Embryo Development in a Stochastic Universe

Abstract

Despite the elucidation of the many processes by which a single eukaryotic cell develops into a complex mature organism, it is still puzzling to some biologists how it is that an unvarying, interconnected set of processes becomes coordinated and insulated from a stochastic universe. This article suggests that electromagnetic processes deriving from the chemistry of an organism may provide such coordination. Specifically, the author develops the pacemaker concept, the periodic, autonomous electrical signal to the entire embryo, the result of which, after each pulse, is to alter or enlarge the transcriptome to produce the next level of complexity and maturity of the organism.

Introduction

The last two decades have witnessed an accumulation of information on the dynamics of embryo development. New techniques have connected processes with one another in the development pageant. Yet, intuitively, some investigators have wondered if an organizational principle governs the unvarying evolution in time and space to an “end product” in all of the eukaryotic species.

The author states at the outset that a teleologic doctrine is not being advanced. Such a doctrine postulates that natural phenomena are shaped by ends or final goals. The author does not assert that there is a “deus ex machina” or “mind” at work. Quite to the contrary, the “blind” processes of evolution are operative. For example, the evolution in size and complexity from single-celled organisms to metazoan structures is enabled by the development of a primitive circulatory system, which evolves a pumping mechanism to convey, among other necessities, oxygen and constitutes an evolutionary advantage.

Presumptively, all interactions between molecules are governed by short-range forces, especially van der Waals forces. These forces underlie the complex interactions of biochemical cycles that drive metabolism and development in structures soon to evolve into separate cells and then tissues.

This article suggests that there is a conceptual problem in mainstream embryology that most developmental biologists would not accept as a problem and would consider the proposed “solution” as being in search of a problem. The mainstream view is that if biochemical steps initiate each other and each one takes some “standard amount of time,” they end up in precise temporal order. The critical factor is the standard amount of time. Such a view does not accord with the concepts of statistical mechanics, which preoccupies itself with averages and deviations in the evolution of material systems in time and space. In the author’s view, the issue remains open, and the following paragraphs follow a more thermodynamic or statistical mechanical view.

A biophysical process, an electrodynamic process, projecting long-range forces across clusters of cells and across organ systems could supply the necessary coordination. The cardiovascular and nervous systems are examples in developed organisms. As argued in this article, the cardiovascular system can be projected “backwards” conceptually to a simpler system in the embryo, bits and pieces of which are discernible as far back as the zygote and blastula stages. Such a system is conceived as an early pacemaker, critical to the process of zygotic genome activation (ZGA) through its interaction with the DNA-core-promoter complex. The idea depends on the ability of DNA to conduct current, resulting in the formation of transcription bubbles and RNA synthesis in a choreographed order.

Goldbeter¹ has analyzed the interaction of multiple oscillatory processes in biological systems. These processes make possible the survival of an organism. Examples would include the cardiac cycle, the mitotic cycle, and numerous metabolic cycles.

In systems that are evolving from a “simple” entity, a zygote, into a continuously changing, developing, enlarging structure, having component nascent parts, the integration of thousands of individual reactions across organ systems in precise temporal order, without a “guiding mechanism,” is hard to conceive.

What coordinates the architectural development of the liver, kidney, and central nervous and cardiac systems such that each stage of their development is precisely coordinated in time with the other systems? This process is occurring in organ systems that are increasingly physically compartmented from each other.

This article endeavors to show that there is a process at work to “solve” the problem of the superseding of stochasticity. The process is already partly visible in experimental observations of cell-wide and tissue-wide electromagnetic phenomena.²

Transcription Regulatory Networks

An entrée to the “control against chaos” problem lies in the increased understanding of transcription regulatory networks, further characterized as “transcriptional regulator modules” (TRMs). A TRM is a set of genes that is regulated by a common set of “transcription factor” (TF) proteins,³ as seen in Figure 1.

FIG. 1.

The mapping of physical interactions between transcription factors (TFs) and their target genes has resulted in the discovery of several interesting network features, some of which are shown here. Some TFs (circles) target a disproportionately large number of genes (diamonds) and are referred to as TF hubs. Sets of TFs that share many target genes are referred to as TF modules. Target gene hubs interact with a disproportionately large number of TFs. (The Royal Society of Chemistry has kindly granted permission for the use of this figure, which originally appeared in the journal cited in the reference to Grove and Walhout.³).

By organizing the genome into TRMs, a cell and sets of cells can coordinate the activities of many genes and “orchestrate” their proper locus in time. TFs bind to a core-promoter-RNA polymerase II (RNA pol II) complex that is located at a TF start site (TSS). The binding facilitates the initiation of transcription at the TSS. However, the facilitation is necessary but not sufficient for transcription. The final step in the hypothesis describes how double-stranded DNA is separated, that is, the production of bubble formation, to allow RNA pol II to initiate transcription and synthesis of mRNA. The mRNA authors new TFs, and this is performed in a coordinated fashion with other defined sites to maintain temporal coordination at every step of embryogenesis. We develop the pacemaker concept, the periodic, autonomous electrical signal to the entire embryo, the result of which, after each pulse, is to alter or enlarge the transcriptome to produce the next level of complexity and maturity of the organism.

There develops an orderly sequence of appearance of mRNAs, with an orderly genesis of one structure or function after another, as illustrated in Figure 2. The genesis and nature of the periodic pulse are presented below, after a brief commentary on ZGA.

FIG. 2.

Illustrates the quantized steps in time, times t₁ t₂ t_3, etc., by a sequence of electric field pulses at the horizontal lines. The pulses originate from a pacemaker and spread through the developing embryo. For the spatial context, one can view an embryonic pacemaker as a small spherical object, containing several cells, radiating a spherical potential, and accompanying gradient force, into surrounding concentric layers of embryonic cells. Each pulse results in a change (enlargement) in the transcriptome, integral to the development of the embryo. Cell division times would depend on the transcriptome at each step. The location of the pacemaker after the zygote stage is not speculated upon but would be presumed to remain at a central location in the developing embryo. Pioneer transcription factors (TFs) would segregate to different cytoplasmic locations in the zygote in anticipation of distribution to different descendant cells and subsequent differentiation. Circles are TFs and diamonds are genes. The vast number of intermediate steps are omitted to highlight the temporal sequence of the hypothesis. The model envisions only one pacemaker, which has no intrinsic informational content except its repetition rate. Different parts of the embryo are developed in accordance with the location of the pioneer TFs in the zygote.

Zygotic genome activation

Prior to ZGA, early embryos replicate DNA and divide, with no interlude between early divisions. With the onset of ZGA, the cell cycle lengthens to accommodate ZGA, which is characterized by the generation or activation of mRNA and proteins originating from the embryonic genome. These genome-derived elements “take over” embryonic development as maternal-derived mRNAs and proteins disappear.

There is a unique category of TFs, the “pioneer” TFs, which are found in the maternal oocyte,⁴ having developed over time in the ovaries as a result of maternal hormone stimulation. They are loosely equivalent to the TFs of the TF modules identified in Figure 1. They are unique from all other TFs, which are descended from them. The descendant TFs are generated de novo from the embryonic genome. The TFs of genomic origin can either be used universally in many sites and developing organs or will operate on specific genes unique to specific organs and control the subsequent expression of hundreds of genes during the major and minor waves of genes, which are the architects of defined structures and functions.

In flies, the TF Zelda (ZLD), a pioneer TF, is deposited in the zygote from the female gamete and is the precursor of hundreds of TFs in the developing embryo, which will orchestrate gene expression downstream. Nanog, SoxB1, and Pou5f3 are pioneer factors in zebrafish ZGA.⁴ Some pioneer TFs are known to be maternally deposited as mRNAs and later undergo translation in the zygote.

Of potential importance is that, in distinction from other TFs, pioneer factors physically bind to the genomic DNA in the promoter region for a period prior to the binding of other factors and prior to activation.⁵ They therefore assist in defining gene specificity along with other parts of the promoter. They also assist, as part of the TF hub, in the first or prior activation of genes which will code for TFs downstream involved in the fine structure of organ system development.

Dynamics of the Preinitiation Complex

The specific order of interactions, the binding and stability of enhancers, TFs, general transcription factors, associated cofactors, and RNA pol II are complex and vary with organisms. These complexes are constantly associating and breaking down in a stochastic fashion.^6,7 The proposed electric pulse would have a slower period compared with these stochastic processes.

The postulate is that a cyclic electric field, acting as a pacemaker, interacts with a fully primed core promoter, associated proteins, and RNA pol II to separate core-promoter-RNA pol II-bound DNA strands at the TSS, producing “bubble” formation and RNA synthesis. One previous hypothesis, among several, as to what generates the bubble is that a subunit of TFII acts as a translocase enzyme that drives DNA opening by threading downstream DNA into a Pol II cleft.⁸ The electric field pulse hypothesis has the advantage of coordinating many specific processes at a precise time in embryo development.

RNA pol II dissociates from the TSS just after the initiation of transcription, about 30–50 nucleotides downstream of the TSS, stopping transcription, in various animal systems, a process known as promoter-proximal pausing. This process repeats with continued transcription and may also be influenced by an electric field pulse. It has been observed in early fly embryos that promoters with paused RNA Pol II are activating synchronously across many cells, which may be important for coordinating tissue morphogenesis.⁹

The temporal process proposed would produce the next iteration of the transcriptome, providing temporal coordination. It would be “quantized” by the electric pulse. As developed below, the process would be carried out by the migration of electrons along the DNA backbone; such a migration, also described as a current, is driven by the electric field pulse.

Possible Origin of Repeating Electromagnetic Pulses Operating Across the Embryonic Tissue

If such a process existed in an early embryo, how would it work? Could there be a pacemaker in an embryonic system, composed of one or several cells, which could generate a spreading wave of depolarization to adjacent cells and which could affect genetic expression in those cells? Might there be a connection with calcium waves, which affect genetic expression? Such a process would be different from that of the heart’s pacemaker, whose function is to activate the sequence of compressions of the heartbeat. There is no conceptual difficulty with a process subserving different functions at different times, and with such a precept, one could view an embryonic pacemaker as the earliest beginning of a pacemaker. The author makes the assumption that such an embryonic pacemaker itself evolves into the cardiac pacemaker. Phenotypic properties such as voltage-gated calcium and sodium channels, and especially HCN/f “funny currents,” critical to pacemakers, are already present in zygotes, as described below.¹⁰

Looking at a number of different species reaching the somite-formation stage, one sees a multiplication of sinoatrial (SA) node cells. In the chick heart, one finds 60–150 SA node cells at the seven to nine somite stage. Individual SA node cells exhibit spontaneous and uncoordinated membrane depolarizations and then become entrained into synchronous depolarizations. SA node cells also acquire gap junctions during this period. A picture of the communication of a pulse to adjacent cardiomyocytes would show that an SA node cell cohort depolarizes and Na⁺ flows through gap junctions into cardiomyocytes, causing them to depolarize. At a certain level of cardiomyocyte depolarization, voltage-gated L-type Ca⁺⁺ channels open, triggering endoplasmic (sarcoplasmic) reticulum to open their channels, a process known as Ca⁺⁺-induced Ca⁺⁺ release depolarization.¹¹ This process moves as a wave through the myocardium. A very similar process can be envisaged in the early embryo, where, at first, an “SA node-like” cell can serve as an originator of pacemaker-type sequential pulses.

The Nature of an Embryonic “Action Potential” Wave

In the cardiac cycle, the depolarization in the atrium can, as a practical matter, be interpreted as “instantaneous” as well as the following depolarization in the ventricle. If it were not practically instantaneous, the contraction of the heart muscle would not be optimally coordinated. But, in fact, the depolarization travels at a finite speed governed by the dielectric properties of the myocardium. Unlike the dielectric properties of inanimate matter, the myocardial response is very dynamic, producing currents in cardiomyocytes and making the characterization of an action potential waveform difficult. The properties of a waveform in an embryo would also be difficult to explicate, but such a waveform would travel at a finite speed.

We must differentiate the drop in potential across the cell membrane^12,13 from a drop in potential across the cell itself caused by partial depolarization of a cell as an action potential crosses from cell to cell. As it crosses a cell, a gradient electric field rises and falls with the moving potential. Such an electric field is proposed to be essential to the sequence of mRNA synthesis, as proposed below. Calcium flow, arising first on the SA node-facing surface before passing to the opposite surface, produces a momentary capacitative electric field with the cell.

For simplicity, one can view an embryonic pacemaker as a small spherical object radiating a spherical potential into the surrounding concentric layers of embryonic cells. Each layer is traversed by a longitudinal pulse. At a certain location on the cell membrane, the inward face of the plasma membrane on the pacemaker side goes positive, whereas the inward face of the plasma membrane away from the pacemaker is still negative. This momentary gradient electric field across the chromatin reaches a maximum and then disappears until the pacemaker produces the next in a sequence of pulses with a given period. Ion flow from the first concentric layer of cells to the next layer would transmit the pulse and thence to more distal layers in a manner similar to myocardial transmission of an action potential.

Production of Transcription Bubbles

Two features of DNA are of particular relevance to this discussion. (1). The DNA molecule consists of two single strands, which are wrapped around each other in the form of a double helix. (2). The molecule is capable of long-distance charge transfer along its long axis. The first discovery, credited to Watson and Crick, based on a contribution by Rosalind Franklin, can be dated to 1953.¹⁴ Shortly after, the idea that the molecule could act as an electrical conductor was proposed. Over the years, many experiments have confirmed charge transfer over a distance, but the physical mechanisms are not entirely worked out and continue to be the subject of investigations. In particular, whether DNA behaves as a semiconductor, highly or poorly conducting, is an issue. Much of the work using photooxidants has come from the Barton group.^15,16 Current flow was measured between defined segments of DNA suspended between microelectrodes at given potentials.^17,18 Evidence of the current flow was found. These in vitro studies are cited only to suggest evidence of conduction in DNA and nothing further. No resemblance to a putative mechanism in living tissue is implied. Although the authors proposed a semiconductor or conductor mechanism, other theories of a mechanism appear in the active literature of DNA conduction.¹⁵

From electrodynamics, it is known that parallel conductors carrying currents in opposite directions repel each other. Of course, parallel conductors carrying currents in the same direction attract each other.

The forces required to accomplish a mechanical separation of the complementary strands of B-form DNA have been measured and reported to be in the range of 10–15 pN, varying in relation to local GC or AT content.¹⁹

In B-form DNA, the pitch angle, that is, the angle between the surface perpendicular to the long axis of the molecule and the direction of current flow, which is assumed to be in the “pi-way,” is 29°. The component of the currents in each strand, which is parallel to the long axis of the molecule, is assumed to be in the same direction. By examination of Figure 3 (reproduced from Elson²⁰), it is seen that at the stipulated pitch angle, the current in one strand is moving in the opposite direction from that of the other strand, and therefore, the currents tend to repel each other.²⁰

FIG. 3.

In this simple model, B-DNA is reduced to a cylinder composed of a thin wall in which the “pi-way” current pulse flows at angle θ (base pairs and backbone omitted for clarity). Consequently, the pitch angle described in the text is π/2 minus θ. The model assumes a uniform flow in the wall in order to use its symmetry properties to produce a simple and approximate computation for the radial force tending to separate the base pairs. The dotted area signifies a small element of the outer surface of integration used in the calculation presented in Elson.²⁰

One must then confront the “force-compliance” issue, that is, the electric force, which produces the current in the DNA strands, and then the current-bubble formation issue. Here, the in vitro data cannot be any more than merely suggestive. DNA, in vivo, in chromatin structure is conformationally far different from canonical B-form DNA. If anything, it may be “looser” and more susceptible to unwinding and bubble formation. Pioneer factors may contribute to the “most looseness” in that they must express themselves first in any hierarchical process. They control the expression of the master genes, which, through the expression of mRNA, tRNA, or rRNA, can produce subsequent expression of genes producing finer levels of control of developing structures and functions.

The cause of the “first-in-time” expression must reside in several factors, of which important ones are the unique structures of the pioneer factors themselves, the unique structure of their conjugate promoters with their distinct core-promoter motifs, and a unique property of the pioneer factors, namely, their ability to bind to the promoter itself, in distinction to nonpioneer TFs that do not bind to promoters. These factors, including runs of ATs in the DNA, must contribute to the relative “looseness” of the core-promoter-RNAP II complex, leading to the formation of a transcription bubble. The resulting RNA leads to the next generation of gene expression.

There are TFs that control the processes of respiration and metabolism and act in all tissues. There are TFs that operate to control the formation of structural elements in all tissues, and there are TFs that are directed to the development of specific tissues or organs, all of which must be entrained. The concepts proposed can explain the coordination and timing of embryo development. As embryonic organ systems evolve, the evolution of structures and functions in otherwise distant organ systems can be entrained.

Competence of Embryonic Cell Membranes to Carry out Postulated Functions

In order to lay the groundwork for the central hypothesis of this article, namely the existence of a pacemaker as early as the zygote, information is presented as to the existence of membrane-ionic channels, which could generate a pacemaker structure from the zygote stage onwards.

Calcium and sodium channels can be either ligand, stretch, or voltage activated. Voltage-operated/activated channels (VOC) are present in gametes and zygotes of diverse species.^21–23 The types, structures, and dynamics are different in different species. As an example, male rodent germ cells express voltage-operated calcium channels.²⁴ Voltage-gated calcium channels are found as early as the two-cell stage in zebrafish.²⁵ Significant to later discussion, HCN/f “funny current” channels are found in ovarian cells and zygotes as well as embryonic stem cells. Such channels are believed to be critical to pacemaker function.¹⁰ HCN/f channels are found in zebrafish sperm.²⁶ T-type, low-voltage calcium channels are present in mouse spermatic cells.²⁷

The earliest deployment of voltage-activated channels is not clear, except in some species, such as the frog. It is already there in the unfertilized egg and during the cleavage stages. Florman et al.^27,28 report that stimulation by ZP3 in the zona pellucida activates a voltage-insensitive ion channel in the sperm membrane. With the depolarization of the membrane, a low-voltage-activated T-type Ca⁺⁺ channel is activated,²⁷ and then a sustained Ca⁺⁺ elevation leads to the acrosome reaction.²⁷ This may be the very first utilization of a voltage-activated channel in embryogenesis.

In the 1980s, a report appeared describing action potentials and resting potential shifts in the zygotes of the tunicate Clavelina.²⁹ The action potentials were varied, in that a cell would begin firing after a silence, fire up to 20 spikes over a 10–20-min period, and then remain silent for the rest of the microelectrode recording. Firing was not correlated with other changes occurring in the zygote. In some eggs, action potentials preceded first cleavage by 10–20 min but then became electrically silent during the process of cell division. “Spontaneous” action potentials were observed in blastomeres from the 2-cell stage to the 16-cell stage.

Electrical activity of the SA node can first be discerned in the rat embryonic heart at the three-somite stage using voltage-sensitive optical methods,³⁰ followed shortly by the initiation of contraction. In the chick embryo, spontaneous membrane depolarizations of SA node cells are observed in the prefused cardiac primordia at the six and early seven-somite stages. Contractions are observed in the middle period of the nine-somite stage.³¹ Initially, the action potentials are sporadic and then become more coordinated with a low frequency. The frequency increases in the chick embryo from the six- to the nine-somite stage, becoming very regular by the nine-somite stage. Gap junctions form during this period. The initial incoherent and isolated depolarizations of clustered SA node cells appear to become collectively coherent through a process of entrainment.

It is seen that calcium use can be adapted to several different functions using VOC. In the cardiac system, which is a pacemaker-operated system, it is used in the process of excitation-contraction. Ligand-operated channels are used heavily as part of feedback systems for mRNA and DNA synthesis, especially in growth and development. We postulate that VOC channels play a “supervisory” role in growth and development.

Depending on the organism, a large hyperpolarization or depolarization of membrane potential occurs in the oocyte (nascent zygote) shortly after sperm entry. In zebrafish, which do not exhibit an acrosome reaction, the first developmental calcium signal after fertilization is intracellular in nature and propagates as a fast Ca⁺⁺ wave.²⁴ The fast wave utilizes calcium-induced calcium release from the endoplasmic reticulum.²⁴ This intracellular fast wave-generating ability appears to be a fundamental signaling process and evolves into more complex signaling patterns in subsequent developmental stages. With sperm-egg fusion, there follows a series of hyperpolarizations that continue up to pronuclear formation.

Following the formation of the zygote and fast calcium wave in zebrafish, Silic et al.,³² using a genetically stable zebrafish line and a genetically encoded voltage indicator, and using light-sheet microscopy, observed hyperpolarization of forming cleavage furrows preceding cytokinesis, from the first cleavage onward. This process continues through the blastula and gastrula stages. The link to the associated mitoses is not clear.

In contrast, Webb and Miller report on Ca⁺⁺ spikes³³ that, in the cleavage period, are intracellular waves and, as the size of the cells decreases, display fast intercellular waves with a velocity of around 20 microns/sec.³⁴ During the cleavage period, ooplasm moves from the yolk (ooplasmic segregation) to the animal pole to form the blastodisc. This is an early sign of polarization or directionality in the embryo.

Polarization and Directionality

Specializing in zebrafish, the about-to-be-fertilized egg displays an animal and vegetal pole. The micropyle is a single, specialized, somatic follicle cell that forms a narrow canal attached to the animal domain, which is the location of the preblastodisc of the oocyte.³⁵ The micropyle is the site for penetration of the sperm. Following sperm entry, the blastodisc forms and appears to be the next step in the polarization of the egg. The nascent zygote consists of two parts: the blastodisc in the animal region, containing the pronuclei, and a yolk inclusion body within the cytoplasm of the vegetal region. At this time, the first calcium wave appears in the zebrafish.³⁵

Also, at this time, cytoplasmic regions, characterized by distinct collections of mRNA, can be discerned in the preblastodisc and resulting blastodisc. This process appears to set the stage for the allocation of different sets of mRNAs into subsequently formed blastomeres. How this process occurs is not known. Cytoplasm flows from yolk globules into the blastodisc, along with cytoplasmic streaming into the blastodisc. A contractile actin ring appears just under the blastodisc.³⁵

Although the connections to different maternal mRNAs are partially worked out, the directionality and streaming mechanics producing a polarized zygote and subsequent cleavages are not understood. A clue could be internal electric forces produced by the intracellular potentials associated with the calcium flows, which cause irreversible polarization.

Discussion

The incompatibility of biological growth and development with the second law of thermodynamics was addressed in 1941 by Erwin Schrodinger,³⁶ who attempted to resolve the issue by postulating a periodic organizing principle that could “entrain” stochastic processes. Schrodinger was thinking of a physically repeating structure in the chromosomes, whose structure was not understood in his time (before the repeating units of DNA were characterized). With the knowledge of the time, a more concrete, less abstract process could not be envisioned, and the idea was not pursued. A “structure” repeating in time, such as is suggested in this article, could also have been conceived.

The idea of “action at a distance” has appeared in many articles, a few of which are identified here. An early example is that of Spemann’s “Organizer,”³⁷ which described the diffusion of molecules from a central locus to adjacent areas, directing the formation of a number of embryonic structures. Cifra³⁸ collected and reviewed a number of theories in the years preceding 2011. Polesskaya³⁹ advanced a theory utilizing the role of DNA conductivity. Savelev⁴⁰ proposed the existence of genomic elements that serve as antennas in resonance signaling and the formation of a “morphogenic field.” Sun⁴¹ presented a theory of the role of the centrosome in intercellular communication via electromagnetic fields. Levin⁴² has reviewed the issue of electrical communication in embryogenesis.

In the spirit of earlier efforts,⁴³ the author presents a hypothesis, which, like the others, is heuristic, that is, encouraging the opening of new investigations. Without devising novel processes for which there is no established evidence, the proposal takes well-known existing mechanisms and adapts them in novel ways.

Potential Importance of this Research

The author has proposed that dynamical processes may be at work in the early embryo involving the transmission of electrical waves, which could “direct” subsequent cellular proliferation, differentiation, and coordinated organ formation. The author suggests that light sheet microscopy and other approaches may be of use in identifying such waves. This could lead to investigating the possible role of such electromagnetic phenomena.⁴⁴

Authors’ Contributions

The author confirms sole responsibility for the following: paper conception and design, background, collection, analysis and interpretation of findings, and article preparation.

Author Disclosure Statement

The author declares that he has no relevant or material financial interests that relate to the work described in this article.

Funding Information

The author declares no sources of funding.