Dev Bio Exam 1

The course of dev bio

molecules within a cell to development of an entire organism. cell movements that lead to complex patterns of tissue are carefully regulated.

Where do we all begin? What is fertilization?

a single nucleus from one parent combining with the other nucleus (gametes; sperm and egg)How d. Fertilization is the beginning of development and a complicated process.

What is differentiation

the process of a stem cell becoming a specific fate. how cells know what to become or how they get told by others. ex: stem cell to neuron

How does pattern formation occur and what is an example of that? (Fated?)

Every tissue starts with pattern formation. A section through the spinal cord shows different groups of neurons in different colors, they are fated to become specific things and depending on pathway it narrows down on what it will become.

What is morphogenesis?

Throughout development, the developing tissues do some intricate folding and cell movements. The cells move and flow to form the tissues of the body, become specific.

Why is growth important?

The mechanisms that allow us to grow to potential and not past determined size are vital to development. Mitosis and meiosis especially.

Why are model organisms important? What are some model organisms?

Model organisms have advantages and disadvantages in being used for research. Examples of this include a ferny plant, drosphilla, hydra (sea squirts which have a good larval stage), C. elegans (3 day lifecycle), Xenopus (large frog), danio rerio/zebrafish, chicks, mice/rats, and humans.

Why is it harder to use model organisms with vertebrates/spines?

Spine has more regulations to the research that can be done. IACUC

Developmental history of the frog

One of the earliest model organisms used for developmental research. Easy to find, externally fertilized embryos, and are large. They go through early development very quickly which is an advantage but then when they reach late juvenile stage (just hatched) it takes two years to reach sexual maturity. Which makes them a bad subject for genetic studies, not enough time! Gastrulation is well studied in frogs though.

How do you mate frogs for dev research?

Large female with smaller male typically. Males or researcher will squeeze females to release eggs. injected with gonadotropin night before to ripen ovaries. The male will fertilize the embryos as releases from female. In lab sacrifice male to acquire testes.

Lifecycle of a frog

gametogenesis to fertilization to cleavage to gastrulation to organgenesis to larval stages to maturity

How do frog eggs appear?

Darker side is animal pole and lighter side is vegetal pole. The cytoplasm has rotated so the darker side is where the nucleus is. In lab we incubate them to break it apart. It is easy to know who wasn’t fertilized because they don’t rotate brown side up.

What is a larval stage embryo that has hatched out of its protective shell called?

A chorion. Which will continue to grow larger before undergoing metamorphosis.

What is a fairly yolk less embryo called?

Isolecithal

What is a moderately yolky egg?

mesolecithal

What is holoblastic cleavage?

When the cell divisions are complete and go all the way through the yolk.

What cell types does holoblastic cleavage occur in?

isolecithal and mesolecithal cells.

What determines the direction of cell division in cleavage

the orientation of the spindles determine the direction.

What are the four types of holoblastic cleavage in isolecithal cells?

radial makes a cube shape, spiral creates s a 4 cell surrounding four more cells, bilateral makes a u shape with more cells inside, and rotational makes a circle with a cavity inside.

Why do yolky cells divide differently?

The yolk inhibits the cleavage furrow from going all the way through the cell. An example is in frog the yolk is on the bottom or vegetal pole and the furrow progresses slower through them leading eventually to an embryo that has more cells in the animal pole on the top.

What is in the yolk of cells?

It’s a thick and goppy substance that contains most of the proteins.

What are cells with thick yolk called?

telolecithal

What are the types of cleavage that can occur in telolecithal cells?

they are meroblastic so incomplete cleavage and it can be bilateral (cell with crack looking divisions in them), discoidal cleavage which is the cells appear protruding from the main cell, a cluster of them. In general the furrow cannot fully make it through yolk and the cells build up on top of a yolk ball.

What is centrolecithal?

A unique incomplete cleavage pattern that happens in drosphila that results in cells surrounding a yolky center.

What is invagination

creating an internal sheet of cells (epithelium) that is an indentation in the animal

what is involution?

inward movement of an expanding outer layer so that it spreads over the internal surface of the remaining external cells.

What is ingression?

Cells lose attachments to their neighbors and enter into the animal cavity

What is delamination?

It results in a second sheet of cells that splits off from a single sheet.

What is epiboly?

Cells migrate as a sheet to encase another cell type or the yolk.

What is the end results of the dividing of a fertilized egg?

3 layers; endoderm, mesoderm, and ectoderm. Animals approach making these differently. After these germ layers it distinguishes.

What is a blastula?

Made up of blastomeres (smaller nucleated cells)

What is a method to determine what cell does what?

Vital dye staining: This is basically fate mapping

Use a tracking dye if there are no intrinsic features or colors you could use. If you put the dye in different locations and at different stages you can mark cells and see where it will end up. External developing embryo makes this easier. An example of this is in zebrafish, when dye was injected in part of the embryo as gastrulation is beginning, the next day you can see those cells became part of the midbrain.

Why is fatemapping so useful?

If you wanted to manipulate a gene expression in a specific tissue you would have to know where those cells come from so you can target them.

Cross species transplants

have been used for ages. easiest organisms to cross transplant were chicken and quail. both being birds meant their development was similar. put cells from one species into another. Could see this because pigmentation differences when grown up.

What is a chimera?

one organism with two different genetic backgrounds.

What is fate mapping with transgenic DNA? Why is it important?

Where you mark cells through the use of genetically encoded fluorescent markers such as GFP. The GFP can be injected in a donor embryo or inserted into a locus on a chromosome so the animal makes it’s own GFP You can follow where cells go in real time. Example of this is tracking neural crest cells which show they control the motility of the gut.

What is a transgene?

A gene that is artificially introduced into the genome of another organism. Like GFP from jellyfish.

Why do vertebrates start development differently?

Because of the difference in the sizes of their eggs. By the beginning of neurulation (folding process in vertebrate embryos) all vertebrate embryos have converged on a common structure. As develop past neurula stage the embryos become less and less like each other.

homologous

structures that have underlying similarity can be attributed to a common ancestral structure. humans to bats

analogous

They perform a similar function without arising from a common ancestor.

A developmental anomaly caused by environmental agent

A few times in history we have learned from mutation. In 1960s pregnant women were given this drug to prevent morning sickness. Children were born with abnormal limbs. Taking the drug at different times in pregnancy caused different limbs to not develop properly. Which allowed us to discover when specific human structures develop in pregnancy.

what is specification?

capable of differentiation (reversible). started down a path but still a bit flexible.

what is determination?

lineage is determined (irreversible). once you become mesodermal, can’t then become a neuron is an example.

what is differentiation?

the generation of specialized cell types, officially become that cell/thing.

what is autonomous?

the cell knows intrinsically what to become

what is conditional?

the cell relies on environmental cues to tell it what to become.

what is cell fate determination? what’s a way to test that?

cell fates are determined early in development. using a fate map you can go back to those early cells and separate them and see what they become. If they become the same thing in a different environment they are autonomous.

How long do zebrafish take to sexually mature

3-4 months

How are fruitfly genes named?

On what they don’t have

How’s the best way to test genes?

Find it lose it move it

What is necessary

without this gene it won’t do that function/make that cell type

what is sufficient

if you move the gene somewhere else it by itself will trigger cell fate and do the same function. Does not need other players.

what’s a way to understand if differentiation is autonomous?

to see if the role of the environment changes the cell fates. If you place a muscle cell in a culture of neurons do you still get a muscle cell (autonomous) or do you get a neuron now (conditional)

what are limitations of cell fate determination?

time dependant, age factors, this has to happen early on and some cascades don’t occur till later

What is an example of autonomous development

in a snail embryo the red cells in the embryo go on to make ciliated trochoblast cells, and if you remove these and culture them they still become ciliated cells alone. Similar thing happens in the eye

development of a tunicate sea squirt (conklin)

cells in early organism have a distinct yellow to them called the yellow crescent. when cells divide they don’t evenly distribute and stay on one side. conklin made the first fate maple. yellow color protein is only muscle cells “macho”, this area later becomes the notochord. The proteins get moved with transport proteins and microtubules and actin. To determine if this fate map was autonomous the blastoderm cells of an embryo were dissected and cultured. Some made mass of ectoderm and some made notochord or muscle with no ectoderm. Which implied they are autonomous for a specific germ layer.

How would you test if macho is needed for muscle

Isolate it and culture it and if muscle occurs without it, it is not necessary.

What is gastrulation

a time in development where an embryo transforms from a one dimensional layer of cells a blastula to a more compex and multilayered structure a gastrula

What is conditional specification

where cells adopt the cell fate by interacting with their environment. if cells from one region are moved into a new location they adopt the cell fate of the new location. Similarly if the cells are removed, neighboring cells can recognize that and regenerate. This results in a normal embryo but if enough is removed the embryo may be smaller.

Roux attempt to show autonomous specification

By destroying one cell of a 2 cell frog embryo by stabbing it. the experiment resulted in the development of only one half of the embryo and his conclusion was that each cell once fully divided contains all information to make an entire organism.

Driesch conditional specification

When he dissociated a sea urchin embryo into single cells, each of the individual cells was capable of creating a larvae. There was discrepancy based on when in development they influenced the cells.

syncytial specification

unique to drosophila where cells undergo nuclear division without cytokinesis. After enough rounds of this you have one cell with 1000 nuclei in it (syncytium). All cells then migrate to periphery and cell membranes begin to form to isolate these nuclei into separate cells. All cells experience the same environment before division.

What are morphogen gradients?

They are during syncytial specification and they are unique because the gradients of these molecules are detected by every single nuclei in the syncytium. Bicoid is one of them on the anterior posterior axis that tells if it is anterior (head and thorax) and caudal tells if posterior (abdomen and tail). There is different information based on location. One represses the other as one goes up the other goes down. They are transcription factors.

How can we identify all the different genes that are turned on in all these different cells as developmental processes

cell fate maturation

an example in zebrafish embryos is if different ages are dissociated into single cell suspensions these cells are then passed through a machine that will insert a barcode on all the cdna molecules in the cell. the end result is a visual map of similar gene expression patterns across many cells and across time.

what is a transcriptome

full range of mrna expressed by an organism

by the time a zebrafish is 24 hr 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. more differentiated cells outward toward ectoderm and then meso then endo.

what is the central dogma of biology

transcription - in nucleus a region of genomic dna is seen by rna polymerase II which transcribes an exact complementary copy of the gene in a single pre mrna

processing - the pre mrna undergoes processing to make a finalized mrna strand

which is then transported out of the nucleus

translation - the mrna complexes with a ribosome and its info is translated into a polymer of AA

protein folding and modification - the polypeptide turns into secondary and tertiary structures through folding

the proteins are said to now be expressed and can carry out function

what are two techniques that detect where the mrna or protein are localized

in situ hybridization and immunohistochemistry

processing removes introns

The first exon is not where protein making always happens. it can happen further downstream.

what makes mrna stable

the 5’ cap and poly a tail

the bridge between enhancer and promoter can be made by what…

transcription factors.

certain transcription factors called basal transcription factors bind to DNA on the promoter (where rna polymerase II initiates transcription) whereas other transcription factors bind to the enhancer (which regulates when and where transcription can occur)

other transcriptional regulators do not bind to the dna rather they link the transcription factors that have bound to the enhancer to the promoter.

If the brain specific enhancer and limb specific enhancer are both active where would the gene be expressed?

gene would be expressed in both tissues, because the enhancers came before the Gene sequence and no specific transcription factors were interfering.

Example: brain specific transcription factors can bind to the brain enhancer which then causes it to bind to the mediator, stabilize rna pol II at the promoter and modify nucleosomes in the region of the promoter. the gene is transcribed in the brain only, limb enhancer does not function. Basically the mediator cuts off the limb enhancer part.

genes are not transcribed in any cell type whose transcription factors the enhancers cannot bind.

Why is knowing where the enhancers and promoters are in a gene important?

For making promoter lines. If you clone the promoter or enhancer before a marker like gfp you can identify which cells are using the enhancer.

ex beta galactosidase

Not all parts of the 5’ utr enhance or promote gene expression. repressor seq. prevent gene expression. what is this called?

a silencer! and if you remove this, then that gene would turn on.

what is the structure of dna and proteins that form nucleosomes and chromatin?

the dna helix is wound around the protein core, the histone tails that extend from the core are sites of acetylation and methylation, which may disrupt or stabilize the formation of nucleosome assemblages.

what regulates gene expression

closing dna up, winds dna around histones making sure it is not accessible.

what is a histone

protein that provides structural support for a chromosome.

how many base pairs of dna circle each histone octamer

147bp and 60-80 bp link nucleosomes together.

histones come together to wrap the dna. when its in its tightly wrapped conformation there is little transcription happening. modification on the tail regulate how tightly coiled the histones are around the dna.

What do condensed nucleosomes look like

the histone tails are largely methylated

what do uncondensed nucleosomes look like

histone tails largely unmethylated and acetylated

What do methyl groups do

they condense nucleosomes more tightly preventing access to promoter sites and thus preventing gene transcription

What does acetylation do?

loosens nucleosomes exposing the dna to rna pol II and transcription factors that will activate the genes.

Why is the tail of histone h3 so special

it is capable of being methylated or acetylated. certain lysines are associated with gene activation and some with repression

how does dna methylation decrease gene transcription?

MeCP3 recognized methylated cytosines of dna. it binds to the dna and is thereby able to recruit histone deacetylases which take acetyl groups off the histones and decrease availability of the DNA. it also recruits histone methyltransferases, which both promote stability of nucleosome and the tight packing of DNA thereby repressing gene expression in these regions of DNA methylation.

what is dnmt3 de novo methyltransferase

it is crucial and wants to shut gene off with new methyl group on unmethylated cytosines.

what is dnmt1 perpetuating methyltransferase

it recognizes methylated Cs on one strand and methylates the C on the CG pait on the opposite strand.

What are imprinting disorders

dnmt methylation effects are heritable. this epigenetic effect can be detrimental. example is prader-willi which is due to paternal imprinting and angelman syndrome which is due to maternal imprinting.

what is an isoform (method of differential gene expression in pre mrna processing)

variant of same gene product that is spliced differently, they code for the same protein they have slightly different functions but same enzymes.

what is differential pre mrna splicing?

a way to regulate different subsets of genes being made. it can process the same pre mrna into different mrnas by selectively using different exons. 38000 splice variants.

what does differential mrna longevity mean (method of differential gene expression in mrna translation)

how long the transcript is in the cell. some are degraded faster than others. the longer they are there the more protein can be made.

how can mrnas be stabilized

length of poly a tail, or by other rna binding proteins (Hu)

what are stored oocyte mrnas used for (method of differential gene expression in mrna translation)

this means maternal contributions have enough for the first few cell divisions (splicer proteins rna pol etc) all in the egg. This was tested in zebrafish because transcription does not turn on till 1000 cells but the futile cycle mutant with only pronuclei survives for a bit because of maternal.

what is ribosome selectivity (method of differential gene expression in mrna translation)

selective activation of the translation of mrna. hox gene translation leads to normal vertebrate.

what do miRNAS function in? (method of differential gene expression in mrna translation)

RNA interference and blocking translation. It blocks the sequence and ribosome from translating. miRNAS also play a role in clearly out the maternal transcripts the areas of influence in the blastula.

what and why cytoplasmic localization of mrnas (method of differential gene expression in mrna translation)

for example in the syncytium it will have to have localized transcription to set up the body plan where body parts are. This is done by diffusion and anchoring certain mrna transcripts.

how mrnas get places in cytoplasm

with dynein and kinesin and microtubules