Chc1 embryos demonstrate over-elongated dorsal trunks.
The paracellular barrier integrity of Chc1 embryos is compromised, as injected dye leaks into the trachea in 36% of analysed mutants.
The neuromuscular junctions of Chc4/Chc1 third instar larvae show similar levels of membrane uptake (dye internalisation) to controls in response to nerve stimulation. However, unlike in controls, the dye is often concentrated in subsynaptic structures, rather than the typical donut shape seen in controls. The size of the boutons is similar to wild type and no additional satellite boutons are seen.
Flies expressing ChcN.4C.T:Zzzz\FLAG,T:Zzzz\TC in a Chc1 mutant background are viable. They display no behavioural defects, the morphology of the third instar larval neuromuscular junctions is indistinguishable from controls, postsynaptic receptors are clustered normally and neurotransmitter release in response to low and high frequency nerve stimulation is similar to controls. These phenotypes are seen both in the presence and absence of Chc photoinactivation by FlAsH-FALI.
When flies expressing ChcN.4C.T:Zzzz\FLAG,T:Zzzz\TC in a Chc1 mutant background are pretreated with FlAsH-FALI to photoinactivate Chc, they show similar amounts of membrane uptake (dye internalisation) to controls in response to nerve stimulation. However, unlike in controls, the dye is often concentrated in subsynaptic structures, rather than the typical donut shape. A similar phenotype is seen when flies are treated with chlorpromazine, but the phenotype is not enhanced by combining treatment with FlAsH-FALI and chlorpromazine together. When flies are stimulated for a second time in the absence of dye, the previously internalised dye is not unloaded, indicating that membrane internalised in the absence of Chc is not released.
The synaptic boutons of third instar larvae expressing ChcN.4C.T:Zzzz\FLAG,T:Zzzz\TC in a Chc1 mutant background and subjected to FlAsH-FALI to photoinactivate Chc are almost devoid of synaptic vesicles and show giant membrane invaginations, with some measuring >1μM in cross section. There is a decrease in vesicle number per area and an increase in vesicle size. The remaining round or oval shaped vesicles in these boutons show heterogeneity in size and a population of larger vesicles and cisternae is observed. However, active zones mitochondria and subsynaptic reticulum are all present in both control and FlAsH-treated synapses.
FlAsH-FALI treatment has no effect on the excitatory junction potential (EJP) amplitude seen in flies expressing ChcN.4C.T:Zzzz\FLAG,T:Zzzz\TC in a Chc1 mutant background during low frequency stimulation. The same result is seen at all Ca[2+] concentrations tested. However, when EJPs are recorded during intense stimulation, FlAsH-FALI photoinactivation reduces the relative EJP amplitude compared to controls, with the amplitude dropping to <25% of the initial value.
Flies expressing Chc4C.T:Zzzz\FLAG,T:Zzzz\TC in a Chc1 mutant background are viable and don't show any obvious developmental defects. No defects are seen in climbing, negative geotaxis or flying. Under normal conditions electroretinogram recordings in response to a light flash are comparable to wild type. However when FlAsH is micro-injected under the photoreceptor layer these flies lack the normal "on" and "off" transients seen in controls.
Mutant embryos show an extension of tracheal tube length compared to controls, show a failure of luminal chitin extracellular matrix organisation, and show a loss of lumen clearance which results in a failure of gas filling.
No defects are seen in axon pruning during metamorphosis, or defects seen in the mushroom body.
Strong loss of function allele.
Germline clonal analysis demonstrates an autonomous cell-lethal mutation in the female germline. Most embryos succeed in hatching, unhatched embryos show no regular cuticle defect.