Early Invertebrate Development (3) Flashcards
1
Q
What is a pluteus?
A
- a feeding sea urchin embryo with all three defined germ layers
- after about one day of development
2
Q
Where is B-catenin expressed?
A
- through correlative data, found expression in micromeres and veg2 layer
3
Q
What occurs if the wnt pathway is activated?
A
- increase in nuc-beta-catenin
- more endomesoderm
4
Q
What occurs if the wnt pathway is blocked?
A
- decrease in nuc-beta-catenin
- no endomesoderm
5
Q
How is beta-catenin signaling activated in the veg2/micromeres?
A
- maternal nuclear b-catenin that is initially independent of the wnt ligand
6
Q
What is found in the cytoplasm of micromeres?
A
- activated form of disheveled which prevents the degradation of b-catenin by inhibting GSK-3
7
Q
What prevents skeletogenic differentiation genes from being activated?
A
- Hnf6
- HesC: repressor
8
Q
What are the 3 types of control mechanisms within the micromeres-PMC regulatory network?
A
- repressor of a repressor or double negative gate: HesC is inhibited, disallowing it to inhibit
- positive feedback loop: B-catenin activates pathway but also activates wnt8 which produces more b-catenin (?)
- “feedforward”: allows circuits to build additional mechanisms of regulation
9
Q
Why are control mechanisms important during development?
A
- control of balance of gene expression is critical
- having a double negative gate allows for genes to be turned off
10
Q
What is the endo16 gene?
A
- endoderm specific gene
- expression comes on late in gastrulation when the gut is being formed
- expressed in gut
11
Q
What experimental approach could be used to identify the transcription factors that directly regulate endo16?
A
- look at cis-regulatory elements (on the same strand of gene may have regulatory elements in close proximity)
- the easiest place to start would be looking upstream from endo16 (but does not necessarily need to be)
- use GFP to see if gene is being expressed
12
Q
What exactly was done to look at transcription factors for endo16?
A
- inject a sequence with a reporter gene into an egg
- fertilize the egg
- examine reporter gene expression
- examine the expression of different “deletion constructs”: take out different upstream areas to see effect
13
Q
What did the researchers find when looking for transcription factors for endo16?
A
- removal of some upstream areas resulted in no expression meaning they are important for expressing endo16
- removal of some upstream areas caused expression in areas outside of the gut meaning they are important for inhibiting expression
14
Q
After identifying the important upstream areas of transcription factors for endo16, what would be the next step?
A
- to determine what is binding to these sites
- use bioinformatics approaches to identify predicted transcription factor binding sites
15
Q
What areas were identified on the endo16 gene?
A
- A: promotes expression in vegetal half
- B: promotes late expression in midgut
- C, D: shuts off expression at skeletogenic mesenchyme boundary, PMCs
- E, F: shuts off expression at ectoderm boundary
- G: boosts expression of A and B
16
Q
What are the putative “direct Tx regulators” that were identified?
A
- Spkrl ->E, F
- UI -> B
- Otx -> A
17
Q
What experimental approach would be used to show that the TF’s are direct regulators of endo16 transcription?
A
- label the transcription factor or use antibodies
- use a gel shift assay: if transcriptor is binding, a shift in the gel assay would be seen
- block the binding site by mutating the binding site
- negative control: not having the transcription factor at all or a different piece of DNA
18
Q
What exactly was done to show that the TF-s are direct regulators of endo16 transcription?
A
- transcriptional transactivation/repression assay
- have a reporter gene (e.g. luciferase, GFP, CAT)
- transfect SpKrl and reporter into cell line that does not have SpKrl
19
Q
What is a weakness of the transcriptional transactivation/repression assay?
A
- does not tell if physiologically relevant in vivo
20
Q
What is ChIP?
A
- chromatin immunoprecipitation
- dissect gut tissue
- crosslink DNA/protein with formaldehyde
- fragment DNA to 100-200 bp pieces
- immunoprecipitate with antibody specific to SpKrl
- PCR region where SpKrl is expected to bind and see if band is present
21
Q
What does the sea urchin endomesoderm regulatory networks (big map) provide?
A
- provides mechanistic basis for cell fate determination
- reveals cell autonomous and non-autonomous interactions
22
Q
What occurs when the a sea urchin embryo at the 4 cell stage has its fertilization envelope removed and is separated into 4 cells?
A
- 4 plutei are developed from each single cell
- each resulting embryo “regulated” its own development
- this was the first experiment to provide evidence in support of regulative development
23
Q
What is regulative development?
A
- “conditional specification” by interaction between cells
24
Q
What is mosaic development?
A
- development strictly adheres to the fate map
- e.g. removal of cells early in development leads to loss of structures fated by those cells
- cell fate specification would be directed by cell-autonomous events
25
What kind of cellular/molecular mechanism could generate this mosaicism?
- perhaps cell polarity, differences in expression at a transcription level, asymmetrical cell division
26
What model organism is C. intestinalis?
- tunicates
- have a notochord
- closer to humans then flies
- deuterostomes and chordates
- usually the swimming larval stage is studied (contains nerve cord, sensory vesicle, notochord, muscle cells)
27
What does correlative data show about the tunicate embryo shortly after fertilization?
- asymmetry is present in one cell stage as there is a darker yellow crescent made of cytoplasm that segregates after fertilization
28
What does functional data of tunicate embryos show?
- isolation studies
- top two left cells: ectoderm
- top two right cells; ectoderm
- bottom two left cells: notochord and endoderm
- bottom two right cells: muscle, mesenchyme and endoderm
29
What hypothesis and experimental approach does the correlative and functional data of the tunicate embryos lead to?
- hypothesis: localized (maternal) muscle inducing factors are present in the egg and/or early embryo
- gene discovery via cDNA differential screen
30
What exactly was done to look at if localized muscle inducing factors are present in the egg/embryo?
- take embryos at 8 cell stage
- use water and needle to separate poles
- isolate RNA from both poles
- use reverse transcriptase enzyme to convert RNA to DNA
- cDNA A gets biotin added to it and acquire concentration of cDNA A 100 fold that of cDNA V
- lower heat to denature and then hybridize to get cDNA V-ubi and b-cDBA A-ubi as well as cDNA veg-specific
31
What are ubiquitous genes?
- genes that are expressed in two different DNA
| - such as in the vegetal DNA and the animal DNA
32
What specific veg gene was found?
- macho-1
| - "candidate gene": could be localized muscle inducing factor
33
What is in situ hybridization?
- uses a labeled complementary DNA or RNA to localize a specific DNA or RNA sequence
34
What did in situ hybridization show about macho-1?
- as an unfertilized egg, macho-1 is already asymmetrical by being highly localized
- it remains highly localized after fertilization
- at the 8 cell stage, it is still highly localized and is only present in one cell
- correlative data
35
Which cell is fated to become muscle?
- B4.1
36
What experiments can follow the correlative findings about macho-1?
- loss-of-function experiment
- using macho-1 antisense RNA injection
- assay: look for muscle using myosin immunostaining (muscle-specific protein)
- with macho-1 antisense RNA injected = no muscle is formed
37
What is antisense and what does it do?
- antisense: complimentary sequence to RNA 'sense' strand
- can be used in loss of function experiments since it will pair up with the RNA 'sense' strand and block transcription of that specific section
38
What gain of function experiment can be done to examine macho-1?
- macho-1 RNA mis-expression
- assay: look for muscle myosin immunostaining (muscle-specific protein)
- inject macho-1 into different cells
39
What was found in the gain-of-function experiment done to examine macho-1?
- injected into B4.1: still get muscle
- injected into A4.1. a4.2, b4.2: get muscle in wild type 25 pg and wild type 125 pg
-
40
What controls were used for the gain-of-function macho-1 experiment?
- negative control: they observed an uninjected embryo
| - positive control: uninjected B4.1 since it should produce muscle
41
What do gain-of-function experiments tell you? loss-of function?
- gain of function: tells us that something is sufficient
| - loss of function: tells us that something is necessary
42
Summarize the experiments done on macho-1?
- 1. gene discovery using cDNA differential screen
- 2. correlative data using macho-1 in situ hybridization
- 3. loss-of-function using macho-1 antisense RNA injection
- 4. gain-of-function using macho-1 RNA mis-expression
- macho-1: early muscle specification factor
43
What is Tbx6?
- transcription factor involved in muscle specification in both tunicates and mammals
- across evolution remarkably high degree of conservation
- activates other genes required for muscle development
44
What is the "master control gene"?
- macho-1
- zinc finger transcription factor
- controls myosin, muscle actin, tbx6 and snail
45
How can single/small group transcription factors have such a big impact on cell fate?
- many downstream target genes that are regulated up or down
- can be examined using microarray analysis
- can be direct or indirect
46
What is one of the most important underlying mechanisms in developmental biology?
- establishing asymmetry
| - polarity: body axis specification, tissue regionalization and subdivision
