Courtship Flashcards
(24 cards)
Describe the courtship display of Drosophila Melanogaster
Tapping, Following, wing extension, singing, licking then copulation
What were gynandromorph experiments and what did they show?
These experiments involved females with ringed x chromosomes. Randomly this would cause one of the x chromosomes to be removed and so masculinising that cell line. Could then identify areas of the boyd involved in the courtship display.
This showed that some parts of the brain were submissive or domineering. The most important regions for, tapping, following and wing extension were the mushroom bodies and the antennal lobes, the mesothroax was important for song and the abdominal ganglion for copulation.
Which gene is the master regulator for courtship behaviour?
XX tra/tra show normal male courtship behaviour
XY dsx/dsx are also normal. So tra must be important for the nervous system and dsx for the soma.
How can we further the evidence from the gynandromorph experiments?
Take Gal4 UAS enhancer traps to find specific enhancers for areas of the drosophila nervous system. Cross the UAS-tra to feminise these regions and assess results.
Feminising MB and AL = bisexual male
Feminising AL = bisexual male
Feminising lateral AL = heterosexual
Note this could be due to blocking of the normal male inhibitory center or stimulating the female stimulation center.
Describe the fruitless mutant
Fruitless mutant originally from an inversion event that disrupted two genes. One was a pheromone then other resulted in bisexual males with a defective song and MOL that chain.
Suspicion that this gene might be affected by tra. So screened tra bidning site from dsx within the genome. This localised to this same gene.
Gene contains sex specific and non specific enhancers, 4 promtors (2-4 and non sex specific and required for viability). mRNA mostly seen in the brain. Tra causes alternate splicing from p1 resulting in fru-m and and non translated fru-f transcript. Fru-m is a TF.
How did we work out where fru-m binds?
Fuse to a DAMID which methylates adjacent adenines. Then do a methyl PCR to identify regions of the genome that it binds to. Fru-m appears to change the splicing of its targets and can influence itself.
Fru is expressed in 2000 neurons and fru-m in around 200.
What happens when we switch on and off fru-m production in males and female?
Created a constituitive fru-f, fru-m and fru-delta-tra.
Fru-f in males reduces female courting, increases male courting and increased chaining.
Fru-m in females increases female courting, decreases male receptivity and causes chaining, also reduced egg laying and fertility.
What was the fru-GAL4 mutants used for?
Knocked in Gal4 to inactive P1 fru but maintain P2-P4. Homozygotes phenocopy fru mutants. Can then cross this to UAS-tra or UASshi(TS) to feminise all fru neurons.
Shibire experiments showed no courtship of either males or females - so neurons required in both males and female for courtship.
Cross to UAS-GFP lights up MB, AL, Johnston’s organs, taste pegs and chemo receptors.
How did they realise that there was sex specific expression of Fru. How was this sex specificity being achieved?
Used a randoms GAL4 insertion and crossed to UAS-GFP. Can see more optic neurons in males than in females. Also these neurons have a different branching pattern in males.
If you knock out the cell death genes in a female (GRIM, REAPER and HID) then the number of female neurons increases. In fru KO males - much fewer neurons - like females. So Fru must be supressor cell death genes.
Are there any sex specific differences other than in the optic lobes - why was this important?
Yes - use MARCM to individually label groups of cells expressing fru-m. This shows again that the P1 cluster is specific to males. If you add in tra to this model and masculanise the P1 neurons in females then they court other females.
Again the cell death genes are mediating this. This time DSX is required to activate them. Without DSX-f the P1 neurons follow a male fate.
Fact that DSX also required for a nervous system related sex specificity breaks down the fru-mind dsx-dualism.
What did fly pods tell us?
Flypod = take off the head of the fly and express fru-Gal4 –> UAS-P2X (light sensitive channel). Stimulate fru neurons and you get singing from both males and females. This song isn’t perfect - IPI is off and males were closest.
Fru-m females have slightly closer song but DSX-m females have no song.
If fru-m is all that is needed for male courtship then fru-m female flypods should have the same as male flypods. All suggests that neither fru-m nor dsx-m neurons are sufficient for normal fly song but perhaps both together gets us close? Also that descending tracts from the brain are important
fru-m and dsx-m co-localise to the MSG and mosaic studies showed that the MSG is important for song. There are more MSG neurons in the male. Female neurons only express dsx-f which is what causes the reduced number of neurons.
The number of neurons appears to correlate with how normal the song is, so song required dsx-m function - again there is no mind body dualism.
What happens if you mess around with the function of dsx-m neurons?
Fuse dsx to GAL4 - cross to UAS-GFP and it is seen in the abdomen, and msg, it is sexually dimorphic and colocalises with fru-m.
Use UAS-TNT and males become sterile have reduced courtship and no mating. Females have no egg laying, little copulation and lots of remating.
How can we analyse the sex specific neurons expressing dsx
Cut up the DSX non coding region and hook to GAL4 then express UAS-GFP. Different lines showed sex specific expression. pCd, PC1 and PC2 are all increased in males, TN1 is only seen in males.
Then crossed 41A01 to UAS-dTrpA1. When temperature is increased in females there is increased receptivity and copulation. Then use UAS-shiTS or TNT and see a decrease in receptivity and copulation
What was the issue with the DSX experiments where they cut up the dsx regulatory regions?
Some neurons may be expressing dsx that don’t normally - so must be some important inhibitory regions that were separated off. Overcome this using an intersectional strategy.
41AO1-LexA –> LexAop2-FLP
Dsx-GAL4 –> UAS->STOP>YFG
Where ever there is crossover shows genuine expression. This identified the pCd (41A01) and PC1 (71G01) neurons as responsible for the changes in copualtion.
Can then use a calcium reporter to show that pC1 responds to cVA and song whilst pCd responds to cVA only in females. In males these neurons co express fru-m in females they don’t. So in females Dsx influences courtship independent of fru.
What experiments identified cells important for males to respond to females?
If you activate fru neurons in a lone male he will start courting. If you use MARCM to do this in specific cells then the p1 and p2b cells are seen to be important for tapping, and wing extension. These two cells are close so could form a circuit?
If we tether a male and show him a female we can see that tapping is very important to initiate courting, that cVA inhibits the male from courting a female. Finally using a calcium reporter shows that the MB and LPR (lateral pro cerebrum) activate when a male is shown a virgin female.
How have we discovered neurons that are important for getting the right IPI?
When fru neurons are expressed they generate song but the IPI is wrong. If you test lots of GAL4 lines for overlap with fru expression using an inter-sectional strategy then you can find drivers that improve song.
NP2361 has expression in P1 and activation with dTrpA1 results in pulse song. Many other lines identified that influence wing extension, IPI and cycles per pulse. All have fru-m expression and some are descending neurons - can then begin to build a circuit controlling song.
What organs are involved in detecting sound and what do deafened males and females do?
Johnston’s organ neurons respond to sound (JON’s). These neurons innervate the AMMC which included the aPN1 neurons and the aLN neurons. Both also express fru. Females with no aristae - don’t mate, males with no wings have reduced mating.
Can genetically deafen females using JOGAL4 eyFLP and UAS>STOP>ShiTS, again these females have reduced mating. If you repeat but use fru-GAL4 instead get the same result.
What is SIL and how does it relate to IPI?
Normally when females hear song they slow down to allow mating whilst when males hear it the have song induced locomotion to try and find mates - SIL. In genetically deafened males - SIL is reduced whilst in females they don’t slow down.
SIL is also IPI sensetive
Can use Flylight drivers to inhibit or over stimulate these neurons. Whichever you do males and females act as if deaf i.e. reduced mating and altered SIL.
Are there any sex specific neurons involved in the response to sound
To find out test Flylight lines and use intersectional strategy with fru-m and look for expression in AMMC.
aPN1 in both males and females whilst vPN1 is in males only. Expressing fru-m in females causes them to gain vPN1 neurons.
Normally males chain when by themselves and hearing song. If vPN1 and aPN1 neurons inactivated then this chaining is reduced. vPN1 is sensetive to IPI - see using Ca reporter. Another neuron PC1 shows a similar pattern for chaining and IPI.
How can we show whether PC1 and vPN1 neurons might synapse?
Use optogenetic stimulation and calcium staining - confirms connection.
What does sex peptide do and what genes are involved?
Screen RNAi using elav-GAL4 –> UAS-RNAi for mutants that will take a second mate and wont lay eggs. Find one gene but SP does not reverse so must be the receptor.
SPR localises to fru neurons in brain and oviduct. If you reverse this screen and use GAL4 lines can find where it needs to be expressed. - X-GAL4 –> UAS-SPR RNAi. This identifies PPK.
What is PPK and what might it do?
Expressed in reproductive tract, brain and looks like a stretch receptor in uterus.
Both ppk and spr expression pattern can be used to rescue SPR mutant with a new SPR allele.
If PPK neurons are silenced then virgins don’t mate as much and virgins start to lay eggs. PPk neurons extend to SOG where auditory sensory neurons also project - does SP make females deaf?
What does Or67d do
It is expressed in the T1 sensilla and is the only neuron to project to the DA1 region. DA1 is bigger in males and expressed fru-m. Can use an electrode to see that it responds to cVA.
Knockouts of Or67d are bisexual (as they don’t detect cVA. and KO in females don’t want to mate even though males still want to mate with them.
If females are covered in cVA this inibits male mating.
This is all conserved between insects - moth receptor can be used to rescue Or67d mutants.
Can we identify anymore of this circuitry in the fly brain?
Yes - using a special photoactive GFP can seen that after the DA1 the next neurons go the lateral horn in both males and females. These neurons have slightly differing ventral projections whihc is fru-m sensitive but show the same response to cVA (with a Ca reporter) so any sex specificity must be further up.
Again using photo active GFP and you can see that in males there are DC1 and 2 neurons and in both males and females there are LC1 and 2 neurons. DC1 looks like it might connect with the ventral nerve cord.
Using an electrode and a Ca reporter can show that
In males: Or67d neuron –> DA1 –> DC1
In females: Or67d neuron –> DA1 –> LC1
There is a flick of a switch mediated by fru-m
