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Molecular Biology L-211 > Exam 5: Lecture 4 > Flashcards

Exam 5: Lecture 4 Flashcards

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1
Q

Drosophila Body Plan

A
  • established in early embryogenesis
  • egg/oocyte can’t initiate body plan formation and must rely on instructions sent from mother
  • instructions for patterning the A/P axis are provided by subset of cells from maternal ovary
  • many of these mRNAs are localized to specific regions of the embryo
  • translated proteins will diffuse away from the source forming concentration gradients within the embryo
  • differing levels of these TFs will activate expression of target genes in concentration dependent manner
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2
Q

Maternal Effect Genes

A
  • tasked with subdividing embryo into large domains

- do so by activating “Gap” genes

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3
Q

Gap Genes

A
  • expressed in large swathes of the embryo
  • loss results in embryo that is missing anywhere from 25%-40% of tissues
  • in turn directly control expression of “Pair Rule” genes
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4
Q

Pair Rule Genes

A
  • expressed in seven stripes

- removal leads to loss of tissues in alternating segments

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5
Q

Segment Polarity

A
  • embryo further subdivided by these genes

- act to determine identity of each of the fourteen different embryonic segments

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6
Q

Homeotic

A
  • confer specific fates upon groups of segments
  • Mutations lead to transformation of one segment into another
  • ex: loss of Antennapedia leads to conversion of legs into antennae while overexpression of antenna will lead to its conversion into a leg
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7
Q

Drosophila Ovary

A
  • female ovaries located within abdomen of adult fly
  • each contains set of sixteen ovarioles
  • most anterior tip contains germarium where germline stem cells are kept
  • stem cells divide and produce cells which give rise to developing egg/oocyte
  • 14 distinct stages to development of each egg/oocyte
  • at end of stage 14 egg/oocyte will first pass through the lateral oviduct, then through the common oviduct, and finally through uterus and vulva
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8
Q

Patterning of Egg/Oocyte

A
  • while it’s developing it is also being patterned in dorsal-ventral and anterior-posterior axes
  • A/V patterning does not involve secretion of ligand
  • gradients of mRNA and protein localization will be established in the early embryo
  • ultimately leads to differential gene expression along A/P axis
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9
Q

Follicle Cells

A
  • ensheath developing egg during development

- one cell gives rise to developing oocyte

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10
Q

Nurse Cells

A
  • set of 15 cells that will lie adjacent to the anterior quadrant of the oocyte
  • connected to each other via ring canals and also to the oocyte
  • allows proteins and mRNA transcripts to be passed between nurse cells
  • these cells deposit large quantities of mRNA transcripts and proteins into the developing egg/oocyte
  • necessary to get development of embryo started prior to onset of zygotic transcription
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11
Q

mRNAs Made in Nurse Cells and Deposited in Egg/Oocyte

A
  • said to be transcribed from maternal effect genes

- appropriate term because transcription of genes in parent has effect on next generation

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12
Q

Deposition of mRNAs and Proteins into Oocyte

A
  • factors manufactured in adjacent nurse cells and are dumped into oocyte via ring canals
  • 3 key mRNA transcripts correspond to bicoid, oskar, and gurken genes
  • these mRNAs are deposited into anterior pole of oocyte
  • concentration gradient doesn’t happen as one would expect
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13
Q

Bicoid mRNA Transcripts

A

-localized to anterior pole

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14
Q

Oskar mRNA Transcripts

A

-localized to posterior pole

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15
Q

Gurken mRNA Transcripts

A
  • localized to anterior dorsal pole

- this localization is important for patterning the embryo in anterior-posterior axis

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16
Q

Localization of Bicoid, Oskar, and Gurken

A

-to discrete positions within developing oocyte is dependent upon activity of several motor proteins and a microtubule meshwork

17
Q

Alpha and Beta Tubulin Subunits

A
  • organized into polymers called microtubules
  • each tubule has two polarized ends (+ and - ends)
  • generally plus end oriented toward posterior of oocyte and minus oriented toward anterior pole
18
Q

Transport of Cargo

A
  • vesicles, proteins, and mRNA transcripts
  • can be transported along microtubule in direction dependent manner
  • this movement is dependent upon motor proteins dynein and kinesin
19
Q

Dynein

A
  • moves cargo towards minus end of microtubule
  • localization of bicoid mRNA transcripts tells you they’re transported by this
  • composed of several different subunits
  • one subunit contains ATPase domain
  • divided into two classes: cytoplasmic and axonemal
20
Q

Kinesin

A
  • moves cargo toward plus end of microtubules
  • localization of oskar mRNA transcripts tells you they’re transported by this
  • comprised of several different subunits
  • one subunit contains ATPase domain which allows it to generate movement via hydrolysis of ATP
  • known to play roles in mitosis, meiosis, and transport of cargo
  • division of these proteins into separate classes is based on overall structure and known biological functions
21
Q

Cytoplasmic Dynein

A
  • used to properly position organelles such as the nucleus and Golgi
  • also used to transport vesicles, proteins, and mRNA transcripts
22
Q

Axonemal Dyenins

A
  • found in flagella and cilia

- used for movement of both structures

23
Q

3’ UTR of Bicoid

A
  • contains regulatory element that is bound by members of dyein complex
  • because dyenin walks cargo to minus end of microtubules the bicoid mRNA transcript will be transported and localized to anterior pole of oocyte (where minus poles are located)
  • as kinesin walks cargo towards plus end of microtubules, oskar transcripts will be localized to posterior pole
24
Q

Experiment: Generate Oskar With Bicoid 3’ UTR

A
  • if you generate oskar mRNA that contains bicoid 3’ UTR and inject it into normal embryo, you will create embryo with two tail ends
  • because endogenous oskar mRNA will be localized properly to posterior pole while mutant oskar that you created will now localize to anterior pole and transform that tissue into posterior tissue
25
Q

Nanos mRNAs

A
  • oskar transcripts not only ones localized to posterior pole after being deposited into anterior pole of oocyte
  • these mRNAs behave in same manner
  • also contain regulatory element within their 3’ UTR that is bound by kinesin
  • if you generate mutant nanos mRNA that contains bicoid 3’ UTR and inject it into normal cell you will create embryo with two tails
26
Q

Oskar and Nanos Experiments

A

-indicate that correct localization of these transcripts are critical for anterior-posterior patterning

27
Q

Concentration Gradients

A
  • maternal bicoid and nanos mRNA transcripts translate early in development of embryo
  • Bicoid and nanos then diffuse creating classic concentration gradients
  • highest levels of Bicoid found at anterior pole
  • highest levels of nanos found at posterior pole
  • decreasing amounts of proteins found towards center of embryo
28
Q

Bicoid: TF

A

-roles in development: to activate expression of orthodenticle (otd) and hunchback (hb)

29
Q

otd

A
  • otd expression restricted to regions of embryo with highest levels of bicoid
  • due to presence of low affinity bicoid sites within embryonic enhancer
30
Q

hb

A
  • hb expression expands into middle of embryo where there are intermediate levels of bicoid
  • due to presence of both high and low affinity bicoid sites within the embryonic enhancer
31
Q

Free Movement

A
  • during early development mRNAs and proteins must be able to move freely throughout egg/oocyte
  • maternal mRNAs being localized to anterior and posterior poles which requires microtubule tracks to be able to run across entire embryo unhindered
  • also after maternal transcripts are translated, encoded proteins will diffuse away from mRNA transcript source
  • for these events to happen early embryo can’t be cellularized
32
Q

Regulating Diffusion Gradients in Embryo

A
  • proteins diffuse across the embryo nuclei bathed in different concentrations of each factor (bicoid, oskar, nanos)
  • different concentrations must be maintained so transcription of zygotic Gap genes can be activated differently across embryo
  • just prior to Gap gene transcription, embryo will undergo cellularization (process by which single cell embryo becomes multicellular organism)
  • traps maternal proteins with each cell at a concentration appropriate for that cellular position along A/P axis
33
Q

Example of Regulating Diffusion Gradients

A
  • cells at most anterior pole now contain highest amounts of bicoid while those at midsection will have lower levels and cells at posterior pole will completely lack it
  • differences lead to transcription of otd in anterior pole and Kruppel in midsection
  • both genes remain off in posterior pole
34
Q

Cellularization of embryo

A
  • prevents proteins like bicoid, nanos, and oskar from diffusing any further across embryo
  • if cellularization is delayed or prevented, these proteins will continue to diffuse and ultimately their concentration across the embryo will be equal in all cells
  • slope of gradient will be a flat line
35
Q

Anterior Half of Embryo

A
  • there are high levels of Bicoid (bcd-activator) and Hunchback (Hb-genes that are necessary for the formation of the head segments
  • one gene shut off in anterior section is Kruppel (Kr) which is required for the formation of the midsection
  • even though Kr embryonic enhancer contains both Bcd and Hb binding sites the activity of the rpressor dominates at the higher concentrations
36
Q

bcd Mutant

A
  • in bcd mutant Kr expression is activated in the anterior section of the embryo
  • this is due to loss of Hb repressor
  • also indicates that other activators are also used to initiate Kr expression
37
Q

Midsection of Embryo

A
  • the levels of both proteins begin to taper off
  • Kr expression is activated since Bcd activity dominates over Hb at lower levels
  • thus anterior border of Kr expression is set b combined activities of Bcd and Hb
  • posterior boundary of Kr expression is set by two repressors called Knirps (Kni) and Giant (Gt)
  • expression of these tow genes is activated by gradients that are initiated by oskar and nanos at the posterior pole
  • combined efforts of Bcd, Hb, Kni, and Gt restricts Kr expression to embryonic midsection
  • Kni and Gt proteins also play important roles in regulating the fate of cells within posterior section of embryo
38
Q

Gap Genes Examples

A

-hunchback, Kruppel, Knirps, and Giant are considered Gap genes since they are expressed in large domains and determine the fates of cells within these domains

Molecular Biology L-211 (38 decks)

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