6 Early development


photo of the sun
Figure 6.1: From this photo, the polarity of the Sun is probably not recognizable. Image credit: "The Sun photographed at 304 angstroms" is in the public domain CC0

Overview of early development

Human is the process where a single cell, a visually unremarkable cell except for perhaps its size, slowly changes into thirty seven trillion different cells. is the study of the phases of development, especially the early phases where most of the interesting stuff happens. A round cell, just a ball with no sides and no special parts, grows into an organism with different organs in different places. Fig. 6.1 is the Sun. You see it every day, can you tell whether the photo is right-side up? Is there even a right-side up? We are not astronomers, but the Sun looks a bit like a fertilized egg, and in Fig. 6.1 there are no obvious landmarks to orient our view. An egg has a similar (without shape) appearance, but it (smoothly changes shape) into the embryo shown in Fig. 6.2, which has a head-end and tail-end, and clear beginnings of arms and legs. The first part of this chapter focuses on concepts common to many developmental events seen in chapters 7 through 11. The second half of the chapter covers the details of early development.

scientific photo of a human embryo
Figure 6.2: Seven-week-old human embryo. Image credit: "Human embryo 7 weeks", by GoldenBear is licensed CC BY SA 3.0

Pregnancy is divided into three equal time periods named . During the first two weeks of the first trimester, a single cell multiplies into a multicellular organism with no discernible shape (other than it is a round blob, not an icosohedron). Starting at week 2, the blob of cells develops into an with different shapes. The embryo keeps developing new shapes until it has all of the basic human shapes, such as a head and arms and heart. At this time (week 8) it is a , and the first trimester has ended. The next two trimesters are relatively boring, the fetus grows bigger and the shapes mature, but nothing terribly exciting happens until birth. That’s why this is a Histology and Embryology book, not Histology and Fetology.

In this book, we list all times in days, weeks, months or years (e.g. Fig. 6.3). In a developmental biology class, times may be listed in carnegie stages, which are less useful to us (unless you want to discuss crown-rump lengths).

rought tiumeline of early development
Fig. 6.3: A rough outline of a few major developmental landmarks (not to scale)

photo of Niko von Glasow
Figure 6.4: Director of the film NoBody’s Perfect, a documentary on surviving the teratogenic effects of thalidomide. Image credit: "Filmmaker Niko von Glasow", is licensed CC BY SA 2.0

As human cells divide during the first , they make decisions as to their , which ultimately determines the number and location of arms and legs, head and heart. These decisions involve signals being sent between cells. Errors in these signals lead to (or congenital malformation, or birth defects (but see below)). These can happen without known cause. Or, chemicals may interfere with developmental signals, and we call these chemicals . Probably the most famous teratogen in the U. S. is the medication thalidomide. If taken by pregnant women at the wrong time, thalidomide interferes with the decision-making processes embryos use to decide where to grow limbs. This causes phocomelia, or malformation of the limbs. For many other countries, the teratogenic effects of agent orange (dioxin, which is also found in cigarette smoke) and atomic radiation (pdf download) are well known, but this information is notably absent in most American school classes (agent orange and atomic radiation also have adverse effects on adults, and the rate this information reaches the American public is subject to similar biases). We will cover a small number of signals involved in important decisions that are made during face and tooth development. 37 trillion decisions are made between the single-cell stage and birth, errors are inevitable. Some errors are minor, changing the appearance but not the function of an organ; we might refer to this as a birth mark or human variation. Other mistakes are more severe, interfering with a person’s health, and we refer to those as congenital disorders (or perhaps inborn errors of development, but preferably not birth defects, because our choice of words influences the amount of health care people seek, it depends on the patient).

Much of what we cover in this chapter comes from studying the development of animals, some of which don’t even have teeth, like sea urchins, tunicate worms and flies. These model organisms are useful to us for two major reasons. One, they develop quickly, in large numbers, externally (no uterus), and may even be see-though. Two,  early developmental processes are highly conserved across metazoans (there have been few mutations to DNA instructions over millions of years). If you don’t think you share much in common with a fly, take a look at your larval stage in Fig 7.1 in the next chapter. We do have significant differences. As a result, sea urchins are much less useful when we discuss traits like a head. Worms and flies are less useful when discussing traits unique to vertebrates, such as teeth and our “new head” (covered in chapter 7).

But for early development (this chapter), experiments on what those of us with new heads often call simpler organisms yields a wealth of useful information. Experimenting on other organisms allows researches to do  -mapping. In essence, if you inject a with a dye, you can follow it as it goes through to produce a large number of adult cells. Any adult cell that contains the dye must have come from that stem cell. This allows us to map the of adult cells. Instead of using dyes, some developmental biologists use chimeras, a technique invented by Nicole Marthe Le Douarin,  to identify the diverse roles of . Others transplanted cells from one location to another to see if that altered the fate of those cells, or if those cells altered the fate of their new neighbors.

If you at any time feel like you need more information on , these links are helpful:

Patterns

In arts and crafts, a pattern is a repeated form or design. The same is true in developmental biology. This section covers the basic patterns that occur during development (patterning, or ). These patterns are repeated, often with slight changes (the way theme music is repeated with changes in different scenes of a film). Multiple patterns can overlap. However, it is useful to think about developmental patterns individually, the way you might focus on just the vocals– or cello, or  accordion– in a song.


animated illustration of clones
Figure 6.5: Illustration of cell cloning, followed by signaling.

Cloning

produces clones, or identical cells with identical . However, cells and different to become different tissues and organs. In the first 2 weeks of , cells divide and produce identical (but smaller) cells. Very soon, cells make decisions as to what they are going to become, and these decisions are coordinated with other cells. Without coordination, an embryo could wind up with two butts and no head.


animated illustration of differentiation
Figure 6.6: Differentiation is caused by changes in gene transcription that leads to cells looking (and behaving) different from other cells. These changes are usually triggered by morphogens.

Differentiation

is the process where begin to different , causing them to appear and function differently from other cells. All cells in the human body have the same , because all cells copied their DNA from the zygote (a few exceptional cells change their DNA a bit). The zygote is a toti-potent stem cell, can potentially bind to and activate any gene. As cells differentiate, they limit their by putting some genes into deep storage (not actually changing the genetic code, but changing what may or may not be accessible to RNA polymerase).

animation of differentiation
Figure 6.7: Differentiation alters cell fate, which is passed on to daughter cells in the form of conserved patterns of DNA methylation and histone packaging.

Differentiation is triggered upon receiving chemical signals. These signals activate , which cause target cells to the of un-necessary , pack those DNA regions around , and permanently shut those genes off. For instance, cells to become ovaries never synthesize insulin, the for insulin is packaged away long before ovaries  secrete estrogen or ovulate. DNA methylation and histone packaging limits the fate of these cells. The methylation pattern is copied during and passed to both daughter cells (and to their daughter cells). The significance of this is that causing just one cell to differentiate can affect the fate of millions of cells in the future.

Stem cells in the skin repair skin damage easily. Why can’t we use those stem cells to repair damage from a heart attack or Alzheimer’s Disease? That is the basic question we are addressing. In Fig. 6.7, imagine a signal instructs one cell to methylate green genes, leaving  red genes available for . Imagine a different signal does the opposite to the other cell, causing mehtylation of the red gene but leaving green genes open. As the red cell duplicates its DNA, it copies the methylation pattern, so that all of its daughter cells cannot express green genes. Conversely, none of the daughter cells of the green cell can express the red gene. We now have 4 red cells, who belong to one , and 4 green cells who belong to a different lineage. All 8 cells have the same DNA (), but not the same DNA methylation pattern (). Further arises as cells transcribe un-methylated genes. In the cartoon, as red cells the red gene, the red protein acts as a short-range signal which nearby green cells to express the dark-green gene. And if expression of the dark green gene made those cells look different, we would say those target cells further. This organism has become more complex, starting as 1 cell type but now having 3 different cell types. This continues until we have 200 or so different cell types. Generally speaking, as a cell becomes more specialized, its fate becomes more limited (more DNA methylation and histone packaging), possibly to the point of being limited to just one cell fate.


illustration of induction
Figure 6.8: Green cell releasing a morphogen to induce the differentiation of a dark blue cell into a lighter blue cell.

Induction

One cell may signal to another cell and (instructs) that cell to . Chemicals that induce another cell to change its shape (to ) and change are called (or sometimes , because cells are both growing and differentiating during early development). Morphogens are similar to neurotransmitters or , they often bind to cell-surface proteins. Activation of morphogen receptors leads to activation of in the cytoplasm, which in turn can either activate or inhibit specific to alter the of different . Changes in gene expression cause the target cell to look and function differently. Morphogens also change the methylation of genes and their packaging around histones, which is a more permanent change to gene expression. This is passed down to daughter cells, which we learned in Chapter 1 is an example of inheritance. By regulating gene expression through both transcription factor activity and methylation, morphogens change not only the the target cell, but their cell fate as well.

There are often several closely-related molecules that belong to a family of morphogens, each chemical having a name like morphogen-1, morphogen-2a, morphogen-2b, and morphogen-3. There are a large number of different morphogens. In this textbook we discuss 4 by name, ones directly related to tooth development, such as the Bone Morphogenetic Protein () family, the Wingless/Int-1 family, and the Fibroblast Growth Factor () family. Morphogens can act over different distances listed in Table 6.1. The names of specific morphogens are very important on embryology exams, much less so on dental hygiene exams.

Table 6.1: Examples of different type of signals seen during embryogenesis.
Distance Type Examples
Direct Cell-to-Cell Epithelial-to-neuro-mesenchymal stem cell (Odontoblast differentiation)
Cell-to-ECM Integrin-to-collagen (Ameloblast differentiation)
Short-range Diffusion through ground substance Bone Morphogenetic Protein (BMP), Wnt, Fibroblast Growth Factor (FGF) (tooth bud induction)
Body-wide signals Hormones PTH, Calcitonin (tooth eruption)
illustration of a morphogen gradient
Figure 6.9: Illustration of a morphogen gradient having different effects on cell fate, dependent on the morphogen levels being above or below certain thresholds at different distances away from the source.

Morphogens are secreted into , creating a concentration gradient of morphogen levels as they diffuse slowly away from the source. The amount of morphogen molecules produced, the relative stickiness of the ground substance, the distance away from the source and the number of receptor proteins on the target cell determine level of a signal cells receive. Sometimes, a morphogen produces visible effects that fall along a linear gradient (or spectrum), such as skin color (from lighter to darker) or body size (from shorter to taller). However, sometimes the effects of a morphogen are more discrete (tooth bud or no tooth bud, there are no half tooth buds). To help explain these observations requires a little knowledge of how work. For example, in the French flag model (Fig 6.9), imagine receiving high levels of the morphogen signal triggers the transcription of a blue gene and inhibits a red gene, causing those cells to into blue cells. Moderate amounts of the morphogen signal may not be enough to recruit RNA polymerase to the blue gene, but enough to bring inhibitory transcription factors to the red gene, causing cells in regions of moderate morphogen levels to differentiate into white cells. Lastly, cells that receive little or no morphogen signal do not inhibit the red gene, causing them to differentiate into red cells. There are other models that help explain the link between induction and morphogenesis– between a chemical and an organism. For instance a morphogenetic field is less Francophile in nature but harder to illustrate. These models all share an important concept: chemical signals can communicate in a developing embryo.

illustration of Hox genes
Figure 6.10: Hox (homeobox) gene expression in fruit flies, mice and humans. Image credit: Hox genes in various species" by Stefanie D. Hueber, Georg F. Weiller, Michael A. Djordjevic, Tancred Frickey is licensed under CC BY 4.0 / cropped

During very early development, regional expression of controls , not (further reading). Homeobox genes are that specify which part of the body a particular cell is now a part of along the anterior-posterior axis, even if that area has no specific shape yet (Fig. 6.10, upper left). They do not encode the proteins which cause cells to become part of a specific organ. Instead, homeobox genes activate or inhibit a collection of other (a program), setting the stage for future changes. Such programs include morphogens like and , which activate genes that alter cell and tissues morphology (Fig. 6.10, upper-right).

Homeobox gene expression represents a very specific plan in embryology. It is highly conserved across animal species, and the pattern is very predictable in embryos. The pattern is also relatively simple and linear (Fig. 6.10). Think of homeobox genes like surveyors who determine the locations where houses will be built in a subdivision, while morphogens are the contractors who follow the blueprints for each house, which are built by cells. We can extend this metaphor one step further: in a subdivision, houses are similar, derived from one basic blueprint, but given slight modifications with each re-use.

Lastly, it is important to note that in embryology, the anterior-posterior axis means head-to-tail, the same way it is used in all forms of animal biology except adult humans, where it means front-to-back. You may also hear the term rostral-caudal applied to human embryos, but not human adults (except their brains).


animation of patterning
Figure 6.11: Illustration of a stable pattern arising in response to two antagonistic morphogens.

Self-organization

The ability of a tissue to create its own is called self-organization (in contrast, maternal effect genes from mom’s cells guide patterns for the embryo). The French-flag model describes how one morphogen can multiple cell fates. But what if you want a flag with repeating stripes? Self-organization involves multiple , initially expressed randomly. Interactions between these morphogens leads to a stable pattern of , and a complex pattern of .

For instance, some morphogens block cells from producing the same morphogen. In Fig. 6.11, imagine a blue morphogen induces one cell to differentiate into a blue cell, but blocks neighboring cells from becoming blue cells. These neighbors would be free to differentiate into red cells. At the same time, a distant cell produces red morphogen, which induces it to differentiate into a red cell, but blocks its neighbors from doing the same. Its neighbors would be free to produce blue morphogen and differentiate into blue cells. The initial pattern that arises is alternating rings of red and blue cells. However, rings produce regions of red-next-to-red and blue-next-to-blue, which violates our make-believe rules, and this starts a battle. To minimize the number of red-next-to-red and blue-next-to-blue, cells change their until a stable pattern arises, in this case stripes. It would be hard to predict from the start which cells would be red versus blue, but you could predict the development of red and blue stripes. If these were pigment-producing cells in a zebra, we’d have a zebra with stripes (the best type of zebra). But this pattern could also be alternating regions of oral mucosa that grow a tooth bud or don’t grow a tooth bud, ensuring each tooth bud has enough space to grow (if you want to go down a rabbit-hole, these interactions can be modeled mathematically). When a morphogen blocks another signal, we call that , and antagonist signals are just as common as induction signals. They must, after all, it is fundamentally just as important to grow one head as it is to not grow two heads.


animated illustration of growth types
Figure 6.12: Cell proliferation can occur appositionally or interstitially

Proliferation

Growth of a tissue is called . Proliferation occurs either by (adding cells) or by adding (synthesizing proteins or other molecules). When new growth happens next to a region of older growth, this is , whereas if growth happens from within and pushes older tissue outwards, that is . Dense tissues, such as , enamel and dentin, undergo appositional growth. Softer tissues like grow interstitially.

aanimated illustration of a bud
Figure 6.13: Interstitial growth in an epithelium can cause budding or invagination

The growth of an organ is regulated by hormones, such as Growth Hormone, which coordinates the rate of growth of tissues body-wide. On the other hand, local-acting cause small regions to grow faster than neighboring regions. To make room for new cells while still maintaining a single-layer of cells connected to neighbors by , and , an epithelium bulges inwards or outwards if it can’t grow wider. An outward growth is known as a , and an inwards growth is an . Arms and legs grow from buds, while teeth, hairs and the brain grow from invaginations. These are both forms of interstitial growth.

When we learned about the histology of epithelia, we said epithelia are located on the outer or inner surface of the body, with the side facing a space and the side next to connective tissue ( being an exception). It is simpler to form a new epithelium by folding an old one, as the process of folding creates a new space. Otherwise, if you tried to form a new epithelium in the middle of other stuff, that would either require re-programming the cells’ , or first digesting a new space and then guiding epithelial cells to its location. Folding is simpler, it creates a space right where you need it (apical), and as a result you will see a lot of invaginations in the next three chapters.


histoic drawing of dinoflagellates
Figure 6.14: An example of different morphs (shapes) of the same basic pattern in dinoflagellates. Images credit: “Kunstformen der Natur (1904), plate 1: Phaeodaria“, by Ernst Haeckel is in the Public Domain CC0

Morphogenesis

is the appearance of new shapes in an organism. It means to go from no shape to some shape. The word can mean the study of these shapes, or be used as an adjective to mean the shape of something (For example, a is a change to the healthy morphology of the palate). From the zygote to the blastula stage (sometimes referred to as the germinal stages) the ball of cells is . At the start of , however, the embryo undergoes numerous morphological changes. New shapes arise in the developing embryo. It develops an inside and an outside, a tail end and a head end,  a left and right, and a front and back. After that, a nervous system and a circulatory system become visible. How long do you think it takes before teeth develop? The answer may surprise you.

On Growth and Form illustrations of fish transformed on Cartesian grids
Figure 6.15: D’Arcy Wentworth Thompson proposed that what may look like different shapes in related species may be transformations of one basic pattern. Image credit: "Fig 150-153" by D’Arcy Wentworth Thompson, On Growth and Form is in the Public Domain, CC0

Fig. 6.14 is a famous drawing of tiny sea creatures with mineralized exo-skeletons. This image was later analyzed by a mathematical biologist named D'Arcy Wentworth Thompson, who noticed different biological shapes can be different transformations of the same shape. Transformations, he proposed, occurred by different localized rates of growth– something our current knowledge of signaling supports. This is what he illustrated in Fig. 6.15. He is regarded as the first person to describe morphogenesis. When Thompson looked at the tiny sea creatures, he didn’t see different shapes, he saw variations on one shape. These variations arose over evolutionary time, and led to different-looking sea creatures (who share ). In this book, we look at different body parts that share the same lineage but change over developmental time, such as hair follicles and tooth buds. Thompson’s ideas were expanded upon by the mathematician Alan Turing, who coined the term morphogenesis. His other achievements include inventing computer science and breaking the Nazi code during WWII (with the help of many female mathematicians at Bletchley park). Sadly, he committed suicide after his arrest, conviction and imprisonment for homosexuality by the British government he helped save. Their government’s view on homosexuality has developed since then, we can understand the current view better by following its lineage.

Morphogenesis and genetics

To have a well-rounded understanding of how morphogen signaling works, it helps to understand some concepts of genetics. When we say understand, we don’t mean memorize the specific details, instead focus on the concepts. First, pay attention to when we use the word morphogen, we likely mean the signaling molecule itself. We at times discuss the morphogen signal, and that extra word signal makes a big difference. Mutations that alter morphogen signals include mutations to the gene for the extra-cellular morphogen molecule itself (if it is a protein such as or ) or enzymes that synthesize the morphogen (such as the conversion of into Retinoic Acid), cell-surface for the morphogens, cytoplasmic second messengers and nuclear . Discussing morphogen signaling levels saves us from the complexity of having to mention the entire . Next, some mutations might be , such as a mutation to a receptor that prevents it from shutting off, and these are often inherited in an autosomal dominant fashion (it only takes 1 faulty to create a signal where it shouldn’t be). Alternatively, mutations prevent proteins from functioning properly, and are often inherited in an autosomal recessive pattern (it takes both copies to be faulty to lead to a loss of a signal where it should be, one good allele would be enough). Some mutations fully stop, or fully start, morphogen signals. and in such cases we would say theses mutation have complete penetrance. Other mutations might partially block or partially start morphogen signals, and have partial or incomplete penetrance, the severity of the symptoms fall on a spectrum.


illustration of maturation
Figure 6.16: Maturation involves the changing of already present patterns. Image credit:  "Generalized scheme of embryonic development" by JMW Slack is licensed CC BY SA 4.0

Maturation

is the process by which new shapes created by continue to and take on a more mature (adult-looking) form. By the end of week 9, all of the basic organ shapes have formed, and we say the period is over and the stages begin. The fetus undergoes maturation, and maturation involves bits of the previous processes (another way of saying that is maturation other developmental processes).


animated illustration of fusion
Figure 6.17: Illustration of the fusion of two separate structures and their tissues.

Fusion

Sometimes, two separate bits of the same type of tissue come into contact with one another and into a single tissue. For an epithelium, this requires the cells share the same type of  . For a connective tissue, this requires the correct binding to the correct . In Fig 6.17, the basic pattern of green being on the outside and red in the middle is initially disrupted by the fusion of two structures. There are three ways to remove green cells from the middle area. The first way is for cells to receive and cause cells to migrate. Some guidance cues are repulsive, such as the lack of correct , or high concentrations of a repulsive (if you are at a party and leave the living room because someone there smells bad, you are acting on a repulsive guidance cue). Other guidance cues are attractive, such as the correct cell-to-cell contact or a positive guidance morphogen (at the same time, you  smell delicious hors d’oeuvres in the kitchen and head there, you are acting on an attractive guidance cue). Whether a cell-to-cell connection or morphogen is attractive or repulsive depends on how the target cell has been programmed (what it is ). The second way to remove cells from the wrong location is to induce them to undergo (see below). Lastly, another way to re-organize cells is to induce them to (revert to a ) and then into the correct cell type. This is called a transition; both early development and tooth development involve examples of and (discussed later in this chapter).


scientific diagram of of apoptosis
Figure 6.18: Growth of fingers occurs by apoptosis of regions of a hand plate, guided by morphogen gradients. Figure legend: E and G: adeauate apoptosis, F and H: reduced apoptosis. Image credit: "Depletion of BMP Signaling Causes Interdigital Syndactyly", by Bandyopadhyay A licensed CC BY SA 4.0

Apoptosis

Cell death is necessary for multicellular life. In the construction of tall buildings or large ships, is built first to allow construction workers to get where they need easily, and the scaffolding is removed after construction is complete. Similarly, during , is an integral part of the construction of body parts. Many tissues grow more cells than are needed in the mature form. To undergo , un-needed cells undergo . For instance, during the growth of , tissue is made to act as a scaffold for . Shown in Fig. 6.18, the hands and feet start off as limb paddles, and only with apoptosis in alternating regions does the of fingers and toes occur. Without enough apoptosis, people are born with webbed fingers or toes, or suffer (their tongue is anchored to the floor of the mouth by a pronounced lingual frenulum).

The first trimester

illustration of the fallopian tube
Figure 6.19: The per-implantation period generally occurs in the fallopian tube. Image credit: "Fertilization in humans", by Ttrue12 is licensed CC BY SA 3.0

Pre-implantation period

The begins with fertilization, spans a week or so of development, and ends with an embryo implanting into the uterus. is the union of one sperm cell and one egg– thousands of sperm are needed, but only one is allowed into the egg. This union joins 23 maternal with 23 matching paternal chromosomes, forming a . Fertilization most often occurs towards the distal end of a fallopian tube. Over the next week the zygote undergoes and increases in cell number, but does not grow in size. All of the amino acids and nucleotides for making new cells during this first week come from the of the zygote, which is why the egg is a very large cell. After , the uterus provides the raw materials for an embryo to grow larger.

illustration of nondisjunction
Figure 6.20: Errors separating chromosomes during mitosis generally triggers cell-cycle arrest. Image credit: "Non-disjunction of sister chromatids", by Wpeissner is licensed CC BY 3.0

During , the 23 pairs of are duplicated and divided between 2 daughter cells evenly. If mistakes are made and an odd number of chromosomes wind up in a daughter cell, this fails a and triggers . If a chromosome breaks, it can lead to the same result.

photo of a boy with trisomie 21
Figure 6.21: Cells with an extra copy of chromosome 21 can survive and develop. Image credit: "Boy with Down Syndrome using cordless drill to assemble a book case" by Rob Kay is licensed CC BY SA 3.0

There are exceptions, however. A cell with an odd number of chromosome 21 or 23 can survive. If that cell is the egg, then the and all subsequent cells will have an odd number of chromosomes. This leads to congenital disorders such as . If the chromosomal segregation error happens at the 2-cell stage of development, then only half of the cells in the adult would have an extra chromosome, and the condition would be less severe. Extra chromosomes interfere with decision-making processes in . People with Trisomy 21 frequently develop , and may suffer from hypo-salivation and bruxism. They therefore need to engage in extra care to maintain healthy oral hygiene.


animation of earely divisions
Figure 6.22: The early cell divisions produce identical cells, from zygote to blastula stage.

Early divisions

During the first week of development, the zygote undergoes 3 where occurs in a synchronized fashion. You can watch a cool video here, as you do see how the embryos are oriented in the same direction. Is the lower half of zygotes heavier? To answer that, frog astronauts helped NASA scientists study early development on the space shuttle. Both on earth and in space, cells duplicate along  single planes, 3 times, producing an 8-cell embryo (suggesting density and gravity are correlated with early cell polarity but do not cause polarity). Synchronizing mitosis requires cell-to-cell communication between to coordinate . Synchronized mitosis leads to the embryo developing symmetrically. As mitosis continues, the number of cells continues to double, but the cells do not divide along the same plane. This forms a solid ball of cells called a . As mitosis continues, cells continue to get smaller, until the solid ball of cells becomes a hollow ball of cells named a (or blastocyst). From the zygote to the blastula stage, cells appear no different except in size. A fancier way to say this is that there are no changes to . During the blastula stage, a group of cells named the move away from the others. The fate of the inner cell mass is to become the embryo, while the outer cells, known as the , are fated to become extra-embryonic structures such as the placenta. The blastula implants in the endometrium about a week after fertilization and continues developing.

Gastrulation

It is not birth, marriage, or death, but gastrulation, which is truly the most important time in your life.Lewis Wolpert.

animation of gastrulation
Figure 6.23: Illustration of gastrulation and differentiation of the three basic germ layers, in cross-section.

Gastrulation overview

During , the embryo is reorganized from a single layer of undifferentiated  cells into three layers of cells. Some of the cells from the single-layered   , forming a layer of cells on the inside and a layer of cells on the outside of the embryo. The significance of this is that this is the first instance where embryonic cells look different from one another. It is also the first instance where cells  limit their . But, wait, if all the blastula cells are identical, why did only some of them migrate? generates 2 cells with identical , but not necessarily identical . If an egg has for a clustered into one spot in the cytoplasm, the first cleavage division produces two non-identical cells, one with the transcription factor mRNA and one without. Later, when the mRNA is , it could induce that cell to migrate inwards, while other cells stay on the outside. NASA’s frog astronauts taught us that transcription factors don’t sink in response to gravity to gastrulation. Instead the difference is a maternal effect. An mRNA for a transcription factor, not transcribed from the zygotes’ DNA but made by mom’s ovary cells, is deposited into a specific part of the egg’s , and it induces some cells to invaginate during gastrulation. In fact, up through the blastula stage, the embryo hasn’t used any of its own DNA. Because the embryo’s DNA is not guiding gastrulation, this is yet another example of an trait.

Let us return to gastrulation: it first generates two layers of cells (outside and inside). Imagine holding a tennis ball and pushing your thumbs inwards, forcing some of the outer rubber to fold inwards. The tube that forms becomes the gastrointestinal tract, and the opening (can you guess?) becomes the anus. The embryo therefore now has a distinct tail direction and head direction. Fig. 6.23 looks more like the way a sea urchin undergoes gastrulation. Human embryos look different because it is only the that undergoes gastrulation, not the entire blastula. But the 2 layers are the same, human gastrulas simply look more squashed. Oh, and sea urchins don’t form anus-first. We are sacrificing accuracy here to focus on important concepts shared across our species. If you are curious, watching movies of these processes in humans is not possible, but it is for organisms like xenpous laevis (frogs).

animated illustration of gastrulation, superficial view
Figure 6.24: Illustration of gastrulation and differentiation of the three basic germ layers, superficial view.

During gastrulation, another important event occurs, cells from the outer layer migrate between the two layers that formed by . This now gives the embryo 3 layers, which are the 3 (Fig. 6.23). The outer layer of and the inner layer of remain epithelial in appearance, while the middle layer of cells () undergoes a change, and becomes . This migration begins at a single location called (or the Spemann organizer). which moves down the embryo forming the behind it (Fig. 6.24). The side of the embryo with the node is fated to become the dorsal side, and the streak divides the embryo into a left-half and a right-half. You have now witnessed, in cartoon form, how something (all the basic body directions) arose from nothing. Don’t let the significance of this sneak past you. Scientists have a hard time explaining what there was before the universe formed, or how the Earth went from having no life to having life. Embryologists, by contrast, explain how embryos change from having no shape to having shape in great detail (we have the benefit of watching and manipulating this process).

Table 6.2: The three germ layers develop during gastrulation
3 embryonic layers Cell fate
Ectoderm Epithelium of skin and oral mucosa, neural tissue
Mesoderm Connective & muscle tissue
Endoderm Epithelial lining of hollow organs
animated illustration of epithelial to mesenchymal transition
Figure 6.25: During gastrulation, and epithelial-to-mesenchymal transition creates the mesoderm layer.

Epithelial-to-mesenchymal transition

The formation of from was a simple matter of . The process by which ectodermal cells quit being ectoderm and migrate to the middle to form is called an (EMT). The epithelial cells of the ectoderm first lose with neighboring cells, which makes them less epithelial. They lose their polarity, into , then into . This process is not only required to form mesoderm during , it occurs during migration, wound healing, and cancer metastasis. There is a reverse process, called a (MET), which also occurs during wound healing. EMT allows epithelial stem cells from a healthy region of to turn into mesenchymal stem cells, migrate away from the healthy region into the damaged region. MET then allows the mesenchymal stem cells to turn back into epithelial stem cells, which divide and differentiate into new . Because this mimics what happens during gastrulation, we say “wound healing   development”.


Differentiation of ectoderm

animation of neurulation 1
Figure 6.26: Illustration of neurulation from a cross section of an embryo. Legend: blue = ectoderm, red = mesoderm, yellow = endoderm, light-blue = neuro-ectoderm, green = neural crest cells.

Neurulation

gave the embryo its first polarity, as well as the beginning of the GI tract. One of the next organ systems to develop is the central nervous system. The basic process used in is when other hollow organs form (watch another cool video here). During the 4th week of development, underlying known as the releases which signal to nearby cells. This induces the nearby region of ectoderm to into , followed by local . Because these epithelial cells cannot easily spread side-to-side, they . Ultimately, invaginating neuro-ectodermal cells lose their contacts with the ectoderm and to other neuro-ectodermal cells, creating a new structure called the . This tube develops into the brain and spine. If you are wondering about the notochord, its is to mostly undergo , although some remains as the nucleus pulposis of the vertebral discs. In headless fish-like  chordates with no backbone called amphioxis, the notochord remains. This wont be the last time you see a human embryo make a structure only to remove it.

animation of neurlulation 2
Figure 6.27: A second illustration of neurulation, focusing on the anterior-to-posterior direction of neural tube folding. Legend: Blue = ectoderm, light-blue = neuro-ectoderm, red = mesoderm, yellow = endoderm.

The neural tube begins folding from the anterior portion of the embryo, and zips up in an anterior-to-posterior direction. The zippering is not perfect because the anterior end is growing wider as it is folding to form the primitive brain. For almost all developmental processes, the anterior end is the region that develops first, followed by more posterior regions. Zipping the neural tube requires adequate levels of folic acid (which is needed for the and ), so hopefully mom’s did an adequate job protecting it.

Scientific diagram of neural crest cell fate
Figure 6.28: Origin of cranial neural crest cells and their target migratory locations. Images credit: "Cranial Neural Crest Cells - migration" by BiolCNC is licensed under CC BY-SA 3.0

Some cells do not . Instead, these cells undergo an transition and migrate away from the neural tube. These cells are , they are to become a number of important cells and tissues throughout the body including , , dental pulp, , and the of the . These cells are sometimes referred to as the fourth embryonic tissue (in addition to the three that arise during ), which suggests some people think neural crest are important. Even after neural crest cells migrate to new and distant tissues, they often retain visible signs of their neural lineage, such as the dendrites on melanocytes or the of odontoblasts. To make migration easier, neural crest cells   a enzyme. This enzyme digests proteins found in the . By now we hope remember the phrase wound healing development, because matrix metalloproteinase enzymes are re-used to heal certain types of tooth wounds. When neural crest cells reach the pharyngeal arches, they secrete and , which each other, producing a striped along the . Some regions are induced to form , the in-between regions form .


Differentiation of mesoderm

historical drawing of somites
Figure 6.29: Somites in a neurula-stage embryo. Image credit: “Plate 20” by Henrey Grey is in the Public Domain CC0

Somite formation

During the same time as , undergoes . Mesoderm starts off as an layer of . Then, regions of mesenchyme pinch off in repeating spherical structures called , forming segments along the anterior-to-posterior axis of the embryo. The of the somites is to become solid organs, either repeating units of connective tissue such as the ribs and vertebrae, or repeating units of muscle tissue such as the rectus abdominus and intercostal muscles. The formation of each somite involves a , some differentiate into an epithelium that separates one somite from the next. Because they form in left/right pairs, the production of matching lateral structures is relatively simple (such as left and right biceps brachii muscles). On the other hand, formation of a single structure from somitic mesodem (such as one sternum) requires of two bilateral structures.

There are other regions of mesoderm besides somites and , such as  splanchnic, lateral plate and paraxial mesoderm. Differentiating between all  types of mesoderm is not essential for understanding clinical concepts in dental hygiene.


illustration of heart formation
Figure 6.30: Development of the heart from two heart tubes. Image credit: “illustration” by OpenStax, is licensed CC BY 3.0

Formation of the heart

In the 3rd week, begins to develop into blood, the heart and the circulatory system. first differentiate into blood islands. Next, begins. Two large blood vessels fuse– with some help from — to form the primitive heart, which begins beating.

Differentiation of endoderm

scientific photos of endoderm formation
Figure 6.31: Different embryonic stages highlighting the endoderm. Legend: yellow = endoderm, red = liver, green = gall bladder. Image credit: "Time line of mouse liver development emphazing it" by Zorn, A.M., Liver development, StemBook is licensed under CC BY 3.0

Formation of the pharynx

The that migrates interiorly during extends its way towards the anterior end of the embryo. This forms the , which later becomes the pharynx. The term “primitive” is used because it has only one opening, there are no oral or nasal  cavities. Those develop later from . Formation of the face and pharynx are covered in detail in Chapter 7.


illustration of the early face
Figure 6.32: You, negative 8 months old.

4th week

We are now only up to the 4th week of development. A lot has happened! At this time, the embryo continues to undergo local regions of , , and the other processes covered at the beginning of the chapter. This leads to the formation of rudimentary (basic) structures. When a structure first becomes visible from the surface of an embryo, it is often called a (an thickening). The cells in a placode are to become an organ, but their overall shape is no more than a raised bump or shallow pit. By the 4th week, head-related organs listed in Table 6.3 begin to develop.

Table 6.3: Primitive structures formed during the 4th week of development.
Structure Fate
Eye placodes primitive eyes
Otic placodes primitive ears
Nasal placodes primitive nose
Pharyngeal arches so many things they get their own chapter
Oro-pharyngeal membrane primitive mouth (not open yet)

Clinical applications of early development

Disturbances in these early stages of development are usually so severe the embryo does not survive to become a fetus, let alone be born. However, clinical conditions exist related to these early stages and to oral health.


radiograph of a hand with polydactyly
Figure 6.33: An example of body plan duplication. Image credit: Polydactyly 01 Lhand AP" by Drgnu23 is licensed under CC BY-SA 3.0

Homeobox gene mutations

Mutations to can cause entire structures to be missing, or to develop in the wrong location. Early experiments on flies led to observations of legs where antennae should be located, or antennae where wings should be located. There are a few homeobox-related genetic disorders in humans. Homeobox-related disorders are rare because the mutations are usually lethal, but when they aren’t the conditions are often severe. A small list is found in Table 6.4

Table 6.4: A few examples of homeobox mutations in humans.
Syndrome Homeobox gene involved Symptoms include
Axenfeld-Rieger Syndome FOXC1, PITX2 Mostly eye-related, sometimes hypodontia and microdontia
Autosomal dominant syndactyly HOXD13 Syndactyly (fused fingers or toes)
Guttmacher syndrome HOXA13 Polydactyly (extra fingers or toes)
Ectodermal dysplasia HOXC13 See below

photo of a hand with syndactyly
Figure 6.34: Lack of apoptosis leads to syndactyly. Image credit: "Hand of a child with Apert syndrome" by Gzzz is licensed under CC BY-SA 4.0

Apert syndrome

Apert syndrome is caused by a mutation in the for the . It is categorized by a wide range of symptoms, including cranial deformations and syndactyly (fusion of digits). FGF is involved in the formation of the (covered in the next chapter), which explains the craniofacial abnormalities. Relevant to this chapter is the ability of FGF (like many ) to inhibit . Having a mutation that causes an FGF receptor to be on all the time inhibits apoptosis in the hand and foot paddles. Regions of apoptosis are required to produce fingers and toes, hence this mutation leads to syndactyly. Partial disturbances to FGF signals can lead to partial syndactyly, or webbing of the fingers or toes.

Otherwise there aren’t many mutations to apoptosis that lead to . Does this mean apoptosis isn’t important to development? No, quite the opposite, it is essential. As a result, humans have redundancy when it comes to triggering apoptosis. We mentioned earlier that mutations require mutating both for a physical change to occur, like only needing one of your two feet to operate the brakes on  a car. Now imagine having 8 legs. Studing the roles of apoptosis in the development of mice shows that removing both alleles of the pro-death signal BAX has no effect, nor does removing both alleles of the pro-death signal BAK, but removing all 4 causes mice to die during the embryonic period after failing to form a functional nervous system and heart. Removing BOK, BAX and BAK is even more lethal (if you are wondering how something can be more lethal, here is further reading). The important concept here is that apoptosis is absolutely esential to development, and as a result human cells are really good at doing it. And people who study apoptosis must be very good at tongue-twisters.


photo of michael berryman
Figure 6.35: Image credit:: "Michael Berryman, actor"  by Stefan Borggraefe is licensed under CC BY 4.0

Ectodermal Dysplasia (part 1 of 2)

Problems with the of during leads to disturbances in the formation of teeth, , salivary glands and other structures. What these have in common is they are all specialized structures of the , induced by neural crest cells to . In a healthy embryo, ectodermal cells receive which activate or inactivate the correct to trigger differentiation into neural crest cells. Neural crest cells migrate to distant regions of the body, determine their location by interacting with morphogens in the , and release other morphogens to induce regions of ectoderm to differentiate into sweat glands, salivary glands, tooth buds or hair follicles. Disruption of this process leads to a condition named .

This group of syndromes is rare, with only 7,000 cases worldwide, but there are at least 40 different genes  implicated. Compare that to Sickle Cell Disease, which currently affects over 100,000 Americans (predominantly African Americans, Hispanic Americans, Greek Americans, Turkish Americans and Italian Americans), all due to mutations in a single , Hemoglobin-Beta. The point of this comparison is to highlight when these genes are . The induction of ectodermal stem cells to and differentiate into different appendages is complex and occurs during embryogenesis. Mutation in the morphogens, the morphogen receptors, the second messengers or and downstream genes that are activated to induce differentiation are all possible targets that cause Ectodermal Dysplasia. Mutations in these genes in a neural crest cell leads to a disruption in any of the subsequent cells induced by this new cell type, similar to the way tackling the ball carrier in soccer (football) disrups the gameplay of any of his or her potential passing targets. By comparison, only red blood cells express hemoglobin-beta, and they are cells, they do not become any other cell type. Obviously red blood cells are an important cell type, one that develops early in embryogenesis, but the symptoms of Sickle Cell Disease present less of a spectrum than the types of diseases we have been discussing, such as Ectodermal Dysplasia.

We discuss Ectodermal Dysplasia further in Chapter 8 as we cover .


photo of an actor with cleidocranial dysostosis
Figure 6.36: Gaten John Matarazzo III, actor and CCD activist. Image credit: "Gaten John Matarazzo III"  by Gage Skidmore, is licensed CC BY SA 3.0

Cleido-cranial Dysostosis

(CCD) is a caused by a mutation to a required for the of and teeth. It is required to trigger osteo-chondro-progenitor cells to exit the cell cycle and differentiate into . It is also re-used to the differentiation of . Furthermore, after teeth have formed, this transcription factor is re-used to activate the of a enzymes necessary for remodeling of the alveolar sockets. Without this enzyme, retention of deciduous teeth occurs. Dental implants or dentures (such as the ones the actor and CCD-philanthropist Gaten John Matarazzo III received in Fig. 6.36) are the preferred treatment. In addition, a person with CCD may have small clavicles and changes to shape of the skull– those are bones that form by . This illustrates two major concepts in development. First, many structures form one way, but are later to serve a different function (teeth form by folding inwards, they later move outwards). Secondly, many different patterns in embryology are re-used (), such as the removal of tissue during migration or the removal of tissue during  .


photo of  Medical Animation Spina Bifida
Figure 6.37: Illustrations of Spina Bifida, which occurs due to incomplete closure of the neural tube. Image credit: "3D Medical Animation still shot of Spina bifida in an infant" by scientific animations is licensed under CC BY-SA 4.0

Spina bifida

Incomplete closure of the , or , can result from a lack of adequate levels of folate during pregnancy. There are other less-common risk factors, such as taking certain anti-siezure medications or poorly managing diabetes during pregnancy. Folic acid is used in a wide variety of biological processes, but it is believed its role in during is most important in spina bifida. Because occurs so early in development (week 4), waiting until a woman knows she is pregnant to prescribe folic acid supplements is often too late to be effective. Prescribing supplements to women who beleive they could get pregnant is better, or supplementing common foodstuffs like flour is even more effective for a populace as a whole.


scientific diagram of stem cells
Figure 6.38: Differentiation of stem cells limits their cell fate (e.g. neuromesenchymal stem cells of dental pulp can potentially differentiate into odontoblasts or heptatocytes, given the right morphogens, but odontoblasts can’t become hepatocytes and hepatocytes can’t become odontoblasts). Image credit: “Figure 3” by Beatriz A. Ro Isdas-Junco et al, is licensed CC BY 3.0

Stem cell therapies

When cells , they permanently inactivate un-needed by and storage around . Researchers are learning ways to reverse this process, and guide differentiated cells to revert to a state. This raises the possibility of promoting of tissues that do not otherwise regenerate, removing the need for tissue grafting or transplantation. Because some dental tissues do not regenerate well, there is potential for these technologies to be applied to the oral cavity, such as growing biological dental implants instead of using metals and ceramics. However, more interest has been placed on acquiring from dental tissues. For instance, stem cells isolated from maxillary third molars have been used in clinical trials to improve healing and reduce the need for transplanted tissue in maxillofacial surgery. Because of their potential to into a wide array of cells, great interest has been placed on collecting dental stem cells to treat diseases unrelated to the oral cavity. With the correct and plenty of dental stem cells, it may be possible to reverse the damage caused by disease such as Alzheimer’s Disease (AD) and Parkinson’s Disease (PD), spinal trauma, myocardial infarct (heart attack), and Muscular Dystrophy (MD). The of the cells that produce dentin, pulp, cementum and the periodontium helps explain the link from teeth to neurodegenerative disoders. What else will you do with those extracted third molars?

Unfortunately, there is no legal definition in the U.S. for what constitutes safe, effective stem cell therapy. Even if good rules are put in place, medical-tourism does not abide. In addition to honest and good research in stem cell therapies (the Institute for Stem Cell and Regenerative Medicine at the University of Washington is a good example), there are a large number of dubious-to-downright-quack stem cell clinics operating in the grey market, using risky, untested methods and making absurd claims to take as much money as possible from vulnerable people.

So, who’s who? Therapies that use or boost a patient’s own  () stem cells have the best possible chance of providing positive outcomes with minimal risk of side-effects– especially if that therapy is targeted, such as by adding a specific to a specific outcome. On the other hand, throwing stem cells at a problem– cells from other people, other animals, or from non-animal sources– carries the biggest risk of serious adverse effects with minimal potential for benefit. Also keep in mind that 19 out of 20 potential drugs that have been shown to work in animal models fail in human trials. That means there is scientific evidence in publication for treatments that we know don’t work, but other people can cherry-pick the positive information and use it to sell a product.


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