“The GRN [Gene Regulatory Network] model illustrating the genomic control of 2D expression pattern formation in the sea urchin ectoderm. This model is a BioTapestry presentation of all interactions among regulatory genes governing ectoderm regulatory state diversification up to the onset of gastrulation. The circuits show that domain-specific repressors are commonly used to define the boundaries along both embryonic axes.” From [2] with blanket permission for use “in a review article” per http://www.pnas.org.uml.idm.oclc.org/site/aboutpnas/rightperm.xhtml

Transplantation of a piece of ectoderm containing a portion of the dorsal lip of the blastopore from lightly pigmented embryo a to darkly pigmented embryo b results in a second neural plate ( c ). The result is a double embryo, i.e., conjoined twins ( d , e ). This shows that any portion of the ectoderm is capable of producing a neural plate and subsequent development. In an ordinary, single embryo the dorsal (top) part of the ectoderm produces the brain and spinal cord, while the lower (ventral) hemisphere produces the epidermis, which becomes skin. This sketch, which depicts the original experiments by Hilde Mangold and Hans Spemann [30], is by Victor Twitty. From [182] with permission from Macmillan Education

The nucleus has areas of chromosome territories (colored clouds) and regions that are open containing RNA polymerases (central orange cloud). A specific set of transcription figures creates an enhanceosome which binds to a specific enhancer. It also binds to a realisator gene which is on its own loop divided into introns and exons. If the gene and enhancer is open, as in realisator loop 1, the DNA can be bound to the enhanceosome if the correct transcription factors are present and form a regulatory archipelago. The gene is then transcribed. If the DNA is sequestered, as in Realisator Loops 2, the DNA cannot be bound the enhanceosome even if the correct transcription factors are present. If the DNA is condensed (as in Realisator Loops 3 and 4 and their enhancers) it completely inaccessible

Each cell has the same set of all genes encoded in the DNA (Red circle). All cells in an organism express a set of “Housekeeping” or “Reference” genes. These genes represent certain elements such as ribosomes and components of the DNA polymerases as well as some of the cytoskeletal elements (Brown circle). There are also genes for specific transcription factors that are required for turning off and on gene expression (Blue circle hatched). Transcriptions factor gene products (i.e., proteins that are transcription factors) may be expressed in many different cell types. Many transcriptions factors require the presence of specific regulatory cofactors (Pink circle) such as small RNAs to perform some (or even all) of their functions. Expression of specific transcription factors and their regulatory cofactors often changes during development and thereby changes function of tissue specific genes. Each cell also has a set of genes that are generally specific to the tissue type that the cell belongs to but which may also be expressed in other cell types (Green circle). A Regulon is the group of a single transcription factor and all of its regulatory cofactors required to regulate a specific gene or set of genes. A regulon is defined by the genes affected by it and the regulatory cofactors associated with it and can be different in various cell types or when studied from different perspectives. A given regulon may also be functional in more than one cell type. A Differon is the set of all genes that can be expressed in a specific cell and the differon defines the cell type. All the cells with the same differon are of the same type. (The differon differs from the transcriptome, the set of all mRNA expressed in a cell, because not all genes that can be expressed are always being expressed in a cell. For example, the liver may express certain genes only in response to the presence of specific toxins and so those genes will not appear in the transcriptome if the toxin is absent but those genes are still part of the differon)

The cell state splitter is an organelle predicted [9] and then at first observed at the apical end of ectoderm cells in axolotl embryos at early gastrulation [11]. It consists of an upper microfilament ring with an intermediate filament ring below it, subtended by a mat of microtubules. As shown in the thumbnail in the upper right, the cell state splitter occupies only 1 % of the height of an ectoderm cell. (Below and in the thumbnail, the vertical and horizontal scales are compressed, as 50 μm = 100 × 0.5 μm)

In a cell state splitter, since the outward force due to the apical mat of microtubules (mt) is approximately independent of cell diameter, and the inward acting microfilament ring (mf) force falls of hyperbolically with cell diameter, there should be a cell diameter at which they are in mechanical balance, i.e., F _mt = − F _mf . We assume that the cell state splitter is set up at this equilibrium point. However, this is an unstable equilibrium. At higher diameter F _mt > − F _mf and at lower diameter F _mt < − F _mf . Thus, if the diameter goes higher than the equilibrium diameter, it will keep getting bigger, flattening the cell. If the diameter goes lower than the equilibrium diameter, it will keep getting smaller, turning the cell into a tall, narrow one. Adapted from Fig. 10 in [9]

This signal transduction model is general. There will be replacement or alternate proteins for some of the proteins in our model in some tissues or organisms. As embryogenesis proceeds there will be more feedback loops amplifying the contraction and expansion signals and additional inhibitory or excitatory interactions down the signal transduction pathways. In some cellular differentiations, highly specialized versions of these signal transduction pathways will exist such as where cells only one cell state splitter reaction can actually take place. Nuclear State Splitter Participants: wnt - Name is derived from “Wingless” and formerly considered “The Morphogen”, wnt acts primarily as an autocrine protein which binds one or more transmembrane protein and affects their conformation and phosphorylation and thereby affects signal transduction pathways, often amplifying other signals. FzR-Frizzled transmembrane protein. Cdc42 –a cell cycle protein that takes part in signal transduction pathways and can signal the cell to enter mitosis. It is a member of the small Rho-kinase family that is known to affect microfilament contraction and also can stimulate pathways promoting cell division. A round of determination is often followed by cell proliferation as part of differentiation and this could be via Rho-kinase. PKC – Protein Kinase C is a protein normally found in the cytosol of the cell in an inactive form but when phosphorylated by other signaling molecules, in particular calcium ions and diaglycerols, translocates to the cell membrane where it interacts with other kinases, especially RACK (receptor for activated C Kinase). PKC continues to signal long after the calcium flux has faded and the diaglycerol signal has ended and so can be considered a signal amplifier. CaN - Calcineurin which is known to dephosphorylate the transcription factor NFAT . This causes a conformation change which exposes the NFAT nuclear import signal allowing it move to the nucleus and bind to specific DNA sequences and change gene expression. β-catenin -Caderin associated protein beta one, a dual function protein that is active in cell to cell adhesion as well as gene expression. JNK-c -Jun terminal kinase first found because they bind and phosphorylate c-jun forming a transcriptional activator domain on DNA. JNK is a form of mitogen activated protein. PLC -An enzyme which cleaves PIP2 to form two products, inositol 1,4,5-triphosphate (IP3) and diaglycerol both of which are second messengers that commonly affect opening and closing of membrane channels. Diaglycerol and calcium combined can trigger Protein Kinase C translocation. PIP2 -Phosphatidyinositol 4,5-biphosphate, a phospholipid that is the major constituent of membranes that is cleaved by PLC. Such cleavage can activate PKC. Dvl – Dishevelled, cytoplasmic phosphoprotein that is required for canonical and noncanonical wnt pathway signal transduction and wnt signaling. CK1 - Casein Kinase 1 involved in phosphorylation of Dishevelled. Axin- A dual domain cytoplasmic protein, with one domain binding the disheveled receptor and the other binding G proteins. APC- Adenomatous polyposis coli, so called because when one version is mutated in humans it is connected to development of colon cancer. It is a negative regulator which reduces β-catenin response and is also directly connected to e-cadherin. GSK3 - glycogen synthase kinase 3, phosphorylates β-catenin (thereby signaling for it to be degraded) in response to signaling from Axin. DAAM1 -Disheveled-associated activator of morphogenesis 1, a protein associated with microfilament polymerization, possibly by acting as a scaffold protein. It is activated by Rho. Rho – A member of the Ras homolog gene family, Rho is a kinase that is activated during microfilament contraction by directly stimulating microfilament polymerization undertaken by the formins. Formins recruit free actin monomers which are then used to elongate microfilaments. (They also capture and stabilize free ends of microtubules required for ruffling in forward movements of cells.) ROCK – Rho associated protein kinase, this protein works downstream to Rho and can either trigger stabilization or destabilization of microfilaments. It can also trigger contraction by activating specific myosins. Rac - a serine/threonine-protein kinase that interacts with multiple other kinases. MAPK – Mitogen activated protein type K. MAPs were originally called microtubule associated proteins because when they phosphorylate another set of proteins, MAP, that bind to microtubules. MAP - microtubule associated protein, bind to microtubules and can either stabilize or destabilize microtubules depending on phosphorylation signals from other proteins. When signalled by wnt signal transduction they generally stabilize and elongate microtubules

This diagram summarizes how the ectoderm contraction wave moves across an axolotl embryo, as seen from the left side. It starts at a point that spreads to a circle (hour #1), breaking into an arc (hour #3) that moves over one hemisphere, finishing off (hour #10) as a circle that comes down to a point and vanishes. From [10] with permission of John Wiley and Sons. This figure is reproduced and discussed in [183]. This wave is a furrow 0.1 mm wide and deep on this 2 mm diameter embryo propagating around 3 μm/min

This differentiation tree indicates tissues determined from cleavage to the end of gastrulation. All tissues types should be considered “presumptive”, meaning that their names refer to the kinds of tissues that form from them. Red, shift to right and the letter C indicate a contraction wave and green, shift to right, and the letter E an expansion wave. Staging numbers and hours since fertilization on the left and right are from [184] and indicate the duration of each wave. Note each wave may pass through one tissue and continue into an adjacent tissue and so, for example, E3 begins endodermal tissue and then continues into mesodermal tissue