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BIOL 309 > Early Invertebrate Development (3) > Flashcards

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
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2
Q

Where is B-catenin expressed?

A
  • through correlative data, found expression in micromeres and veg2 layer
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3
Q

What occurs if the wnt pathway is activated?

A
  • increase in nuc-beta-catenin

- more endomesoderm

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4
Q

What occurs if the wnt pathway is blocked?

A
  • decrease in nuc-beta-catenin

- no endomesoderm

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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
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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
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7
Q

What prevents skeletogenic differentiation genes from being activated?

A
  • Hnf6

- HesC: repressor

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8
Q

What are the 3 types of control mechanisms within the micromeres-PMC regulatory network?

A
  1. repressor of a repressor or double negative gate: HesC is inhibited, disallowing it to inhibit
  2. positive feedback loop: B-catenin activates pathway but also activates wnt8 which produces more b-catenin (?)
  3. “feedforward”: allows circuits to build additional mechanisms of regulation
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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

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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
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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
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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
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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
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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

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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
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16
Q

What are the putative “direct Tx regulators” that were identified?

A
  • Spkrl ->E, F
  • UI -> B
  • Otx -> A
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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
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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
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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
Q

What kind of cellular/molecular mechanism could generate this mosaicism?

A
  • perhaps cell polarity, differences in expression at a transcription level, asymmetrical cell division
26
Q

What model organism is C. intestinalis?

A
  • 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
Q

What does correlative data show about the tunicate embryo shortly after fertilization?

A
  • asymmetry is present in one cell stage as there is a darker yellow crescent made of cytoplasm that segregates after fertilization
28
Q

What does functional data of tunicate embryos show?

A
  • 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
Q

What hypothesis and experimental approach does the correlative and functional data of the tunicate embryos lead to?

A
  • hypothesis: localized (maternal) muscle inducing factors are present in the egg and/or early embryo
  • gene discovery via cDNA differential screen
30
Q

What exactly was done to look at if localized muscle inducing factors are present in the egg/embryo?

A
  • 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
Q

What are ubiquitous genes?

A
  • genes that are expressed in two different DNA

- such as in the vegetal DNA and the animal DNA

32
Q

What specific veg gene was found?

A
  • macho-1

- “candidate gene”: could be localized muscle inducing factor

33
Q

What is in situ hybridization?

A
  • uses a labeled complementary DNA or RNA to localize a specific DNA or RNA sequence
34
Q

What did in situ hybridization show about macho-1?

A
  • 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
Q

Which cell is fated to become muscle?

A
  • B4.1
36
Q

What experiments can follow the correlative findings about macho-1?

A
  • 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
Q

What is antisense and what does it do?

A
  • 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
Q

What gain of function experiment can be done to examine macho-1?

A
  • macho-1 RNA mis-expression
  • assay: look for muscle myosin immunostaining (muscle-specific protein)
  • inject macho-1 into different cells
39
Q

What was found in the gain-of-function experiment done to examine macho-1?

A
  • 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
Q

What controls were used for the gain-of-function macho-1 experiment?

A
  • negative control: they observed an uninjected embryo

- positive control: uninjected B4.1 since it should produce muscle

41
Q

What do gain-of-function experiments tell you? loss-of function?

A
  • gain of function: tells us that something is sufficient

- loss of function: tells us that something is necessary

42
Q

Summarize the experiments done on macho-1?

A
    1. gene discovery using cDNA differential screen
    1. correlative data using macho-1 in situ hybridization
    1. loss-of-function using macho-1 antisense RNA injection
    1. gain-of-function using macho-1 RNA mis-expression
  • macho-1: early muscle specification factor
43
Q

What is Tbx6?

A
  • 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
Q

What is the “master control gene”?

A
  • macho-1
  • zinc finger transcription factor
  • controls myosin, muscle actin, tbx6 and snail
45
Q

How can single/small group transcription factors have such a big impact on cell fate?

A
  • many downstream target genes that are regulated up or down
  • can be examined using microarray analysis
  • can be direct or indirect
46
Q

What is one of the most important underlying mechanisms in developmental biology?

A
  • establishing asymmetry

- polarity: body axis specification, tissue regionalization and subdivision

BIOL 309 (14 decks)

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