Like Prometheus discovering fire, Archimedes in the bathtub, Copernicus declaring that the sun is the center of the solar system, or Einstein’s Relativity Theory, we need an ‘aha!’ moment for biology. This essay describes just that.

The ‘Problem’

When we think of evolution in terms of contemporary biologic phenotypes, we make the systematic error of reasoning backwards from the present to the past. Yet reasoning after the fact, by definition, is illogical. All of biology is formed from and by cells, which emerged from the primordium 3-4 billion years ago, likely as primitive micelles formed from lipids. Such structures are semi-permeable, generating intracellular chemical gradients, a process referred to as chemiosmosis, ultimately allowing for the reduction of entropy within the cell, transiently circumventing the second Law of Thermodynamics. It was under these conditions that life began on Earth, initiated and perpetuated by the perennial competition between prokaryotes and eukaryotes, a battle which rages on to this day.

The Solution to the Problem

We have identified a mechanism that integrates development and physiologic homeostasis- cell-cell interactions. Why not apply that mechanism to Evolutionary Biology as the long-term basis for phylogenetic change? Using that approach at the cell-molecular level offers the opportunity to determine how cellular composition has accommodated adaptation. In a recently-published book, entitled Evolutionary Biology, Cell-Cell Communication and Complex Disease, we exploited this approach to understand how the lung evolved to accommodate metabolic drive, based on the role of surfactant in facilitating both the developmental and phylogenetic increases in lung alveolar surface-area for gas exchange. By reducing this process to ligand-receptor interactions and their intermediate down-stream signaling partners, we are able, for example, to envision the functional homologies between such seemingly disparate structures and functions as the lung alveolus and kidney glomerulus, the skin and brain, and the skin and lung.

Using such a reductionist approach to functional genomics has led to a mechanistic understanding for how internal selection pressure, brought on by physiologic stress within Claude Bernard’s milieu interieur, may have given rise to such disparate diseases as Goodpasture Syndrome and asthma. By linking together the cell-molecular pathways for basic physiologic mechanisms, independently of their overt structural and functional appearances, particularly as they relate to extrinsic ecologic selection pressures, one can discern the ‘how and why’ of evolution. By literally starting from the ‘middle’ of the mechanism, tracing the signaling pathways linking genes to phenotypes, one can see how such pathways evolved across the space and time of biology as ontogeny and phylogeny.

One fundamental insight from such molecular analyses is that the time and space dimensions for evolutionary processes are artifacts of descriptive biology; once the underlying mechanisms are identified, the dimension of time falls out of the analysis, other than as the sequence of events. Once achieved, vertical integration literally and figuratively eliminates time and space. This point of view opens up to a very different perspective on life actually being simple, rather than complex. Moreover, it begs the question whether metazoans are merely a further extrapolation of such prokaryotic, pseudo-metazoan traits as lateral inheritance, biofilm and quorum sensing. Perhaps protozoans evolved their metazoan phenotype as a way of monitoring the environment iteratively. After all, H.G. Wells wanted to teach us such humility by having bacteria save mankind in The War of the Worlds.

Putting Humpty Dumpty back together again based on epigenetic principles

A systematic error in the reductionist approach to Evolutionary Biology is our failure to recognize that it is a mechanism, not a ‘thing’, namely DNA. In order to understand how and why evolution functions, one must first reduce it to its smallest functional unit of activity- the cell. In contrast, evolutionists describe the process dichotomously as mutation and selection, overarching the cell. That is why Cell Biology is not part of the conventional analysis- it is not considered to be necessary, yet it is the fundamental mechanism of ontogeny- it is only in the recent past that we have been able to determine the mechanisms underlying morphogenesis based on cell-specific production of soluble growth factors and their cognate receptor signaling partners on the surfaces of neighboring cell-types. These developmental mechanisms culminate in homeostatic control, providing a unified functional basis for physiology, repair and regeneration. And since such processes are amenable to modification under selection pressure, they are also the mechanisms for phylogeny. Such cellular signaling mechanisms common to both ontogeny and phylogeny provide insights to the mechanisms of evolution, complying with the ‘emergent and contingent’ nature of the evolutionary process.

We humans have succeeded as a species because of our highly-evolved central nervous system. We have an obligation to both our ancestors and offspring to use our minds effectively so that we don’t destroy ourselves and the environment in the process. If we understood where we evolved from perhaps we would act in more socially responsible and humane ways. The key is to deconvolute Evolutionary Biology, which has become so complicated as to be useless in utilizing the Human Genome for the prediction and prevention of disease. The solution to the puzzle of evolution is right under our noses, but instead we generate more and more neologisms and metaphors that allow us to circumlocute and evade the solution.

Paracrine Growth Factors- from morphogenesis to homeostasis

Contemporary molecular embryology is based on growth factors signaling via their cognate receptors, depending upon spatio-temporal relationships that determine morphogenetic patterns. As such, these mechanisms provide a predictive magnitude and direction for the formation of structure and function. In this sense, it is no different from what we expect of a mechanistic basis for Evolutionary Biology, which is also trying to comprehend the magnitude and direction of biologic change, though the time scales are (seemingly) very different. But perhaps that’s just an artifact of the descriptive modality. Once we transition to a mechanistic approach, such time and space considerations are independent of the mechanisms of interest, other than providing the nominal sequence of events.

So what is the value added in using a cellular-molecular mechanistic approach? We have been able to envision this continuum, and how it has fostered the evolution of the lung, for example. Based on our working knowledge of how paracrine growth factor-receptor interactions have mediated the development of the mammalian lung, we considered the overall ontogeny and phylogeny of the lung phenotype, i.e. its evolution, as an overall selection pressure for increased surface area, from fish to man in service to the metabolic drive underpinning the water-to-land transition. This has been realized by a progressive decrease in the size of the alveoli, increasing the gas-exchange surface area-to-blood volume ratio over phylogeny and ontogeny (see Schematic, Fig. 2).

This process could not have occurred without an increase in the net production of lung surfactant, which must physic-chemically compensate for the increased surface tension resulting from the decrease in alveolar diameter (by the Law of Laplace, that the surface tension is inversely related to the diameter of a sphere). The cellular regulation of surfactant production is orchestrated by interactions between the alveolar epithelial lung cells that synthesize the surfactant, known as Alveolar Type II cells, and the adepithelial connective tissue fibroblasts that underlie them within the alveolar wall. The cell-cell interactions that regulate surfactant production have evolved from the secretion of cholesterol, the simplest form of surfactant, into the lumen of the swim bladder of fish to prevent the walls from adhering to one another, to a progressively more efficient means of synthesizing and secreting a more complex biochemical surfactant mix of lipids and proteins in order to accommodate the increase in surface area, as the lung has evolved phylogenetically. Along with the decrease in the diameter of the alveoli, the alveolar walls also became progressively thinner, further facilitating the gas exchange between the alveolar space and the lung microcirculation. The ‘invention’ of tubular myelin, an extracellular latticework of surfactant proteins and phospholipids generated from the lamellar bodies secreted by the Alveolar Type II cell, provides an extracellular homolog of the lipid barrier formed by the stratum corneum of the skin, including both the lipids and the antimicrobial peptides packaged within the lamellar bodies.

Tracing the changes in structure and function that have occurred over both the short-term history of the organism (=ontogeny), and the long-term history of the organism (=phylogeny), and how the mechanisms shared in common account for both biologic stability and novelty, will provide the key to understanding the mechanisms of evolution. Like solving a fraction problem in math, the cellular-molecular approach determines the ‘least common denominator’ for both ontogeny and phylogeny, eliminating the artifactual temporal-spatial differences between these processes.

It is important to bear in mind that there are certain gene-phenotype homologous relationships that are fairly readily apparent because of their position as ‘barriers’ at the interface between the environment and the organism, such as the lung, skin, and gut, likely having originated from the cell membrane in unicellular organisms as their ‘common denominator’. And then there are other homologies that are ‘derived’ from those more readily apparent properties that must be deciphered based on their short- and long-term histories, particularly as they derive from those primary mechanisms. Instead of taking a ‘top-down’ or ‘bottom-up’ approach to understanding physiologic evolution based on superficial appearances, we have advocated for a ‘middle-out’ approach based on the underlying cell-cell communication by which to determine the evolutionary origins of cell-molecular traits.

We have demonstrated the utility of a cell-molecular developmental physiologic approach in deconvoluting lung evolution, providing a cell-molecular mechanistic continuum from development to physiologic homeostasis and regeneration. Moreover, this tack allows for understanding the interrelationships between tissues and organs at a fundamental cell physiologic level, independent of their contemporary appearances and functions, effectively replacing the need for illogically reasoning after the fact. This approach has provided novel insights to the mechanisms of evolution for both the more directly evolved structures/functions of the lung, namely skin and bone, as well as for the deeper homologies of the kidney and brain, based on cell-cell signaling as the integrative mechanism, for the first time.

We have learned from cell culture experiments that normal metazoan cells are not structurally or functionally autonomous; over time, differentiated cell-types lose their phenotypes. They exist within microenvironments created during development by cell-cell interactions between cells of derived from different cell lines. The underlying mechanisms of development, physiologic homeostasis and regeneration are mediated by soluble growth factors and their cognate receptors, which signal through second messengers to determine the metabolic and proliferative status of their surroundings. We maintain that these mechanisms are the basis for the evolution of complex biologic traits, and that by systematically analyzing these diachronic signaling mechanisms over time within and between species, the mechanistic basis for evolution can be discerned.

The Water-Land Transition, PTHrP Amplification, and the Adaptation to Land

The evolution of PTHrP signaling known to have occurred during the water-land transition (Fig.4) would provide a mechanistic explanation for the morphing of fish into land vertebrates, like Neil Shubin’s Tiktaalik, the fossil remains of the transitional tetrapod discovered in 2004.

All of the essential water-land adaptations—lung, skin kidney, gut, and brain—would have been facilitated . At first glance, this event may seem like a Just So Story for vertebrate adaptation to land, yet we know that there were at least 5 separate attempts by vertebrates to breach land based on skeletal fossilized remains; this could not have occurred independently of the evolution of the visceral organs, particularly because many of the same genetic mechanisms are common to both bone and visceral organ development (PTHrP, Wnt/βcatenin, TGFβ, PKA, PKC, Shh), so these events should also be viewed in the context of hypothetical internal selection mechanisms for cellular adaptation.

Mechanistically, the PTHrP Receptor gene is known to have duplicated during the water-land transition, amplifying the PTHrP signaling pathways for the adaptive morphing of the lung, skin and bone- all of these organs are dependent on the PTHrP signaling pathway for their development and homeostasis. Though the literature suggests that this occurred by chance, it could well have happened as a direct consequence of the generation of excess oxygen radicals and lipid peroxides due to vascular shear stress within the microcirculations of these very same tissues. On the one hand, these tissues and organs would have constrained land adaptation, but on the other, increased PTHrP signaling would have been advantaged by such gene duplication events. This process is formally known as the Baldwin Effect.

In fact, if adaptation is thought of in the context of internal selection caused by vascular shear stress, the concept of plasticity becomes much more relevant, not to mention being experimentally testable; constitutive genes are the ones that were most vulnerable to mutation, since they were the genes being targeted by such selection mechanisms. And perhaps such unconventional internal selection was followed by classic Darwinian population selection for those members of the species that were best fit to regulate those constitutive genes to survive, rendering the newly evolved homeostatic mechanisms regulatable. Theoretically, this may have been due to the fact that regulated mechanisms would be more resilient, and therefore less likely to generate mutagens than non-regulated constitutive genes. And this may also explain why humans have fewer than the predicted number of genes based on descriptive instead of mechanistic biology.

Those members of the species best able to up-regulate their PTHrP signaling in support of any one or all of the land adaptive traits- bone, skin, lung- would have had a higher likelihood of surviving on land. In turn, other tissues and organs would also have been positively selected for their amplified PTHrP signaling capacity, making them more likely to survive. This is particularly relevant to the glomeruli of fish kidneys, which range from large (salt water), to small (fresh water) to being absent in some species, but are ubiquitous in land vertebrates. Shear stress within the renal vasculature could have given rise to PTHrP signaling for glomerular function- PTHrP-mesangium signaling for water and electrolyte flux. Similarly, PTHrP is expressed in the pituitary and adrenal cortex of land vertebrates, making for a more robust physiologic stress ‘fight or flight’ mechanism since the corticoids stimulate epinephrine secretion as they course their way from the adrenal cortex through the adrenal medulla. But this amplified epinephrine response to stress is only applicable to amphibians and beyond phylogenetically since fish have an independent adrenal cortex and medulla. Such an evolved stress mechanism would have been advantageous for various physiologic adaptations to land, not the least of which would have been the positive selection for brain evolution- epinephrine inhibits flow through the blood-brain barrier, generating more neuronal interconnections within the Central Nervous System due to increased epinephrine and norepinephrine production within the brain.

This is not merely a tautologic rationalization of the data. If you experimentally delete the PTHrP gene in the embryonic mouse, the bone, skin and lung fail to develop the self-same characteristics for land adaptation- Phylogenetically, the PTHrP signaling pathway has been amplified through gene duplication, fostering stronger skeletal support, skin barrier function and lung gas exchange.

In further support of the causal relationship between the water-land transition and the evolution of specific physiologic traits that actively accommodated the adaptation to life on land, there were two other gene duplications that occurred during the water-land transition: the βAdrenergic Receptor (βAR), and the glucocorticoid receptor. The evolution of the βARs was necessitated by the demand for independent regulation of the systemic and pulmonary blood pressures to accommodate the expanding surface area of the evolving lung. The evolution of the glucocorticoid receptor from the mineralocorticoid receptor was necessitated by the increase in blood pressure due to the increased effect of gravity on land, causing increased blood-pressure, generating further selection pressure for the βAR mechanism in alleviating the constraint on the expansion of the lung surface area; the effective stimulation of  the βARs by glucocorticoids caused further positive selection pressure  for the co-evolution of both genes. Again, as in the case of the duplication of the PTHrP Receptor, the specific effects of the physiologic stress due to land adaptation on shear stress in the lung and kidney may have specifically precipitated gene duplications in these capillary beds, functionally alleviating the physiologic constraints on these tissues and organs through internal selection, further fostering these physiologic adaptations through external selection. For example (Fig.5), the episodic bouts with hypoxia due to the unmet physiologic needs of the organism as it attempted to adapt to land would have caused physiologic stress since hypoxia is the most potent stressor known,  stimulating the Pituitary-Adrenal Axis (PAA), ACTH stimulating glucocorticoid production by the adrenal cortex, and epinephrine production by the adrenal medulla; acutely, epinephrine would have alleviated the hypoxic stress by stimulating surfactant secretion by the evolving alveoli, and the glucocorticoids would have increased βARs, acting synergistically with epinephrine. As a result, the increased distension of the alveoli would have stimulated PTHrP production by the alveolar type II cells, promoting further alveolarization (Rubin et al, 1994), alveolar capillary perfusion, and angiogenesis of both the capillaries and possibly the lymphatic vessels- taken together, the evolution of alveolar PTHrP signaling coordinates the secretion and homeostasis of surfactant with gas exchange across the microvasculature at both the macro-level, and at the micro-level,  since it co-regulates calcium in the alveolar fluid hypophase with the regulation of surfactant removal from the alveolus via lymphatic drainage.  In the aggregate, this adaptive integration of the PAA and the pulmonary system would have fostered the phylogenetic adaptation of land vertebrates. And this cascade of physiologic adaptations may explain the evolution of PTHrP signaling for pituitary ACTH and adrenocortical glucocorticoid, since it would have further facilitated the positive selection for land adaptation by PTHrP Receptor gene duplication.

Bear in mind that these events didn’t occur all at once- it took place over eons of land vertebrate evolution, both within and between species. Consistent with this scenario, elsewhere we have shown that in the course of lung evolution, there were alternating intrinsic and extrinsic selection pressures for the genes that facilitated the increased surface area of the lung. This pattern may atavistically reflect the original mechanism by which the cell membrane of unicellular organisms facilitated the adaptation of the cell to the environment.

As added evidence for the interrelationship between key genetic changes that occurred during the water-land transition and physiologic stress causing internal selection, type IV collagen also evolved novel polymorphisms in the basement membranes of the lung and kidney phylogenetically from fish to humans during this period. The NC1 domain of Type IV collagen forms a natural physicochemical barrier against fluid exudation from both the lung and kidney due to its molecular electrostatic and polar properties, preventing the loss of fluid across the alveolus and glomerulus that would otherwise have occurred due to the increased physiologic demand on these structures during the water-land transition.

Contrast Evolutionary and Developmental Biology as Descriptive vs Mechanistic

 If the ‘key’ to understanding evolution is as a mechanism for spatial-temporal relationships of genes as determinants of phenotypes, and these relationships are mediated by soluble growth factors and their cognate receptors, then by following the latter we can understand the former. After all, how can you generate an ‘arrow of time’ without a mechanism for the magnitude and direction of its trajectory? Ironically, the Evolutionary Biology literature has virtually no orientation to growth factors as the mediators of evolution, or their signaling to their cognate growth factor receptors, which are the determinants of the ‘arrow of time’ described by evolutionists. As a result, Evolutionary Biology is purely descriptive, offering no biologic mechanism to explain Natural Selection.

On the other hand, as mentioned earlier, contemporary developmental biology is predicated on the functions of growth factors and their receptors as the determinants of morphogenesis. The big breakthrough in molecular embryology occurred in the late 1970s with the discovery that soluble growth factors and their receptors underlie and mediate the patterns of development. And developmental physiology as the outcome of embryonic development acknowledged that the denouement of development is integrated homeostasis. Recognition of such developmental and homeostatic mechanisms as a continuum provides deep insight into the mechanisms of evolution. By superimposing cell-cell signaling on conventional ways of thinking about descriptive evolution, one can begin to understand such otherwise nebulous terms and concepts as Survival of the Fittest, Descent with Modification, Natural Selection, the Biogenetic Law, Spemann Organizers, Canalization, Genetic Assimilation, Exaptation, Modularity, Evolvability, Systems Biology, Developmental Systems Theory, Pleiotropy, etc, etc.

Conrad Waddington invoked Canalization, aka homeostasis, in the context of evolution. When a Cell Biologist looks at Waddington’s adaptive landscapes, which resemble tents, supporting poles and all, they want to look under the canvas and see what has caused those hills and valleys (I know I do). In so doing, they have been able to determine the cellular/molecular basis for morphogenesis, which is where evolutionists began in the 19th century, but were unable to provide the mechanistic basis for Haeckel’s Biogenetic Law or Spemann’s Organizer. So the geneticists wrested the subsequent inquiry into evolution from the embryologists, and have been reducing Evolutionary Biology to mutation and selection ever since. Cell biology has literally been eliminated from Evolution Theory for these historic reasons, yet it has revealed how single cells can create whole organisms, much the same as evolution has. And suffice it to say that evolutionists are not trained in Cell Biologic methods. Therefore, it would seem productive to let the Cell Biologists back into the tent. How would this advance our understanding of the mechanisms of evolution? Perhaps by addressing some of the major concepts in Evolution Theory in cellular terms (see above), we may see how developmental biology would facilitate our thinking in this field, which has the potential for being the basis for a unifying theory of biology in practice, as well as in principle- science is deductive, not inductive. We suffer from too many metaphors and too few experimentally refutable hypotheses.

One often reads of molecular biologists alluding to the highly conserved nature of genes of interest as validation for their relevance to some biologic process or structure, but what does that mean functionally? That it is expressed far back in the history of the organism, inferring that it has been present through much of the evolution of the species. But rarely if ever is this pursued mechanistically in order to determine how and why such a conserved gene was involved in the evolutionary mechanism. Other than the process of development, there is no system in which to test such mechanisms.

Although this is a simple concept, there was considerable difficulty in actually executing studies based on the idea. Development and evolution certainly offer a facile sort of analogy to each other: both are processes of change. Although this analogy was compelling during the nineteenth century, it was sterile until the developmentalists discovered soluble growth factors and their cognate receptors, which were able to mediate the spatiotemporal aspects of the developmental process. Development is a programmed and reproducible process. If we accept Darwinian mutation and selection, evolution can be neither. Evolution can consist of internal and external selection, with internal stability being homeostasis, which can exhibit ‘reaction norms’ that are heritable based on the Baldwin Effect. The process of evolution is described as “emergent and contingent”. Canalization can be seen in the context of homeostatic regulation, which, when it fails, can generate cryptic genes that represent the history of the organism, now reprised to provide a physiologic ‘safety net’ that allows for the healing to occur; as such, it allows for reproduction even in the face of illness. The apparent inevitability of development was daunting. To connect it effectively with evolution , two major ideas had to be accepted. The first, pointed out by Garstang, is that the larval stages also face the rigors of life (reminiscent of the Barker Hypothesis, that adult diseases originate in utero). Mendelian genetics allows new traits to appear at any developmental stage, and natural selection potentially operates upon them as it does upon traits expressed in adults. The second major point is that although ontogeny appears inevitable and inextricably orchestrated in its flow, it is not a single process. There are a large number of processes at work, some more or less coupled to others. It was Joseph Needham who, in 1933, using an engineering metaphor of shafts, gears and wheels, suggested the idea of dissociability of elements of the developmental machinery. He pointed out that it is possible to experimentally separate differentiation from growth or cell division, biochemical differentiation from morphogenesis, and some aspects of morphogenesis from one another. The implication of this idea is enormous: developmental processes could be dissociated in evolution to produce novel ontogenies out of existing processes, as long as an integrated developmental program and organismal function could be maintained.

Epistemology- Maybe We Got it Backwards?

The integrated mechanism for physiology has long been accepted to be a fait accompli, yet we know that there are processes of development, evolution and regeneration-repair that comply with some unknown, underlying bauplan. The recent experimental evidence for the complete metazoan toolkit being present in the unicellular state of sponges provides the rationale for such an integration of structure and function, by definition. Mechanistically, the insertion of cholesterol in the plasma membrane of eukaryotes facilitated endocytosis, locomotion and respiration, providing the impetus for their evolution. Moreover, it is striking that the cytoskeleton collectively mediates homoestasis, mitosis and meiosis alike, suggesting the phenotypic autonomy of these unicellular organisms. The significance of this is evinced by subjecting yeast, the simplest eukaryotes, to microgravity, causing both loss of polarity and failure to bud. Without polarity, there is no calcium flux or reason to locomote- where is up, down, sideways? and budding is the reproductive strategy of yeast- loss of these fundamental traits by ‘disorienting’ the cytoskeleton underscores the adaptation to the one element in the environment that is omnipresent, unidirectional and was there from the inception of the planet. So perhaps multicellularity was merely the eukaryotic ploy used to combat lateral inheritance, biofilm and quorum sensing in our age-old competitors, prokaryotes.


 The multicellular form may merely be a derivative of the unicellular state, acting as a matrix for it to monitor the on-coming environment so that the gene pool knows what epigenetic marks acquired during the multicellular phase of the life cycle to include or exclude in the next generation. For example, Dictyostelium exists in two forms, a free-swimming amoeboid form and a colonial Fruiting Body. Under conditions of abundant nutrients, the Slime Mold remains in its free-swimming amoeboid form; under low food abundance conditions, the amoeboid forms colonies. Logic would dictate that this organism evolved under high nutrient abundance conditions, and therefore its unicellular form is the primary phenotype, the colonial form being derivative.

We need to better understand evolution from its unicellular origins as the Big Bang of biology.

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