Organism Dev Flashcards
What is forward genetics vs backward genetics?
Forward genetics → Starting from the phenotype (weird) and looking for what changes occurred in the genotype
Reversed genetics → Starting from changes in the genotype and seeing how it affects the phenotype
What are 2 important characteristics of model organisms?
- Mimic specific aspects of human biology
- Are (comparatively to humans) easy to work with
What are the main 5 eukaryotic model organisms?
- S.cerevisiae (yeast)
- C. elegans
- Drosophila melanogaster
- Danio rerio (zebrafish)
- Mus musculus (house mouse)
From 1 → 5: Get more like humans, but get more difficult to grow/work with
*Diverged from human more and more recently in history/development
What are the characteristics of S. cerevisiae?
(generation time, advantages, disadvantages)
- Eukaryotic, UNICELLULAR fungus
- Generation time: 2-3 hours
- Can exist as haploid or diploid
- Can reproduce sexually and asexually
- Can be frozen and revived (easy)
What is measured when we talk about the generation time?
It is the time between when the embryo is conceived to when the offspring is capable of reproducing itself (mature enough), of conceiving an embryo
What does the life cycle/reproduction cycle of S. cerevisiae look like?
- Haploid go through asexual reproduction (budding) → haploid offspring
- Haploid can mate to make diploid offspring → mitosis → back to haploid (x2)
- Diploid can bud and produce diploid offspring
What are the advatanges of yeasts in their haploid form vs in their diploid form?
Haploid form → easy to study gene effect (direct)
Diploid form → study gene interaction, patterns of dominance, trans-acting, etc.
What are the characteristics of C.elegans?
(generation time, advantages, disadvantages)
- Invertebrate animal, multicellular
- Generation time: 3 days → 300 progeny (eggs)
- Extremly simple → translucent
- Can trace fate of each cell (1090 total)
- Can be frozen and revived
2 sexes: male (XO) & hermaphrodite (XX):
- Hermaphrodite can self-fertilize → make mostly hermaphrodites
- Can also mate with males → different combinations of genes
What does the life cycle of C. elegans look like?
Normal cycle:
L1 → 12h + 8h + 8h → L4 → 18h → Reproductive adult (can lay eggs) → embryo → 14h → L1
Dauer stage → if exposed to a tough envrionment → hibernation → can later develop and lay eggs when get out of Dauer stage
take ~ 13h from L1 → Dauer (can then stay several months) → L4 (out of Dauer)
What are the characteristics of D. melanogaster?
(generation time, advantages, disadvantages)
- Invertebrate animal, mutlicellular
- Generation time: 10 days → 100 progeny
- More complex than C. elegans
- Share 75% of human disease-causing genes (how they affect specific cell-types)
- Very well studied, many genetic tools
What are the characteristics of Danio rerio?
(generation time, advantages, disadvantages)
Danio rerio = zebrafish
- Vetebrate animal, multicellular → CNS closer to human
- Generation time: 2-3 months → 200 eggs
- Optically translucent embryo & larva
- Relatively simple & inexpensive to maintain (need aquarium)
- Easily treated with small molecules for drug & toxicity screens
What are the characteristics of Mus musculus?
(generation time, advantages, disadvantages)
- Vertebrate MAMMAL
- Generation time: 3 months → 2-12 pups
- Small, easy to house (for a mammal)
- Commonly used to study human biology, perform preclinical testing
**Mice are not always perfect models for human, there are some major differences!!
What is special about Axolotl mexicanum as an emerging model organism?
In can regenerate its limbs
What is special about Planaria as a model organism?
It can regenerate its whole body from any cut part (full of stem cells)
What are the 3 main steps of a forward genetic screen?
- Perturb a lot of genes (randomly or systematically)
ex: chemical mutagen - Look for a specific phenotype
ex: The organism dies, changes in some specific way - Figure out which gene you mutated
How was cell cycle of S.cerevisiae explored using a forward genetic screen?
Why is S. cerevisiae used to study cell cycle?
We can visualize different stages of the cell cycle easily:
G1 phase → tiny bud
S phase → DNA being replicated and pushed into the bud
G2/M phase → bud separation → 2 cells
What is the mechanisms and the importance of temperature sensitive mutants?
Problem: The cell division cycle mutants of interest couldn’t be cultured and replicated because they couldn’t go through the cell cycle
Solution: Temperature sensitive mutants → At low permissive temperatures, the yeast can replicate/divide
At high restrictive temperatures, the mutant phenotype can be observed (blocked at a specific stage of the cell cycle) → can go back to dividing when brought back to lower temperatures
*So mutants can be expanded and studied
What is replica plating used for?
- Put different colonies in aggregates in a master plate
- Stamp the colonies on a sterile velveteen (sticky)
- Stamp the colonies on different plates at different temperatures
- Can see which mutants grow or not at different temperature and compare since all the plates have the same organization
Used to isolate temperature sensitive mutants
What process is affected in cdc1 mutant yeasts?
Cells have a small bud, but won’t start replication, bud stays very small (stops in G1)
Functional gene product: Putative metallophosphodieasterase → involved making cell wall (not helpful for humans)
What process is affected in cdc2 mutant yeasts?
Mutants can form a bud, can see the start of DNA replication, but doesn’t complete it, stops in S phase
Functional gene product: DNA polymerase delta catalytic subunit
What process is affected in cdc3 mutant yeasts?
Cdc3 mutants stop in late stages of mitosis, division of the buds can happen to a certain extent, but the cells can’t separate 100%
Functional gene product: Septin family member, involved in cytokinesis
How did researchers confirm that 2 yeasts with the same phenotype had a mutation in the same gene?
By complementation test:
If mating the 2 haploid yeasts together → diploid mutant → same gene
If matin the 2 haploid yeasts together → diploid WT → different genes bc the WT copy of each gene complemented
*2nd stage of the forward screen
How did researchers proceed to identify which was the mutant gene in the yeast after having identified the different cell cycle mutants? (cdc)
*Final stage of a forward genetic screen
Try to rescue the mutant with different DNA plasmids:
- Yeast dsDNA → cleaved w/restriction endonuclease
- Thousands of genomic DNA fragments
- DNA fragment inserted into plasmids
- Introduce into multiple bacterias → Yeast genomic DNA library
- Introduce different plasmids into yeast → test which plasmids rescue the mutant
Sequence plasmid that rescues → gene that is mutated in the mutant yeast
*plasmids are WT
How did researchers assess if some human genes were related to the mutated cdc genes in yeast?
Made cDNA plasmids from human mRNA → test which plasmid rescuse the yeast cdc mutant
What was the human version of cdc28 identified through forward genetic screening?
cdc2 (S. pombe) = cdc28 (S. cerevisiae) = Cdk1 (human)
*Very conserved in cell cycle regulation
Which model organism was used to analyze apoptotic pathways?
Why?
C. elegans
- Every embryo develops with the same pattern of cell division & migration
- Adults worms have exactly 959 cells generated in the exact same way
1090 cells formed during embryogenesis → 131 die, the exact same always
*Only disadvantage, when cells die, they are difficult to see as they are engulfed very quickly
What was seen in the ced-1 C. elegan mutant?
In ced-1, they could see spots of dead cells → cells could dye, but could not be engulfed
Can use that to see if cell death is affected by other mutants because you can visualize cell death → They did a suppressor screen to find mutants in the ced-1 background
What was seen in the ced-1 and ced-3 double mutants in C. elegans?
They could no longer see visible corps → no cell death → means cell death is an active process as it is regulated/induced by specific genes
What is specific of Egl-1 C. elegans mutants? What explains it?
Egl-1 can make eggs but can’t lay them → fat worms
It is explained by the lack of Hermaphrodite-specific neurons (HSN) which are required for cell death → a bit upreagulated cell death and HSN are more sensitive
*Used to do supressor screens
What gene was found with the C. elegans Supressor screen with egl-1?
Discovery of ced-4 :
Ced-4 induces cell death so by mutating it → mutation in ced-4 leads to less cell death → rescue of HSN → C. elegans can lay eggs
What was seen in C. elegans ced-9 mutants?
What human gene is it similar to?
Excessive cell death
Ced-9 WT must allow cell survival and its absence leads to cell death
*In over expression of WT ced-9 → NO cell death at all → extra 131 cells in C. elegans
Ced-9 has a very similar sequence to human Bcl2 gene → Human Bcl-2 can modulate cell death in C. elegans because so conserved
What is the normal signaling pathway leading to cell death in human?
In none cell death → Bcl-2 inhibits BAX & BAK (by binding and sequestering)
1. In death conditions, BAX & BAK premeabilize the mitochondrial membrane
2. Mitochondrial cytochrom c is released → activates caspase 9
3. Caspase 9 cleaves and activated caspase 3 → irreversible cell death
*Bcl2 = human proto-oncogene
What human genes are similar to C. elegans ced-9, ced-4, ced-3 genes?
What is the effect of a loss of function of these genes?
ced-9 = human Bcl-2 → loss of ced-9 causes more death
ced-4 = human Apaf1 (binds to pro-caspase9 to activate it) → loss of ced-4 causes less death
ced-3 = human Caspase3 → loss of ced-3 causes less death
Which C. elegans gene does Egl-1 inhibit ? What is the effect?
Egl1 inhibits ced-9 (Bcl-2)
*Original mutation on Egl1 was a gain of function → stronger ced-9 inhibition → more cell death
What is lin-4?
What was the results of the Northern blot investigation?
lin-4 C. elegans gene encodes for miRNA that inhibits
Nothern blot results:
Lin-4 encodes 2 small RNAs → lin-4L (60bp, hairpin) + lin-4A (21bp, linear)
By sequencing → lin-4 RNAs are complementary to lin-14 mRNAs
What is lin-14?
C. elegans genes that controls developmental timing in early larva stages
What photypes where observed in development of lin-14 (lf), lin-14(gf) and lin-4?
What is the name of these types of mutants?
lin-14 (lf) → skipped early larva stage of dev. in cell division
lin-14 (gf) → stuck in early larva stage of dev.
lin-4 mutant → stuck in early stage of dev.
*Heterochronic mutants → affect developmental timing
What were the protein levels of lin-14 in the following mutant C. elegans?
WT, lin-14 (lf), lin-14(gf), lin-4
WT ~ medium
lin-14(lf) → None
lin-14(gf) → More than WT
lin-4 mutant → More than WT (same as lin-14 gf)
*lin-4 is a repressor of lin-14
What is another example of gene with similar characteristics as Lin-4 in C.elegans?
Let-7:
- Mutation is heterochronic (affected development)
- Nothern blot detects 21bp product → complementary to other heterochronic RNAs
*Could be common mechanisms for gene regulation?!
How were human miRNA studied ?
- Isolated all 21 bp RNAs from a gel
- Clone them into plasmids
- Sequence them
- For a library of 33 miRNA from human (HeLa) cells
miRNAs all come from similar hairpin-forming sequences
How does miRNA processing and activity occur?
- Drosha cuts pri-microRNA into a ~ 70 bp hairpin in the nucleus
- Exportin 5 bring it to the cytoplasm
- pre-microRNA’s stem loop spliced by Dicer → 21 bp fragment
- Strand selection + Argonaute interaction → RISC complex
Effect:
- mRNA target cleavage
- mRNA deadenylation and decapping
- Translation inhibition
How do miRNAs recognize their targets? How specific are they?
Mostly the 3’ and 5’ end of the miRNA are complementary, variable lengths and places in the 3’UTR of the target mRNA
- One miRNA can bind multiple targets
- One mRNA can be bound & regulated by multiple miRNAs
- In vertebrates, miRNAs tend to be a gentle regulator mechanism → fine-tuning gene expression level, not all or nothing
What general area was studied through seveless gene forward genetic screens?
Signaling pathways and their modulators
what is the structure of the components of drosophila eyes?
1 eye component = ommatidium → each ommatidium has 8 photoreceptor cells (numbered in specific arrangement)
What is the phenotype of the Sev drosophila mutant?
R7 (of the 8 photoreceptors/ommatidium) can’t form
*Seen with immunofluorescence staining, anti-R7 will not bind
What is the effect of a Boss mutant?
What is the function of that gene?
Bride of Sevenless (Boss) mutants for all cells → don’t form R7
Boss mutants in only R7 → R7 forms
Boss mutants in only R8 → R7 forms
*Boss plays a cell non-autonomous role in R7 development
Boss is the ligand for Sev RTK → downstream signaling pathway for formation of R7
What phenotype was observed in Sev (LOF), Sev (GOF) and Sev(GOF), Sos(LOF)?
Sev (LOF) → no R7 cells in ommatidium
Sev (GOF) → extra R7 cells
Sev (GOF); Sos (LOF) → 1 Single R7 cells
What are the downstream components of the sevenless RTK pathway?
- Sev RTK binds Boss
- Sev RTK phosphorylates
- Phosphorylated Sev RTK recognized SH2 adaptor protein (Drk)
- Interact with Ras-GEF (Sos)
- Ras-GDP → Ras-GTP
- Downstream signals for development of R7
What is Sos protein?
Sos is a Ras-GEF → turns GDP to GTP → activate Ras → downstream signaling in development of R7/R7 specification
What are the 2 types of transposons?
(Parasitic DNA elements)
Class I: copy and paste → similar to retrovirus
Class II: cut and paste → more helpful for genetic screening, encode machinery to allow dsDNA to be excised and put back elsewhere in the genome
What is a gene trap?
It is an insertional construct that that consist of flanking regions, a transposase coding sequence, reporter gene, selection gene, etc. → can jump into the genome of the host
- Interrupts a gene by ranodm insertion → loss of function
- Inserts a reporter (GFP) that can be used to visualize where the gene is expressed
- Usually combined with a genetic screen to provide additional information
What is the enhancer trap?
It is a insertional construct that contains a weak promotor upstream from the reporter gene
- Used to identify enhancer that promote gene expression in a specific cell type
- Inserted DNA does not need to land inside a gene
- See tunned expression in different tissues and cells
By which 3 mechanisms does 1 single cell become an organized & functional organism?
- Cell proliferation
- Cell differentiation
- Cell morphogenesis
What are the main steps of the first few days of mammalian development?
- Fertilization
- Cleavage division → no growth, just division
- Major EGA → degredation of maternal RNA, embryo starts relying on its own genome (day 4)
- Compaction → cells interact with adherens, increase in adhesion between cells
- 1st Lineage → trophectoderm + inner cell mass → Epiblast
- Cavitation → pumping fluid inside (helped by compaction so fluid doesn’t leak out) → Early Blastocyst
- Expansion and hatching → Late Blastocyst
- Implantation → blastocyst implants onto the uterin wall
How do cells know wether the differentiate into Trophectoderm cells or inner mass cells?
If cell have contact with other cells on 100% of their surface (inside) vs if they have an area with no contact (outside)
*Before pumping of fluids (cavitation)
1. F-actin accumulates in exposed cell membrane → sequesters YAP kinases (LATS1/2) → can’t phosphorylate YAP → YAP moves into the nucleus → turns on specific genes for differentiation of cells into Trophectoderm cells
*YAP = transcriptional coactivator
- In inner cells, LATS1/2 is not sequestered to the membrane → phosphorylates YAP → phosphorylated YAP can’t enter the nucleus → no transcription of the Trophectoderm factors
What structure emerge from Trophectoderm and Inner cell mass?
Trophectoderm → Placental cells
Inner cell mass → All cells of our body
What are the 2 general effects of gastrulation?
- It generates the 3 embryonic germ layers
- It establishes the 3 main body axes
What cell types are made in the different germ layers?
Ectoderm:
- Skin cells
- Pigment cells
- Neurons
Mesoderm:
- Skeletal, cadiac, smooth muscles
- RBC
- Bone tissues
Endoderm:
- Digestive tract/stomach cells
- Thyroid cells/pharynx
- Respiratory tube/lung cell (alveolar cells)
What are the 3 main body axes?
- Anterior-posterior (head-feet)
- Ventral-dorsal (stomach-back)
- Right-left
What step follows gastrulation in embryo development?
Organogenesis → almost all in 1st trimest
2nd and 3rd trimests are for growth
What did Ubx mutation show?
*Ubx = Ultrabithorax, expressed in a specific region of the embryo → showed homeotic transformation
loss of Ubx → loss of the haltere segment, replaced by a duplication of the wing segment
gain of Ubx → duplication of the halter segment, no wings
What does Hox genes/Hox clusters control?
Hox genes control anterior-posterior (AP) patterning
What differs in drosophila vs human Hox clusters?
*Both derived from 1 ancestral Hox complex, conserved in almost all animals to some level
Drosophila → all in 1 cluster
Human → 4 different Hox clusters on 4 different chromosomes
What is the effect of an over-expression of HoxA10 in all vertebrates vs a loss of HoxA10?
Hox A10 important for lumber part of vertebrates
Over-expression of Hox A10 → All vertebrates are lumbar (no thoracic vertebrates anymore)
Loss of Hox A10 → Lumbar vertebrates become thoracic → can see ribs all the way to the sacral vertebrates (included) (more than 12 ribs)
How is the A-P axis reflected in individual cells?
Through planar cell polarity —> each cells knows where is the anterior and posterior
- Pathway that transduces the global pattern to cellular information
Different components expressed on both sides:
Proximal/Anterior —> Van Gogh/Strabismus, Prickle
Distal/Posterior —> Frizzled, Dishevelled, Diego
What is essential for building neural circuits?
Dorsal-ventral patterning
- Ventral horn —> Proprioception
- Dorsal horn —> Dorsal root ganglion —> Mechanoreceptors, Nociceptors, Propriceptors
What are morphogens (their role)?
Diffusible signals that exert graded effects —> ventral-dorsal gradient of the neural tube for example
- Source of morphogen = 1 cell
- Cells are exposed to a different concentration depending on their distance (diffusion) from the morphogen source —> different cellular response
What are some common morphogens?
- Shh
- BMP
- TGF-beta
- Fgf
- Wnt
*Same morphogens are used in different organs and different stages of development
How is the nueral tube patterned (dorsal-ventral)?
By morphogen gradient:
- Dorsal BMP
- ventral Shh
Different combinations of concentration —> different expression patterns/cellular decisions
What are the steps for formation of the neural tube?
- Neuroectoderm limited by the Neural plate border
- (Neuroectoderm —> Neural plate) + (Neural plate —> Neural fold)
- Both neural folds assemble and dissociate from the non-neural ectoderm
- Neural tube is done —> neural crest cells form gradually
Which 2 mechanisms allow the body to re-use the same signaling pathways over and over for different purposes during development?
- Combinatorial signaling:
Different sombinations of signals/concentrations/contact cells/etc. - Cellular memory
- 2 different cells exposed to the same signal will not respond in the same way
- Usually due to chromatin organization/epigenetic status —> different response to the same signal
What are the main changes in the 2nd and 3rd trimerster of fetal development?
Mostly growth of the organs and tissues
1. Cell growth (single cell)
2. Cell division (make more cells)
3. Modulating cell death (prevents from getting smaller)
What pathway regulates cell division and cell death in fetal development?
Lats1/2 phosphorylates YAP —> Phosphorylated YAP can’t enter the nucleus
Non-phosphorylated YAP —> enters the nucleus —> Myc (growth), Cyclin E (division), Diap and Bantam (survival) —> tissue growth
*Same signaling pathway as differentiation of trophectoderm and inner cell mass
What is the process of gastrulation?
Allows the epiblast to establish the 3 primary germ layers:
- Formation of the primitive streak (near posterior end of bilaminar embryonic disc) → defines anterior/posterior and right/left axis
*Anterior end of the primitive streak (~ middle) → primitive node - Invagination: Cells migrate through the primitive groove, and downwards
- 1st cells to invaginate → replace hypoblast cells → Endoderm
- Invaginated cells that are between the hypoblast and the epiblast form the mesoderm
- Remaining cells of epiblast (don’t invaginate) → Ectoderm
