Developmental Biology Exam 1

What are some of the model systems used in developmental biology? What are they often used to study?

Arabidopsis thaliana (plants), Drosophila melanogaster (flies, helps with development from fertilized egg to grown up developed animal with cell patterning), Hydra vulgaris (whole body regeneration, stem cell dynamics, pattern formation), C. elegans (single fertilized egg division, differentiation, and cell organization), Xenopus laevis (frog, single fertilized egg develops into complex vertebrate animal), Brachydanio rerio (zebrafish, embryogenesis, organogenesis, and vertebrate development in real time), Gallus gallus (vertebrate embryonic development, organogenesis, and cell lineage tracing, external development makes it easy to visualize), Mus musculus (mammalian embryogenesis, organogenesis, and genetic control of development), and stem cells of humans (model early embryonic growth, track how unspecialized cells signal and differentiate into specific tissues, and study the genetic instructions that guide organ formation).

Describe the steps of early development in the frog Xenopus laevis and what can be gleaned from each step.

1. Frogs mate by amplexus, in which the male grasping the female around the belly and fertilizing the eggs as they are released

2. A new clutch of eggs is laid. The cytoplasm rotates such that the darker pigment is where the nucleus resides, making the nucleus visible when looking at the eggs.

3. Eggs develop into 8 cell embryos where each cell can be seen.

4. 8 cell embryos progress to late blastula stage (when a solid ball of cells turns into hollow fluid-filled sphere of cells), which contains thousands of cells.

5. Early gastrula stage is reached, with a visible blastopore lip through which the mesodermal and some endoderm cells migrate.

6. Neurula stage, where the neural folds come together at the dorsal midline, creating a neural tube.

7. A pre-hatching tadpole has formed, with the protrussions of the forebrain beginning to induce eye formation. Brain, gill area, forebrain, stomodeum (mouth), somites, and tailbuds are beginning to be visible.

8. A mature tadpole has formed, having swum away from the egg mass and begun feeding independently, independent structures like the eyes and brain have formed and organism is essentially self sufficient.

What is holoblastic cleavage?

Complete cleavage. Includes isolecithal (sparse, evenly distributed yolk; radial, spiral, bilateral, and rotational) and mesolecithal (moderate vegetal yolk disposition;displaced radial) organism cleavage patterns.

What is radial cleavage?

Occurs in echinoderms and amphioxus. One circular cell goes to two ovular cells, to four ovular cells, to eight spherical cells. Successive cleavage planes are parallel or perpendicular to the vertical axis, resulting in tiers of blastomeres stacked directly on top of one another. Isolecithal/holoblastic/complete cleavage.

What is spiral cleavage?

Occurs in annelids, mollusks, and flat worms. Cells divide at oblique (tilted) angles, creating an offset, twisted arrangement around the pole-to-pole (straight up and down) axis of the embryo. Isolecithal/holoblastic/complete cleavage.

What is bilateral (isolecithal) cleavage?

Occurs in tunicates. The first cell division bisects the zygote into left and right mirror-image halfs. Later divisions use this central axis as a mirrored plane. Isolethical/holoblastic/complete cleavage.

What is rotational cleavage?

Occurs in mammals and nematodes. The first division occurs along the vertical axis, and the subsequent division occurs at a 90 degree angle from that. Results in a solid ball of cells. Isolecithal/holoblastic/complete cleavage.

What is displaced radial cleavage?

Occurs in amphibians. A heavy concentration of yolk at the vegetal pole pushes the third horizontal cleavage plane toward the animal pole, causing unevenly sized cells. (Ex. smaller on top than on bottom). Mesolecithal holoblastic/complete cleavage.

What is meroblastic cleavage?

This is incomplete cleavage in which the entire zygote does not divide. Includes telolecithal (dense yolk throughout most of the cell) and centrolecithal (yolk in the center of eggs) divisions.

What is bilateral (telolecithal) cleavage?

Occurs in cephalopod mollusks. Early embryonic developmental pattern in which the zygote is divided into two mirror image halves (doesnt divide completely). Telolecithal/meroblastic/incomplete cleavage.

What is discoidal cleavage?

Occurs in fish, reptiles, and birds. Mitosis is restricted to a small disk of cytoplasm at the animal pole of a heavily yolked egg. Results in one large cell with many small cells on the top. Telolecithal/meroblastic/incomplete cleavage.

What is superficial cleavage?

Occurs in most insects. Nuclei divide without cytoplasmic division, forming a multinucleated cell before membranes grow inward at the surface. Centrolecithal/meroblastic/incomplete cleavage.

What is invagination?

A cell movement during gastrulation in which the infolding of a sheet (epithelium) of cells occurs, much like the indentation of a very soft rubber ball when it is poked. Occurs in sea urchin endoderm.

What is involution?

A form of cell movement during gastrulation in which there is an inward movement of an expanding outer layer so that it spreads over the internal surface of the remaining external cells. Occurs in amphibian mesoderm.

What is ingression?

A type of cell movement during gastrulation. The migration of individual cells from the surface into the embryo’s interior. Individual cells become mesenchymal (i.e. separate from one another) and migrate independently. Occurs in sea urchin mesoderm and Drosophila neuroblasts.

What is delamination?

A form of cell movement during gastrulation in which one cellular sheet is split into two more or less parallel sheets. While on a cellular basis it resembles ingression, the result is the formation of a new (addition) epithelial sheet of cells. Occurs in hypoblast formation in birds and mammals.

What is epiboly?

A form of cell movement during gastrulation in. which there is a movement of epithelial sheets (usually ectodermal cells) spreading as a unit (rather than individually) to enclose deeper layers of the embryo. Can occur by cells dividing, by cells changing their shape, or by several layers of cells intercalating into fewer layers; often, all three mechanisms are used. Occurs in ectoderm formation in sea urchins, tunicates, and amphibians.

What are the axes of a bilaterally symmetrical animal?

There is a single plane called the midsagittal plane dividing the animal into left and right halves. There are also cross sections bisecting the anterior-posterior axis. Top is dorsal, bottom is ventral, front is anterior, back is posterior, sides are lateral. Cutting the animal in half vertically in between the anterior and posterior sides across the thorax region is the transverse plane (cross section), cutting the animal in half vertically with symmetrical sides is the midsagittal plane, and cutting the animal in half horizontally is called the horizontal plane.

What are the three distinct embryonic germ layers?

Dividing cells of the fertilized egg form three distinct embryonic germ layers, including the ectoderm (outer layer), mesoderm (middle layer), and endoderm (internal layer). Each gives rise to myriad differentiated cell types and distinct organ systems. Germ cells are precursors of sperm and egg and are set aside early in development and do not arise from any particular germ layer.

What are some examples of cell types stemming from the ectoderm (outer layer)?

Epidermal cells of skin (outer surface), neuron of brain (central nervous system), and pigment cells (melanocytes, neural crest).

What are some examples of cell types stemming from the mesoderm (middle layer)?

Notochord cells (dorsal), bone tissue (paraxial), tubule cells of kidney (intermediate), red blood cells (lateral), and facial muscle (head).

What are some of the cell types stemming from the endoderm (inner layer)?

Stomach cells (digestive tube), thyroid cells (pharynx), and lung cells (alveolar cells/respiratory tube).

Describe the evolution of pharyngeal arch structures in the vertebrate head.

Pharyngeal arches (aka branchial arches) can be found in the embryo of the Mexican salamander (ambystoma mexicanum). They form neural crest cells streaming down from the midline of the embryo. They can also be found in adult fish, forming the hyomandibular jaws and gill arches. In amphibians, birds, and reptiles, these same cells form the quadrate bone of the upper jaw and the articular bone of the lower jaw. In mammals, the quadrate has become internalized and forms the incus of the middle ear. The articular bone retains its contact with the quadrate, becoming the malleus of the middle ear. thus the cells that form gill supports in fish form the middle ear bones in mammals. developmental evolution across species.

Describe early cell fate mapping.

Edwin Conklin mapped the fates of early cells of the tunicate Styela partita, using the fact that in embryos of this species many of the cells can be identified by their different-colored cytoplasms. Yellow cytoplasm marks the cells that form the trunk muscles. At the eight cell stage, two of the eight blastomeres contain this yellow cytoplasm. In the early gastrula phase, the yellow cytoplasm is in the precursors of trunk musculature. In the early larval stage, yellow cytoplasm can be found in the newly formed trunk muscles. Using inherent properties of a cell to map where they go during development.

Describe vital dye staining of amphibian embryos.

Vogt’s method for marking specific cells of the embryonic surface with vital dyes includes placing 11 agar chips with alternating dye. Can watch the labeled dye move throughout development, ex. go into the dorsal lip of the blastopore, where cells begin to enter the embryo. When the embryo becomes more developed, you can view the specifically labeled dyed sections to see where those specific cells went during the process of development.

Describe the use of genetic markers as cell lineage tracers.

Ex. an experiment in which cells from a particular region of a 1-day quail embryo have been grafted into a similar region of a 1-day old chick embryo. After several days, the quail cells can be seen in the chick embryo by using an antibody to quail-specific proteins. This region produces cells that populate the neural tube. Shows where cells end up through development. Chick and quail cells can also be distinguished by the heterochromatin of their nuclei. Quail cells have a single large nucleus (dense purple), distinguishing them from the diffuse nuclei of the chick (this makes it easier to delineate between the quail and chick cells). Additionally, a chick resulting from this transplantation of a trunk neural crest region from an embryo of a pigmented strain of chickens into the same region of an embryo of an unpigmented strain shows neural crest cells that gave rise to the pigment migrated to the wing epidermis and feathers (helps with tracking by making the actual section of the animal where the cells migrated a different color).

Describe the use of fluorescent dye for fate mapping.

The fate of cells after development can be tracked through the use of a fluorescent dye. Ex. specific cells of a zebrafish embryo were injected with a fluorescent dye that will not diffuse from the cells. The dye was then activated by laser in a small region (about 5 cells) of the late-cleavage-stage embryo. After formation of the central nervous system had begun, cells that contained the activated dye were visualized by fluorescent light. The fluorescent dye is seen in particular cells that generate the forebrain and midbrain. Can track where seemingly random cells at different locations in early development end up. Can be done on many different parts of the cell to see where each section ends up. Ex. Fate map of zebrafish central nervous system: flurescent dye was injected into cells six hours after fertilization (different color per area) and the results are color coded onto the hatched fish. Overlapping colors show that some cells in these regions of the 6 hour embryo contribute to two or more regions.

Describe transgenic DNA fate mapping and what it has helped to reveal.

Fate mapping with transgenic DNA shows that the neural crest is critical in making gut neurons. Ex. A chick embryo containing an active gene for green fluorescent protein (GFP) expresses GFP in every cell. The brain is forming on the left side of the embryo, and the bulges from the forebrain (which will become the retinas) are contacting the head ectoderm to initiate eye formation. The region of the neural tube and neural crest in the presumptive neck region is excised and transplanted into a similar position in an unlabeled wild-type embryo. One can see the transplanted tissue by itse green fluorescence. One day later, one can see the neural crest cells migrating from the neural tube to the stomach region. In four more days, the neural crest cells have spread in the gut from the esophagus to the anterior end of the hindgut. (essentially, you tag a region of cells of interest expressing GFP from one organism and transplant it into another organism to track where it moves during development).

Why do vertebrates start development differently?

Vertebrates (fish, amphibians, reptiles, birds, and mammals) all start development differently because of the enourmous differences in the sizes of their eggs. By the beginning of neurolation, however, all vertebrate embryos have converged on a common structure. Ex. a lizard embryo is shown next to a human embryo at a similar stage (appear very similar). As they develop beyond the neurula stage, the embryos of different vertebrate groups become less and less like each other.

What is the significance of the tree of life?

Shows that across a geological timescale, all life on earth is related. there are common ancestors between all organisms. Many of the common ancestors of aceols anf flatowrms, insects, vertebrates, and land animals (annelids,arthropods, mollusks, echinoderms, and vertebrates) can be traced to the Cambrian explosion of diversity.

What is the definition of cell specification?

When a cell is capable of differentiation into a more specialized cell (reversible). Has not reached a determined, irreversible stage of differentiation.

What is the definition of cell determination?

A cell who’s lineage is determined. Has reached a point where it has chosen definitively which pathway of differentiation it wants to take. This is irriversible. (often this comes with time, at a certain stage in development cells must be determined).

What is autonomous specification?

When a cell intrinsically knows what to become, if you change its environment, it will still become the same type of cell. Lineage is determined.

What is conditional specification?

When a cell relies on environmental cues to tell it what to become. The cell is not determined, and if you move it to a different location it will likely become a different cell than it originally was going to.

What is a way to study cell fate determination to determine if a cell experiences autonomous specification?

Using a fate map, you can go back to early cells, isolate them in culture, and see what they become. If you wanted to find out if they were autonomous or conditional, you could see if the role of the environment changes the cell fates. If you place a muscle cell in a culture of neurons, two things can happen. If the differentiation is autonomous, the original cell type is irriversible and you get a muscle cell in the dish of neurons. If the muscle cell becomes a neuron, the cell fate was not autonomous and was able to be changed by influences in the environment. These experiments are important because they tell you when a cell fate change is irreversible in development and if the fate is not autonomous, what factors can be applied to the culture to get the cell types that are wanted.

What is an example of autonomous specification?

Autonomous development of blastula cells in snail embryos. Certain cells in the blastula go on to make ciliated trochoblast cells. If you remove one of these cells and culture them,they will still go through the same developmental timeline to become cilliated cells as if they weree still in the organism. This process also happens in the retina where you get different neurons being born in a culture dish as you would have them born in an intact eye.

Describe how the first fate map was done in the tunicate sea squirt.

Was made by Edward Conklinin 1905 at Woods Hole in Massachusetts. Conklin was observing fertilized embryos of the tunicate sea squirt (go through an interesting development to become a larvae that swims around in the ocean and even contains nervous tissue, then once they find a good place to live, they attach to the substrate and become an immobile sea squirt) and he noticed that the cells in the early organism had a distinct color to them and he called this the yellow crescent. He also noticed that when the cells divided, this yellow crescent was not evenly distributed to the daughter cells, but remained on the same side of the animal. Tracing these yellow cells, Conklin made the first fate map. From Conklin’s observations and lineage tracing, you can extrapolate a fate map as early as the single cell. As this cell divides, the cell fates continue to specify with the yellow cell always containing cells that make up the endoderm or the gut of the animal while the blue is ectoderm. Also an example of autonomous specification.

How was the cell map of the early tunicate embryo generated/how was it determined that the early tunicate embryo exhibits autonomous specification?

The blastoderm cells of an 8-cell stage embryo were manually dissected and cultured. Animal pole cells generated a mass of ectoderm without any of the other cell types present. In contrast, vegetal pole cells generated either notochord or muscle with both having some endoderm present but a complete lack of ectoderm. This implied that at the 8-cell stage, they are autonomously specified for a particular germ layer (can be different cell types but still autonomous for germ layers).

Macho gene regulation of muscle development in the tunicate, how do you tell if it is needed.

Typically, it appears that cells with macho are the only ones that can become muscle. By removing macho (through CRISPR) and marking for muscle actin, we can see that there is significantly less muscle actin in removed macho cells, but there is significantly less muscle actin than with macho. This shows that macho is very important for muscle development. In order to test if macho is enough to produce muscle alone, you can move it to a place where it normally does not go to see if there are muscles there in the future (this makes it sufficient). Macho is sufficient.

What is necessary vs. sufficient in terms of proteins?

Necessary proteins are needed to make something that the body needs, sufficient proteins are proteins that can act alone to make that important something. Ex. macho is necessary because without it very little muscle cells are created. Macho is also sufficient because when you move it to a new area, without the other muscle causing proteins, it still develops. It does not need the other muscle causing proteins, so it is thus sufficient. Something can also be necessary but not sufficient.

What is an example of conditional specification.

If you take a chunk of frog cells out that would originally be specified for back cells and put them in the belly region, they will become belly tissue (in the blastula stage, if it was further along in development it is possible that the cell fate would already determined and would not be able to change their cell fate. Can also take a chunk of cells out of the embryo and the embryo will reprogram cells to make sure they still get all of the right structures (although the animal will be smaller).

Describe Roux’s attempt to demonstrate autonomous specification

Roux attempted to demonstrate autonomous specification by destroying (but not removing) one cell of a 2-cell frog embryo by stabbing it with a hot poker. This experiment resulted in the development of only one half of the embryo and his conclusion was that each cell once fully divided contains all the information to make an entire organism. The organism did not recognize that something was missing, thus it was autonomous specification.

Describe Driesch’s demonstration of conditional specification.

Driesch found the exact opposite results than Roux when he dissociated an intact 4-cell sea urchin embryo into single cells. Each of the isolated cells up to an 8 cell stage embryo were capable of generating an entire pluteus larvae. The organism recognized that there were no longer any of the other cells there, and fills in the gaps, creating four smaller fully developed plutei.

Why was there a discrepancy in Roux and Driesch’s experiments?

They are different organisms, could also be a different time period/stage of development (too early for one or too late for another). One was killed and left associated and the other was separated (different experimental design).

What is syncytial specification?

This is the last type of specification we are talking about. In Drosophila, cells undergo nuclear division without cytokinesis (superficial cleavage). After enough rounds of this process, you have essentially one cell with 1000 nuclei in it. This is called a syncytium. All the nuclei then migrate to the periphery and cell membranes begin to form to isolate these nuclei into separate cells. Waves of nuclear division occur before cellularization. Morphogen gradients created during syncytial specification are unique that in that the gradients of these molecules are detected by every single nuclei in the syncytium.Every nuclei in the syncytium along the A/P axis is exposed to a different concentration of protein (ex. bicoid gradient from anterior to posterior, caudal gradient from posterior to anterior, they actually repress each other). The concentrations and ratios of these proteins distinguish each position along the axis from any other position. When nuclear division occurs, the amounts of each morphogen differentially activate transcription of the various nuclear genes that specify the segment identities of the larval and the adult fly.

How can we identify all the different genes that are turned on in all these different cells as development progresses? How do we come up with the transcriptome?

Advances in single-cell RNA sequencing has allowed us to start tackling that question while generating an insane amount of data. In this example, zebrafish embryos of different ages are dissociated into single cell suspensions. These cells are then [assed through a machine that will insert a barcode on all of the cDNA molecules in the cell. The generated tagged cDNA molecules are then sequenced and annotated with databases of known genes. The gene exrepssion profules of each cDNA from that cell are then compared to other cells from that organism. The end result is a visual map of similar gene expression patterns across many cells and across developmental time.

What is an example of the transcriptome sequencing experiments?

Early in development, there are very few clusters of cells that pop out and these are ectoderm and endoderm. At this stage, gastrulation has not started yet and there is no mesoderm. Slightly later in development you start to see additional gene expression profiles that are indicative of specific germ layers or even further into cell type specification. By the time the zebrafish embryo is 24 hours old, all its major organs are patterned and you can see clear separation of populations of cells that all have very similar gene expression profiles. This data does not tell you spatially where in the embryo these came from. The earliest time points are at the images center, with more differentiated cells emanating outward to the ectoderm, mesendodermal, and neural lineages.