microvasculature, while simultaneously providing increased perfusion for remodeling of the adjacent parenchyma in further adaptation to internal physiologic stress. Such a mechanism would be consistent with the progressive expansion of the surface area of the lung, and may help explain the evolution of the glomerulus, which is absent from kidneys of some fish species (7) but is omnipresent in amphibians, reptiles, mammals, and birds.
Perhaps it was not merely fortuitous that there was a PTHrP receptor gene duplication “just in time” for Tiktaalik's water-to-land migration. More likely, there were cumulative, episodic increases in shear stress on the microvessels of the organs that facilitated that transition–swim bladder, lung, adrenals, skin, kidney, skeleton—causing the generation of radical oxygen species (ROS) and lipid peroxides that affected those vascular beds. ROS cause DNA damage (58), giving rise to mutations such as cross-overs and gene duplications. ROS are also embryonic signal transducers (21) that may act to promote structural and functional remodeling through morphogenetic events. The endothelium is known to be highly heterogeneous, each endothelial cell acting like an adaptive nonlinear input/output device (1). The input comes from the extracellular environment, consisting of biomechanical and biochemical forces. The output is the heterogeneity of the endothelial cell population, reflected by cell shape, calcium flux, protein expression, mRNA expression, migration, proliferation, apoptosis, vasomotor tone, hemostatic balance, release of inflammatory mediators, and leukocyte adhesion/transmigration (1).
Such dual mechanisms of development and mutation for evolutionary change would have provided robust pressures for both internal and external selection, depending on the nature and magnitude of the agent, suggesting a mechanistic way of thinking about “evolvability.” More importantly, organisms such as Tiktaalik exhibited the plasticity necessary for the remodeling of vital organs for adaptation to terrestrial life. Tiktaalik's ancestors were thus preadapted for the “great leap forward” during vertebrate evolution, but because metazoans evolved from unicellular organisms (29), Tiktaalik was preadapted for such a critical “sea change” in vertebrate evolution.
Cellular Growth Factors as the Universal Language of Biology
The approach to a fundamental a priori understanding of vertebrate physiology, not as a top-down descriptive process, but as a series of co-optations originating from the cell membrane of unicellular organisms, will lead to understanding of the FPPs based on its evolutionary origins. The actualization of such FPPs would have numerous ramifications, including a predictive model for physiology and medicine (60, 64), as well as a functional merging biology, chemistry, and physics into a common algorithm for the natural sciences. Such a perspective would allow us to deemphasize the human signature from our anthropocentric view of our physical environment, on the scale of the recentering of the solar system on the Sun, which obscures our perception of our universe, and that of other universes.
What Predictions Derive From a Cellular Approach to Evolution
Starting with the premise that ontogeny is the only biological process we know of that formally generates new structures and functions, we should exploit this process to understand evolution, since it does so throughout the phylogenetic history of an organism. By focusing on cell-cell interactions, particularly ones mediated by soluble growth factors and their cognate receptors, one can deconvolute the evolution of the lung, functionally tracking it back to the swim bladder of physostomous fish (62, 63). Since the adaptation of fish to land was contingent on efficient atmospheric gas exchange, the lung can be seen as the cellular/molecular blueprint for the evolution of other physiologic adaptations to land life (62, 64). By systematically tracing the molecular homologies between the lung, adrenals, skin, kidney, gut, bone, and brain across developmental, phylogenetic, and pathophysiologic space and time, the FPPs can be determined (62, 63, 72). Once such relationships are traced back to unicellular organisms, the underlying principles can be used to replay the evolutionary tape, and predict and prevent homeostatic failure as disease (61–64). Perhaps the reduction of biology to ones and zeros can offer the opportunity to merge biology, chemistry, and physics into one common user-friendly algorithm as a “periodic table of nature” (72).
No conflicts of interest, financial or otherwise, are declared by the author.
J.S.T. conception and design of research; prepared figures; drafted manuscript; edited and revised manuscript; approved final version of manuscript.
- Copyright © 2013 the American Physiological Society