hhAC2/hhts2 transheterozygote females (moved to the non-permissive temperature - 29[o]C - after eclosion) show a moderate tumorous phenotype with numerous ectopic germline stem cell/cystoblast-like cells in a portion of the adult germaria.
Germaria in hhAC/hhts2 flies exhibit an enlarged morphologic structure filled with fusome-containing cysts and are defective in correct segregation of individual germline cysts at region 2a/2b.
Mushroom body gamma neurons appear indistinguishable from controls in hhAC/hhts2 animals transferred to the restrictive temperature at the onset of pupariation and analysed at 29 hours after puparium formation.
Shifting hhts2 mutants from 18 to 29[o]C to disrupt pathway activity at any stage after SOP formation gives no alteration in multidendritic neuron number. Shifting the temperature before SOP formation causes a loss of multidendritic neurons. In stage 16 hhts2 embryos, the ventral class IV neuron vdaB is lost in 20% of parasegments. This loss of vdaB is preceded by SOP1a loss (in 23% of cases). There is no loss of v'ada or SOP4a. In hhts2 mutants the other non-class IV MD neurons are lost in the ventral cluster, for example vbd is lost in 62% of embryos.
hhts2 heterozygotes exhibit loss of the vdaB neuron, but not the v'ada neuron, in 17% of parasegments. SOP1a is lost in 15% of cases, while SOP4a is never lost.
When single cell hhts2 clones are made in the wing imaginal disc, cytonemes in the wing primordium are affected. At the non-permissive temperature (29oC) cytonemes emanating from cells at the lateral flanks of discs do not orient themselves towards the anterior posterior organiser (as those see in wild-type do). Cytonemes in these discs are also more numerous than in wild-type, and unlike in the wild-type, are sometimes seen to be bent, curved or crossing over each other. These effects are not observed if mutant larvae are heatshocked and returned to permissive temperatures.
hhts2 larvae that are grown at the restrictive temperature prior to and during cell shape changes in the disc epithelia develop severely reduced wing and leg discs compared to heterozygous controls. These reduced discs fail to form squamous cells.
Stigmatophores are present in hhts2 embryos that have been maintained at the restrictive temperature from 5-8 hours of development despite severe segment polarity defects.
hhts2 larvae have fewer S phase neuroblasts per brain lobe when raised at above the restrictive temperature than wild-type controls or hhts2 larvae raised at below the restrictive temperature.
Ovaries of homozygous females maintained at the restrictive temperature (29oC) for 4-5 days show a number of defects including germaria with disorganised encapsulation. Large groups of disorganised somatic cells are seen at the periphery of the germaria. Ovaries of hhAC/hhts2 females maintained at the restrictive temperature (29oC) for 4-5 days show a number of defects including multicyst egg chambers. Large groups of disorganised somatic cells are seen at the periphery of the germaria and multicyst egg chambers.
Removal of hh during second instar using hhts2 leads to precocious glial cell migration out of the optic stalk. However instead of choosing their normal path along the basal surface of the eye disc, glial cells migrate at the apical surface next to the Bolwig's nerve.
The mirror-image patterning or posterior tergite and posterior hairy zone structures caused by expression of hhScer\UAS.cPb under the control of Scer\GAL4T155 is unaffected if the flies are also homozygous for hhts2 (and raised at the restrictive temperature). The transformation of anterior tergite to intersegmental membrane is partly suppressed in these flies.
Shifting hhts2 animals to their restrictive temperature at various time does not generate individuals with head capsule defects.
Temperature-shift experiments indicate that the phenocritical period for hh function in Bolwig's organ development is between 4 and 7 hours.
hhts2/hhAC embryos have a severe segment polarity phenotype, in which the naked cuticle is lost, at the restrictive temperature. Expression of hhC84S.Scer\UAS under the control of Scer\GAL4en-e16E at 29oC in flies which are heterozygous for hhts2 results in an enhancement of the loss of the L3-L4 intervein region seen in flies expressing hhC84S.Scer\UAS under the control of Scer\GAL4en-e16E in an otherwise wild-type background.
98.9% of hhts2/hhAC ovarioles contain multi-chambered cysts. Branching of the fusome occurs closer to the anterior end of the germarium than in wild type.
Using temperature shifts to 29oC, a dependence of mitosis in the ovariole on hh function has been demonstrated.
Loss of function at the restrictive temperature causes severe disruption of early eye discs. Clones generated during the L1 stage show defects in both the eye disc proper and the peripodial membrane.
When hh function is inactivated at 6 hours after egg laying in hhts2 embryos, the 1o-3o fates in the dorsal cuticle (dorsal denticles, smooth cuticle and thick hairs respectively) are missing and are replaced by excess 4o fates (fine hairs).
When hhts2 females are shifted to the restrictive temperature, budding of egg chambers diminishes after about 3 days and virtually ceases by 7 days. During this period, some egg chambers bud with multiple complements of germline cells. In these compound egg chambers an oocyte is always seen at the posterior. When hhts2 females are held at the restrictive temperature for 7 days and then returned to the permissive temperature, egg chamber budding resumes within 3 days, showing no significant evidence of defects in oocyte positioning or polar cell differentiation in the most recently budded egg chambers.
Flies raised at the restrictive temperature of 30oC during the third instar larval stage lack the entire medial domain of the dorsal head, including the ocelli, interocellar cuticle, and the ocellar, postvertical and interocellar bristles. This is replaced by the ridged frons cuticle.
Homozygous larvae raised at the nonpermissive temperature have only vestigial wing imaginal discs. The overgrowth of the anterior compartment of the wing disc seen in larvae expressing hhN.Scer\UAS.T:Ivir\HA1 under the control of Scer\GAL4en-e16E in a hhts2 background is still seen if the discs are also homozygous for dispS037707.
Without hh, posterior 3/4 fragments of the L1 disc fragments switch from a duplicative to a regenerative program. All missing anterior leg structures can be regenerated. hhts2 larvae shifted to the restrictive temperature late in development (90 hours AED) pupariate but fail to eclose. Pharate legs reveal largely normal cuticle patterns, with the exception of the loss of the anterior claw at the distal tip of L1, L2 and L3.
When hhts2 mutants are raised at the restrictive temperature, the genital discs are very small.
S phase lamina neuron precursors (LPCs) are absent from the lamina in homozygotes that have been raised at 28oC after hatching.
Shifting to restrictive temperature causes failure of axons from photoreceptors to induce neuronal differentiation in the lamina. A hhts2 animal can have large clones of hhαTub84B.PB (i.e. hh+) at low frequency (by a mild heat shock of hhScer\FRT.Rnor\CD2.αTub84B flies carrying Scer\GAL4hs.PB). Shift to restrictive temperature at 84hr AEL arrests morphogenetic furrow progression; only a few retinal axons enter the lamina target field. When such axons enter a hh+ field only those LPCs in the vicinity of the axons show neuronal differentiation. When hhts2 animals are shifted to the non-permissive condition at 90hr AEL and examined 36hrs later, retinal axons grow into the target field in a relatively normal manner but fail to induce hh-dependent events. When flies are maintained at the non-permissive temperature from early larval development R cell development is blocked.
A shift to the restrictive temperature during the first instar stage causes a small wing dics in third instar larvae.
Mirror-image duplication of the anterior tergite is seen in some segments of hhts2 flies shifted to the restrictive temperature after pupariation. This phenotype is not altered in biQd-Fab; hhts2 double mutants.
Homozygous larvae reared at 18oC up to the late third instar stage and then shifted to 29.5oC immediately after pupariation give rise to pharate adults in which many hemitergites have mirror-image anterior duplications. In these cases, the posterior tergite, posterior hairy zone (PHZ) and intersegmental membrane (ISM) are deleted and replaced with a mirror-image duplication of the anterior tergite. Other phenotypes seen include complete or partial deletion of the PHZ and ISM, a missing posterior pigment band and transformation of tergite macrochaetae to microchaetae. Ventrally, the sternites are reduced in size and the sternal bristles are usually absent.
Larvae maintained at the restrictive temperature (29oC) from the end of the first instar stage until the early third instar stage have reduced eye-antennal discs.
At the nonpermissive temperature the germarium shrinks and the few remaining follicle cells envelop multiple germ line cysts.
Normal wings develop at 18oC.
When shifted to the non-permissive temperature at 90hrs AEL, mutants show the "furrow-stop" phenotype. The eye discs contain between 6 and 12 columns of ommatidial R cell clusters. The most anterior clusters appear normal and the axon fascicles from them are found in their proper dorsoventral positions posterior to the lamina furrow. But viewed on their antero-posterior axis the fascicles are collapsed on each other. Glial cells are absent from the optic stalk and lamina. Lamina precursor cells fail to undergo their terminal division, arresting in G1 at the lamina furrow.
Third instar larvae at the restrictive temperature completely lack medial head structures. Ocellar cuticle is replaced by frons spanning the entire head vertex.
Embryos exhibit a ventral cuticle phenotype.
At the permissive temperature adults exhibit reduced eye size and disordered facets, particularly at the anterior edge.
Distal structures of the leg and antenna fail to develop at 29oC.
Two temperature sensitive periods, one in the early embryo and one in the late larva/early pupa. At 18oC the embryonic cuticle is wild type though the adult eyes are slightly rough at the anterior margin. A 12hr heat pulse in the third larval instar reduces the eye and the ocelli. Removing hh functions during eye development stops the morphogenetic furrow.