lec 8 - drosohpila part 2

segments

repeated blocks/repeated unis that our body is built from

segmentation

body patterning into segments

segmentation is [variable/conerved]

conserved

importance of the heidelberg screen

• first and clearest dissection of how segmentation works

• revealed principles that apply far beyond insects

when is the body plan laid out in drosophila

early on in development of pre-fertilized egg

name the axes and the corresponding structure in drosophila

anterior - head

posterior - tail

dorsal - naked cuticle

ventral - cuticle with teeth-like denticles

role of cuticle preps

in-situ hybridization allowed researchers to see gene expression patterns in the embryo

heidelberg screen - researchers didn’t know if the 580 mutation-causing phenotypes were all in different genes. how did they find out

decided to use complementation testing

complementation group

a set of mutations that when crossed to each other, fail to complement and hence are in the same gene

explain how to find complementation groups

• cross flies with the same phenotype - they all have single recessive mutations

• if the offspring show WT, they complement, meaning they have different gene mutations that lead to the same outcome

  • the offspring only has one copy of each defective gene

• if the offspring shows mutant, they do not complement, meaning that they have the same gene mutation

  • the offspring has 2 copies of the defective gene

maternal vs zygotic genes - definitions and fxn

maternal

• mRNA/proteins deposited in the egg by the mother

• establish initial positional info

zygotic

• activated by embryo

• refine and elaborate body planning

what are the 3 classes of zygotic genes

gap genes

pair rule genes

segment polarity genes

gap genes - mutation phenotype, fxn, example

• phenotype = lose a large segment in the body

• fxn - define broad regions

• ex: knirps

pair rule genes - mutation phenotype, fxn, example

• phenotype = lose paired/alternating segments of the body

• fxn - refine gap gene domains into repeating units

• ex: paired

segment polarity genes - mutation phenotype, fxn, example

• phenotype - lose one side of each segment (ex: lose only anterior side)

• fxn - refine pair rule segments into anterior and posterior

• ex: gooseberry (and also Hh and Wnt!)

heirarchy of refinement

maternal genes → gap genes → pair rule genes → segment polarity genes

types of maternal gene mutations

• bicoid - loss of anterior region

• nanos - loss of posterior region

• torso - loss of terminal regions

what is bicoid and what does it determine

• maternal gene - DNA-binding TF maternally loaded into developing oocyte

• determines anterior

how does bicoid pattern the anterior (head)

acts as a morphogen - forms an anterior→posterior gradient

name some experiements that prove bicoid acts as a morphogen

• WT anterior cytoplasm injected into anterior bicoid -/- embryos rescues head development

• WT anterior cytoplasm injected into middle of bicoid -/- embryos produces ectopic head structures (in the middle) and mirror image thoracic segments

• the more bicoid genes you add, the further the segments are pushed back/the larger the anterior head region is

why does injecting bicoid into the middle of a bicoid -/- embryo create 2 heads with mirror-image thoraxes

molecules diffuse both ways, so heads also appear both ways based on the concentration gradient

how is the bicoid gradient read

french flag model

• creates regional gene expression domains

  • lower concentration of bicoid - higher affinity binding sites

  • high concentration of bicoid - low affiniy binding sites (need high concentration of bicoid to be activated

first class of zygotic segmentation genes

gap genes

how do gap genes know where to activate

read maternal gene gradients to define domains of gene expression

where is hunchback expressed

anteriorly (like bicoid)

where is kruppel located

band in the middle

explain how kruppel and hunchback are related

huntchback acts anteriorly, gives an A/P gradient

• high hunchback ⊣ kruppel

• no hunchback ⊣ kruppel

• low hunchback → kruppel

kruppel stays in the middle

what happens to kruppel expression in a bicoid -/- mutant

bicoid mutation partially inhibits hunchback

• low hunchback → kruppel

kruppel is expressed anteriorly

gap genes activate ___ genes

pair rule genes

pair rule genes expression pattern

alternating stripes

expression of pair rule genes is controlled ___

stripe-by-stripe

each pair rule stripe is controlled _____ by _____

• independently

• a different combo of gap genes

  • multiple gap genes with different concentrations throughout the embryo - pair rule genes read the specific combination and concentration of gap genes that exist in that stripe area

pair rule - stripe formation depends on

interaction between positively and negatively acting TFs

segment polarity genes control

patterning within a segment (anterior/posterior of each segment)

parasegment

• the posterior half of one segment + the anterior half of the next consecutive segment.

• will later shift to form adult morphological segments

Wg/Wnt mutation

Hh mutation

no segment polarity, every segment has only anterior regions (covered in denticles)

no segment polarity, covered in denticles, end up with spike-like pattern

how are parasegment boundaries formed

Hh and Wg/Wnt feedback onto each other to maintain each other’s expression and refine segment borders

• engrailed stimulates Hh production

how are denticle patterns formed

Hh maintains Wg expression which supresses denticle development - where there is Wg activation, there is no denticles, but engrailed right next to it promotes Hh signaling afterwards

selector genes

give each segment an identity (“who am i” information)

how do selector genes give segments their identity

they are TFs, turn on a bunch of other downstream genes

• ex: gene specifying a leg turns on all the downstream genes required for making a leg

key selector genes

hox/homeotic genes

hox genes patterning is

HIGHLY conserved, pattern from A→P in a conserved way

one parasegment is controlled by

a specific hox gene

hox genes are expressed along the anterior body in what order

the same order as genes are within a genome

selector genes are controlled by

combo of gap and pair-rule genes

antennapedia experiment

• antennapedia = hox genes for legs

• put it in the head, fly produces legs on the head

selector genes v segment polarity genes

same level

segment polarity tells them which way to go, selector tells them what they will become

germ banding

how much of the egg is pre-patterned

large vs intermediate vs small germ band

• large = whole is pre-patterned

• intermediate = partly pre-patterned, rest of patterning develops during segmentation

• short - not pre-patterned (outside of AP/DV)

drosophila is ____ germ band

long

pros and cons of long germ banding

pro - fast development

con - compliacted, maternal/gap/pair rule control for every segment

intermediate germ band insect - how does it pattern itself

• start with head and thoracic segments, uses an ancestral version of drosophila

  • patterns some but not all of egg

• add abd segments sequentially

  • posterior disc buds off over time

what are the receptors/ligands involved in segment addition

notch/delta - activates downstream pathways to make a segment

describe the segmentation clock

• negative feedback loop with delay

• delta ligand binds/activates to notch (and activates downstream segment patterning pathways)

• active notch activates Her (hairy enhancer of split related) which represses delta production

• repressed delta turns off notch

• delta production starts agin

how does the segmentation clock work

• notch activation causes down regulation of delta

• time lag in response causes oscillation between strong and weak signaling levels

• propagation of signal between cells causes wave of activation

• allows for segment addition

what does the delta mutation cause

specification of somite patterns/segmentation clock to get messed up

what type of organisms are the segmentation clock used for

intermediate germ organisms and small germ organisms

• intermediate germ - most patterning is set up, with segmentation clock in the tail end

  • ex: tribloium (and other beetles)

• small germ - little to no patterning is set up, segmentation clock happens throughout

  • ex: strigamia (and other mirapods)

most ____ use segmentation clocks

vertebrates

genes of the _____ pathway create vertebrate bones

notch/delta

notch/delta segmentation clock - when in evolution was it created

a long time ago - ancient evolutionary idea