shi2/Df(1)sd72b mutants are partially lethal. Surviving adults do not exhibit seizure activity at any voltage range of high frequency stimulation in the brain, unlike wild type.
When shi2 embryos are fixed at the restrictive temperature, metaphase actin rings are often incompletely formed and some rings surround more than one condensed mass of DNA. Approximately 25% of metaphase furrows in shi2 embryos are lost at the restrictive temperature in patches across each embryo, and those furrows present surround many DNA elements, unlike in controls. These two phenotypes overlap: regions of embryos lacking actin rings also lack plasma membrane furrows. Incidences of abnormal tripolar spindles are also seen in temperature-restricted shi2 embryos relative to wild-type embryos.
Temperature-restricted shi2 embryos in interphase both disrupts plasma membrane polarity and leads to loss of plasma membrane compartmentalization across the embryo.
Abnormal sprouting of filopodium-like projections can be seen in some tracheal cells in mutant embryos at the restrictive temperature. Tracheal cells of mutants at the restrictive temperature show moderate ectopic migration defects in 72% of embryos, and show severe ectopic migration defects in 19% of embryos. Mutant embryos at the restrictive temperature have tracheal lumen defects, with ectopic, tortuous tracheal branches.
Homozygous embryos raised at the restrictive temperature for 7 hours (after 7 hours of development at 25[o]C) show ectopic branching in the tracheal network.
At 25[o]C, shi2 embryos show normal salivary gland development. However, if shi2 embryos are shifted to the restrictive temperature of 30[o]C, then embryos are seen in which the salivary gland either fails to turn posteriorly or does not complete its posterior migration.
Shifting 2-day-old shi2 adults to 29oC for 11 hours results in a reduction of F-actin density in the investment cones of spermatid individualization complexes. A 16 hour heat pulse results in actin density being undetectable in these cones.
After 2 minutes at 29 oC, 0% of shi2 flies remain standing, compared to 100% of control flies.
Synaptic vesicle endocytosis (examined in synapses innervating larval muscle fibres) is completely blocked in shi2 flies stimulated at 33 oC.
Unlike wild type, shi2 nerve terminals (recorded from larval bodywall muscle 6 within A2) exhibit synaptic depression (as a result of use-dependent depletion of synaptic vesicles) when stimulated at 10 Hz at 30 oC.
The heartbeat of homozygous or hemizygous pupae is increasingly slower and more arrhythmic than wild type with increasing temperature. The severity of the phenotype increases uniformly as the temperature rises. The phenotype is recessive.
Mutant flies are paralysed at 27oC.
Mutant flies show tight and complete paralysis in 2 minutes at 28oC. High frequency stimulation of the larval neuromuscular junction (NMJ) at 30oC results in a rapid decline in synaptic transmission (wild-type NMJs can maintain synaptic transmission at this temperature for several minutes). At 28oC, the mutant NMJ shows only a slight depression relative to wild type.
At the non-permissive temperature, a decline of synaptic current amplitude is seen during 400 s 10 Hz tetanic stimulation of neuromuscular junctions.
Mutant shi2 flies paralyze within 2 minutes at 28oC; at lower temperatures, shi2 flies take a longer time to paralyze.
Homozygotes and shi2/shi20 flies are paralysed at a temperature of 27.5oC and 26.5oC respectively.
At the permissive temperature (25oC) hemizygous males develop and behave normally. At the restrictive temperature (29oC) hemizygous males become paralysed and uncoordinated movements can be observed. shi2; α-Adaptin3/α-Adaptin+ males are indistinguishable from wild type at 18oC but at 25oC males can neither fly or walk and they show sporadic and uncoordinated movements.
When shi2 pupae are exposed to 31oC for 10-16 hours after puparium formation (APF), the majority of sensory units of the third antennal segment are composed of single or two cell clusters, in contrast to the control flies, which have sensory units mostly composed of three cells. Sensory development in the anlage of the second antennal segment and the wing disc is comparable to control flies. These pupae give rise to adults with a significant reduction in the number of sensilla basiconica, trichodea and coelonica on the third antennal segment. Temperature pulses between 0 and 8 hours APF have no effect on the sensilla founder cell selection process in the antennal discs, although extra sensory organ precursors are seen in the wing and notum, which give rise to ectopic bristles. Temperature pulses between 14 and 24 hors APF result in a conversion of trichogen and tormogen cells of the macrochaetae and microchaetae to neurons.
Sub-anaesthetic concentration of carbon dioxide specifically suppresses the temperature sensitive paralytic phenotype. Sub-anaesthetic concentration of carbon dioxide also rapidly reverses paralysis induced at the restrictive temperature. The effect depends on the absolute concentration of CO2 rather than the ratio with oxygen.
Synapse inhibition of dye uptake is seen at 34oC.
Weak eye phenotype is observed at 32oC, slight disorganisation. A strong eye phenotype is observed at 34oC. Six hour 34oC pulses to pupae aged 32 hours at 22oC cause almost total elimination of the thoracic macro and microchaetae. Heat pulses also cause dramatic disorganisation of bristles at the anterior wing margin, dorsal and ventral bristles appear to be missing or duplicated. 34oC heat pulses to flies 32 hours after pupation results in adults with holes in their wing blades. A 32-38 hour window defines a critical period for a shi-dependent step in wing blade formation.
shi2/shi4 flies become paralysed very slowly at 29oC or 27oC compared to shi2 or shi4 homozygotes. Some shi2/Df(1)sd72b flies survive. They become paralysed at 24oC, and are weak and sluggish at 22oC. They also show bang-sensitive paralysis.
temperature-sensitive temperature of adult paralysis: 29oC temperature of larval paralysis: 31oC temperature causing developmental defects: 31oC viability of allele over deficiency: strong