The Embryo Molecular Mechanics of the Sensory Humans (I)

A BRIEF HISTORY OF EMBRYOLOGY

The process of progressing from a single cell through the period of establishing organ primordia (the first 8 weeks of human development) is called the period of embryogenesis (sometimes called the period of organogenesis); the period from that point on until birth is called the fetal period, a time when differentiation continues while the fetus grows and gains weight. Scientific approaches to study embryology have progressed over hundreds of years. Not surprisingly, anatomical approaches dominated early investigations. Observations were made, and these became more sophisticated with advances in optical equipment and dissection techniques. Comparative and evolutionary studies were part of this equation as scientists made comparisons among species and so began to understand the progression of developmental phenomena. Also investigated were offspring with birth defects and these were compared to organisms with normal developmental patterns. The study of the embryological origins and causes for these birth defects was called teratology.
In the 20th century, the field of experimental embryology blossomed. Numerous experiments were devised to trace cells during development to determine their cell lineages. These approaches included observations of transparent embryos from tunicates that contained pigmented cells that could be visualized through a microscope. Later, vital dyes were used to stain living cells to follow their fates. Still later in the 1960s, radioactive labels and autoradiographic techniques were employed. One of the first genetic markers also arose about this time with the creation of chick-quail chimeras. In this approach, quail cells, which have a unique pattern to their heterochromatin distribution around the nucleolus, were grafted into chick embryos at early stages of development. Later, host embryos were examined histologically, and the fates of the quail cells were determined. Permutations of this approach included development of antibodies specific to quail cell antigens that greatly assisted in the identification of these cells. Monitoring cell fates with these and other techniques provides valuable information about the origins of different organs and tissues.

Grafting experiments also provided the first insights into signaling between tissues. Examples of such experiments included grafting the primitive node from its normal position on the body axis to another and showing that this structure could induce a second body axis. In another example, employing developing limb buds, it was shown that if a piece of tissue from the posterior axial border of one limb was grafted to the anterior border of a second limb, then digits on the host limb would be duplicated as the mirror image of each other. This posterior signaling region was called the zone of polarizing activity (ZPA), and it is now known that the signaling molecule is called sonic hedgehog (SHH).

About this same time (1961), the science of teratology became prominent because of a drug called thalidomide that was given as an antinauseant and sedative to pregnant women. Unfortunately, the drug caused birth defects, including unique abnormalities of the limbs in which one or more limbs was absent (amelia) or was lacking the long bones such that only a hand or foot was attached to the torso (phocomelia; Fig. 1.2). The association between the drug and birth defects was recognized independently by two clinicians, W. Lenz and W. McBride and showed that the conceptus was vulnerable to maternal factors that crossed the placenta. Soon, numerous animal models demonstrating an association between environmental factors, drugs, and genes provided further insights between developmental events and the origin of birth defects.

Figure 1.2 Child with phocomelia (absence of the long bones of the limb) caused by the drug thalidomide.

Today, molecular approaches have been added to the list of experimental paradigms used to study normal and abnormal development. Numerous means of identifying cells using reporter genes, fluorescent probes, and other marking techniques have improved our ability to map cell fates. Using other techniques to alter gene expression, such as knockout, knock-in, and antisense technologies has created new ways to produce abnormal development and allowed the study of a single gene’s function in specific tissues. Thus, the advent of molecular biology has advanced the field of embryology to the next level, and as we decipher the roles of individual genes and their interplay with environmental factors, our understanding of normal and abnormal developmental processes progresses.

In 2011, embryology got the most significant breakthrough. In 1989, Mr. Yuan Lin, who is in charge of the project of human evolutionary researches, discovered the Energy in Absolute Homeostasis and utilized the Absolute Homeostasis Energy Source to activate many kinds of DNA sequences in the body and controlled the process of special amino acid sequences and peptides of protein, and led cellular division, reproduction, and new organelle formation to give sudden rise to the whole functional new human line. Following the functional classification the new human line is: Eukarya Homeostasis/ Dual Synthetic Animalia/ Strong Chordata/Photosynthetic Mammalia/ Control Energy/ Mixotroph Hominidae/ Photoenergy Hominini /Yuan Homo/Sensory Humans.
The discovery of the Absolute Homeostasis Energy Source, the combination of broad activation of Linyuan mechanics with molecular biology and the sudden rise of the new human line open the new area in embryology. Human beings can utilize the Energy in Absolute Homeostasis to activate many kinds of DNA sequences appearance under no molecular weight change, no structure change, no shape change, at temperature 25℃, atmosphere 1.0 atm, pH =7.0, and isolated closed space, no catalyst, no biological active substances, no chemical substances, and no physical functional force to contact. Thereby change the normal development of embryo and modify abnormal development and proceed the producing of the exceedingly perfect new human line; this complete new area is the embryo molecular mechanics of the Sensory Humans.

Before explanation of the embryo molecular mechanics, there are the basic courses of molecular biology in embryology, such as: molecular regulation and signaling, gene transcription, other regulators of gene expression, induction and organ formation, and cell signaling such as fibroblast growth factor (FGF), Hedgehog proteins, TGFβ(transforming growth factor-β) super family, and WNT(wingless in Drosophilia) proteins etc. And signal transduction pathways that include paracrine signaling and juxtacrine signaling are the requisite basic knowledge. They are separately explained as follows:

INTRODUCTION TO MOLECULAR REGULATION AND SIGNALING     

Molecular biology has opened the doors to new ways to study embryology and to enhance our understanding of normal and abnormal development. Sequencing the human genome, together with creating techniques to investigate gene regulation at many levels of complexity, has taken embryology to the next level. Thus, from the anatomical to the biochemical to the molecular level, the story of embryology has progressed, and each chapter has enhanced our knowledge.

There are approximately 35,000 genes in the human genome, which represents only one third of the number predicted prior to completion of the Human Genome Project. Because of various levels of regulation, however, the number of proteins derived from these genes is closer to the original predicted number of genes. What has been disproved is the one-gene-one-protein hypothesis. Thus, through a variety of mechanisms, a single gene may give rise to many proteins.

Gene expression can be regulated at several levels: (1) different genes may be transcribed, (2) nuclear deoxyribonucleic acid (DNA) transcribed from a gene may be selectively processed to regulate which RNAs reach the cytoplasm to become messenger RNAs (mRNAs), (3) mRNAs may be selectively translated, and (4) proteins made from the mRNAs may be differentially modified.

Gene Transcription

Genes are contained in a complex of DNA and proteins (mostly histones) called chromatin, and its basic unit of structure is the nucleosome (Fig. 1.3). Each nucleosome is composed of an octamer of histone proteins and approximately 140 base pairs of DNA. Nucleosomes themselves are joined into clusters by binding of DNA existing between nucleosomes (linker DNA) with other histone proteins (H1 histones; Fig. 1.3). Nucleosomes keep the DNA tightly coiled, such that it cannot be transcribed. In this inactive state, chromatin appears as beads of nucleosomes on a string of DNA and is referred to as heterochromatin. For transcription to occur, this DNA must be uncoiled from the beads. In this uncoiled state, chromatin is referred to as euchromatin.

Figure 1.3 Drawing showing nucleosomes that form the basic unit of chromatin. Each nucleosome consists of an octamer of histone proteins and approximately 140 base pairs of DNA. Nucleosomes are joined into clusters by linker DNA and other histone proteins.

Genes reside within the DNA strand and contain regions called exons, which can be translated into proteins, and introns, which are interspersed between exons and which are not transcribed into proteins (Fig. 1.4). In addition to exons and introns, a typical gene includes the following: a promoter region that binds RNA polymerase for the initiation of transcription; a transcription initiation site; a translation initiation site to designate the first amino acid in the protein; a translation termination codon; and a 3’ untranslated region that includes a sequence (the poly A addition site) that assists with stabilizing the mRNA, allows it to exit the nucleus, and permits it to be translated into protein (Fig. 1.4). By convention, the 5’ and 3' regions of a gene are specified in relation to the RNA transcribed from the gene. Thus, DNA is transcribed from the 5’ to the 3’ end, and the promoter region is upstream from the transcription initiation site (Fig. 1.4). The promoter region, where the RNA polymerase binds, usually contains the sequence TATA, and this site is called the TATA box (Fig. 1.4). In order to bind to this site, however, the polymerase requires additional proteins called transcription factors (Fig. 1.5). Transcription factors also have a specific DNA binding domain plus a transactivating domain that activates or inhibits transcription of the gene whose promoter or enhancer it has bound. In combination with other proteins, transcription factors activate gene expression by causing the DNA nucleosome complex to unwind, by releasing the polymerase so that it can transcribe the DNA template, and by preventing new nucleosomes from forming.

Figure 1.4 Drawing of a "typical" gene showing the promoter region containing the TATA box; exons that contain DNA sequences that are translated into proteins; introns; the transcription initiation site; the translation initiation site that designates the code for the first amino acid in a protein; and the 3; untranslated region that includes the poly A addition site that participates in stabilizing the mRNA, allows it to exit the nucleus, and permits its translation into a protein.

Figure 1.5 Drawing showing binding of RNA polymerase II to the TATA box site of the promoter region of a gene. This binding requires a complex of proteins plus an additional protein called a transcription factor. Transcription factors have their own specific DNA binding domain and function to regulate gene expression.

Enhancers are regulatory elements of DNA that activate utilization of promoters to control their efficiency and the rate of transcription from the promoter. Enhancers can reside anywhere along the DNA strand and do not have to reside close to a promoter. Like promoters, enhancers bind transcription factors (through the transcription factor’s transactivating domain) and are used to regulate the timing of a gene’s expression and its cell-specific location. For example, separate enhancers in a gene can be used to direct the same gene to be expressed in different tissues. The PAX6 transcription factor, which participates in pancreas, eye, and neural tube development, contains three separate enhancers, each of which regulates the gene’s expression in the appropriate tissue. Enhancers act by altering chromatin to expose the promoter or by facilitating binding of the RNA polymerase. Sometimes enhancers can inhibit transcription and are called silencers. This phenomenon allows a transcription factor to activate one gene while silencing another by binding to different enhancers. Thus, transcription factors themselves have a DNA binding domain specific to a region of DNA plus a transactivating domain that binds to a promoter or enhancer and activates or inhibits the gene regulated by these elements.

Other Regulators of Gene Expression

The initial transcript of a gene is called nuclear RNA (nRNA) or sometimes premessenger RNA. nRNA is longer than mRNA because it contains introns that are removed (spliced out) as the nRNA moves from the nucleus to the cytoplasm. In fact, this splicing process provides a means for cells to produce different proteins from a single gene. For example, by removing different introns, exons are “spliced” in different patterns, a process called alternative splicing (Fig. 1.6). The process is carried out by spliceosomes, which are complexes of small nuclear RNAs (smRNA) and proteins that recognize specific splice sites at the 5’ or 3’ ends of the nRNA. Proteins derived from the same gene are called splicing isoforms (also called splice variants or alternative splice forms), and these afford the opportunity for different cells to use the same gene to make proteins specific for that cell type. For example, isoforms of the WT1 gene have different functions in gonadal versus kidney development.

Figure 1.6 Drawing of a hypothetical gene illustrating the process of alternative splicing to form different proteins from the same gene. Spliceosomes recognize specific sites on the initial transcript of nuclear RNA from a gene. Based on these sites, different introns are “spliced out” to create more than one protein from a single gene. Proteins derived from the same gene are called splicing isoforms.

Even after a protein is made (translated), there may be posttranslational modifications that affect its function. For example, some proteins have to be cleaved to become active, or they might have to be phosphorylated. Others need to combine with other proteins or be released from sequestered sites or be targeted to specific cell regions. Thus, there are many regulatory levels for synthesizing and activating proteins, such that although only 35,000 genes exist, the potential number of proteins that can be synthesized is probably closer to three times the number of genes.

Induction and Organ Formation

Organs are formed by interactions between cells and tissues. Most often, one group of cells or tissues causes another set of cells or tissues to change their fate, a process called induction. In each such interaction, one cell type or tissue is the inducer that produces a signal, and one is the responder to that signal. The capacity to respond to such a signal is called competence, and competence requires activation of the responding tissue by a competence factor. Many inductive interactions occur between epithelial and mesenchymal cells and are called epithelial-mesenchymal interactions (Fig. 1.7). Epithelial cells are joined together in tubes or sheets, whereas mesenchymal cells are fibroblastic in appearance and dispersed in extracellular matrices (Fig. 1.7).

xamples of epithelial-mesenchymal interactions include the following: gut endoderm and surrounding mesenchyme to produce gut-derived organs, including the liver and pancreas; limb mesenchyme with overlying ectoderm (epithelium) to produce limb outgrowth and differentiation; and endoderm of the ureteric bud and mesenchyme from the metanephric blastema to produce nephrons in the kidney. Inductive interactions can also occur between two epithelial tissues, such as induction of the lens by epithelium of the optic cup. Although an initial signal by the inducer to the responder initiates the inductive event, cross-talk between the two tissues or cell types is essential for differentiation to continue (Fig. 1.7, arrows).

Figure 1.7 Drawing illustrating an epithelial-mesenchymal interaction. Following an initial signal from one tissue, a second tissue is induced to differentiate into a specific structure. The first tissue constitutes the inducer, and the second is the responder. Once the induction process is initiated, signals (arrows) are transmitted in both directions to complete the differentiation process.

Cell Signaling

Cell-to-cell signaling is essential for induction, for conference of competency to respond, and for cross-talk between inducing and responding cells. These lines of communication are established by (1). paracrine interactions, whereby proteins synthesized by one cell diffuse over short distances to interact with other cells, or by (2). juxtacrine interactions, which do not involve diffusable proteins. The diffusable proteins responsible for paracrine signaling are called paracrine factors or growth and differentiation factors (GDFs). There are a large number of factors, most are grouped into four families, and members of these same families are used repeatedly to regulate development and differentiation of organ systems. Furthermore, the same GDFs regulate organ development throughout the animal kingdom. The four groups of GDFs include the fibroblast growth factor (FGF), WNT, hedgehog, and transforming growth factor-β families.

FGFs

Originally named because they stimulate the growth of fibroblasts in culture, there are now approximately two dozen FGF genes that have been identified, and they can produce hundreds of protein isoforms by altering their RNA splicing or their initiation codons. FGF proteins produced by these genes activate a collection of tyrosine receptor kinases called fibroblast growth factor receptors (FGFRs). In turn, these receptors activate various signaling pathways. FGFs are particularly important for angiogenesis, axon growth, and mesoderm differentiation. Although there is redundancy in the family, such that FGFs can sometimes substitute for one another, individual FGFs may be responsible for specific developmental events. For example, FGF8 is important for development of the limbs and parts of the brain.

Hedgehog Proteins

The hedgehog gene was named because it coded for a pattern of bristles on the leg of Drosophila that resembled the shape of a hedgehog. In mammals, there are three hedgehog genes, Desert, Indian, and sonic hedgehog. Sonic hedgehog is involved in a number of developmental events including limb patterning, neural tube induction and patterning, somite differentiation, gut regionalization, and others. The receptor for the hedgehog family is Patched, which binds to a protein called Smoothened. The Smoothened protein transduces the hedgehog signal, but it is inhibited by Patched until the hedgehog protein binds to this receptor. Thus, the role of the paracrine factor hedgehog in this example is to bind to its receptor to remove the inhibition of a transducer that would normally be active, not to activate the transducer directly.

WNT Proteins

There are at least 15 different WNT genes that are related to the segment polarity gene, wingless in Drosophilia. Their receptors are members of the frizzled family of proteins. WNT proteins are involved in regulating limb patterning, midbrain development, and some aspects of somite and urogenital differentiation among other actions.

The TGF-β Superfamily

The TGF-β superfamily has more than 30 members and includes the transforming growth factor-βs, the bone morphogenetic proteins, the activin family, the Müllerian inhibiting factor (MIF, anti- Müllerian hormone), and others. The first member of the family, TGF-β1, was isolated from virally transformed cells.

GF-β members are important for extracellular matrix formation and epithelial branching that occurs in lung, kidney, and salivary gland development. The BMP family induces bone formation and is involved in regulating cell division, cell death (apoptosis), and cell migration among other functions.

Signal Transduction Pathways

Paracrine Signaling

Figure 1.8 Drawing of a typical signal transduction pathway involving a ligand and its receptor. Activation of the receptor is conferred by binding to the ligand. Typically, the activation is enzymatic involving a tyrosine kinase, although other enzymes may be employed. Ultimately, kinase activity results in a phosphorylation cascade of several proteins that activates a transcription factor for regulating gene expression.

Juxtacrine Signaling

Juxtacrine signaling is mediated through signal transduction pathways as well but does not involve diffusable factors. Instead, there are three ways juxtacrine signaling occurs: (1) A protein on one cell surface interacts with a receptor on an adjacent cell in a process analogous to paracrine signaling (Fig. 1.8). The Notch pathway represents an example of this type of signaling. The Notch receptor protein extends across the cell membrane and binds to cells that have Delta, Serrate, or Jagged proteins in their cell membranes. Binding one of three kinds of proteins to Notch causes a conformational change in the Notch protein such that part of it on the cytoplasmic side of the membrane is cleaved. The cleaved portion then binds to a transcription factor to activate gene expression. Notch signaling is especially important in neuronal differentiation, blood vessel specification, and somite segmentation. (2) Ligands in the extracellular matrix secreted by one cell interact with their receptors on neighboring cells. The extracellular matrix is the milieu in which cells reside. This milieu consists of large molecules secreted by cells including collagen, proteoglycans (chondroitin sulfates, hyaluronic acid, etc.), and glycoproteins, such as fibronectin and laminin. These molecules provide a substrate for cells on which they can anchor or migrate. For example, laminin and type IV collagen are components of the basal lamina for epithelial cell attachment, and fibronectin molecules form scaffolds for cell migration. Receptors that link extracellular molecules such as fibronectin and laminin to cells are called integrins. These receptors “integrate” matrix molecules with a cell’s cytoskeletal machinery (e.g., actin microfilaments) thereby creating the ability to migrate along matrix scaffolding by using contractile proteins, such as actin. Also, integrins can induce gene expression and regulate differentiation as in the case of chondrocytes that must be linked to the matrix to form cartilage. (3) There is direct transmission of signals from one cell to another by gap junctions. These junctions occur as channels between cells through which small molecules and ions can pass. Such communication is important in tightly connected cells like epithelia of the gut and neural tube because they allow these cells to act in concert. The junctions themselves are made of connexin proteins that form a channel, and these channels are “connected” between adjacent cells.

It is important to note that there is a great amount of redundancy built into the process of signal transduction. For example, paracrine signaling molecules often have many family members such that other genes in the family may compensate for the loss of one of their counterparts. Thus, the loss of function of a signaling protein through a gene mutation does not necessarily result in abnormal development or death. In addition, there is cross-talk between pathways, such that they are intimately interconnected. These connections provide numerous additional sites to regulate signaling.(Medical Embryology,p.4 to10).

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