Maternally and zygotically mutant dsh3 embryos (coming from mothers whose whole germline constitutes from dsh3 mutant cells only - created by the OvoD germline clone technique) show a segment polarity phenotype and no naked cuticle between denticle belts on the ventral epidermis (resulting in a lawn of denticles).
Flies expressing dshY473F.T:Avic\GFP-EGFP in a dsh3 mutant background display strong PCP defects in all tissues analysed, including classical PCP defects in the thorax and wings, rough eyes with randomised chirality, symmetrical clusters and misrotation. Co-expression of dshY474F.T:Avic\GFP-EGFP does not enhance these phenotypes.
Flies expressing dshY474F.T:Avic\GFP-EGFP in a dsh3 background display wild type PCP patterns in all tissues analysed.
dsh3 maternal/zygotic clones (expressing dshΔDIX.Scer\UAS under the control of Scer\GAL4arm.PS to remove the mild polarity defect of dsh3) display strong denticle alignment defects.
Maternal dsh3 mutants (expressing dshΔDIX.Scer\UAS under the control of Scer\GAL4arm.PS to remove the mild polarity defect of dsh3) show a loss of denticles.
dsh3 mutants display strong patterning defects, and all epidermal cells make denticle precursors.
dsh3 maternal/zygotic mutant embryos show a ventral epidermis covered with denticles, and the naked regions of cuticle which separates the denticle belts in wild-type embryos are absent in these mutants. Every cell secretes a denticle, and denticle placement is not properly oriented toward the posterior of each cell. All ventral cells display a uniform square shape and lack normal alignment.
dsh3 maternal effect mutants display wild-type cuticles.
In clones mutant for dsh3, the peripheral ommatidia do not undergo programmed cell death and so fail to release their associated 2o and 3o pigment cells to join the pigment rim. As a consequence, the pigment rim in dsh3 clones is significantly reduced in relation to neighbouring wild-type tissue.
The cuticles of embryos from dsh3 germline clones have an anterodorsal hole and are very short with most of the dorsal epidermis missing. At stage 13 in these embryos the leading edge cells fail to polarise or elongate dorso-ventrally.
Germband extension occurs normally in dsh3 homozygous embryos from mothers carrying dsh3 germ line clones. Polarisation of germband cells during germband extension also appears normal in these embryos.
Clones expressing dsh3 that are induced at the dorsal periphery of the eye show a loss of pigment rim tissue and an associated gain of extra ommatidia that extend almost to the head capsule. These extra ommatidia have a variable number of cells and many do not extend the depth of the retina. Some of these clones show an absence of dorsal rim ommatidia, while these are present in other clones.
Homozygous clones in the eye result in large clonal outgrowths. Additional rows of dorsal rim area ommatidia are seen in wild-type tissue adjacent to homozygous clones, probably due to overproliferation.
During dorsal closure in dsh3 mutant embryos, frequent detachments of the amnioserosa from the epidermis occur. The leading edge cells lack filopodial activity, and have only a thin cable of actin, lacking distinguishable actin nucleation centers. In additions, these cells do not undergo D-V elongation or D-V polarization. One manifestation of this is that the microtubule bundles that form in these cells do not align perpendicular to the leading edge.
Homozygous clones in polar regions of the eye show non-autonomous effects, with polarity inversion in tissue lying equatorial to the clone. Polarity errors are also seen within the clone. Clones elsewhere in the eye rarely show non-autonomous effects, but when they do, polarity inversions are seen on the equatorial side of the clone.
When clones are induced in the wing disc, a second set of concentric folds forms in the disc epithelium, which may reflect the formation of a secondary proximo-distal axis. Mutant cells give rise to dorsal rather than ventral structures in the adult and can organize surrounding wild type cells to contribute to supernumerary legs, which branch out from the ventral surface of the normal leg.
Embryos derived from germline clones exhibit an extreme segment polarity phenotype in which naked cuticle and segmental furrows are replaced by a lawn of denticles. Head skeleton and filzkorpers fail to develop and the embryos fail to hatch.
Clones of cells in the wing display different phenotypes depending on the position of the clone within the wing blade. Cells within the wing blade show abnormalities in differentiation related to a function of dsh in tissue polarity. When clones reach the margin they behave as if they were in the middle of the wing blade, developing trichomes and non-pigmented cuticle. Cells outside these clones are induced to form ectopic bristles characteristic of the margin (this also occurs for arm clones. Clones of cells simultaneously mutant for both dsh and sgg do not produce a dsh mutant phenotype at the wing margin, but display the phenotype of cells mutant for sgg alone.
When both maternal and zygotic components of dsh are lacking, dsh embryos display patterning defects identical to those for wg null mutants such as wgl-17 : no segment borders form to define segments, the usual naked regions of cuticle are not seen and filzkorper and head structures are missing. The second midgut constriction fails to form. Clonal analysis reveals that dsh is required for patterning but not viability of ventral cells in the leg, with dsh clones producing supernumerary appendages from the ventral face. Wing clones show supernumerary cells in the triple row.
Clonal analysis revealed polarity defects in adult tissues as for dsh1, and that dsh is required autonomously for cell polarity. Similar mutant leg phenotypes, e.g. duplications and bifurcations, are produced by dsh clones, dsh, wg double heterozygotes and wg mutants. There is allele specificity in these interactions.
Females possessing homozygous germline clones when mated to wild type males produce two classes of embryos: heterozygous females are fertile and morphogenetically normal and hemizygous male embryos lack dorsal cuticle, posterior spiracles and filzkorper material. The ventral cuticle present has no naked cuticle in the thoracic or abdominal segments. Embryos show an aberrant pattern of cell death and lack of parasegmental and segmental boundaries.
Double-heterozygous flies of the genotype dsh/+ together with either kmr1/+ or kmr2/+ display misoriented hair patterns on the wing, thorax and eyes, while single heterozygotes exhibit phenotypes similar to wild-type controls.
dsh3 maternal/l(2)gl4 zygotic double mutants display a cuticle which resembles the l(2)gl4 maternal mutant phenotype. Patches of denticles are observed which lack proper alignment, and the ventral epithelium is continuous and formed. Denticle precursors are not properly oriented toward the posterior of cells and denticle placement appears to be random within each cell.
dsh3 germ-line clones expressing armS56A.Scer\UAS.T:Zzzz\His6.T:Hsap\MYC under the control of Scer\GAL4arm.PS restores some pattern, but a strong denticle alignment phenotype remains.
Embryos expressing armS56A.Scer\UAS.T:Zzzz\His6.T:Hsap\MYC under the control of Scer\GAL4arm.PS in a maternal dsh3 background display a complete absence of denticles.
dsh3fz21/fz21 clones in the pupal wing (32 hours after puparium formation) cause neighbouring cells to point their trichomes towards the clone, as occurs with fz21/fz21 single mutant clones in the pupal wing.
dsh3Vangstbm-6/Vangstbm-6 clones in the pupal wing (32 hours after puparium formation) cause neighbouring cells to point their trichomes away from the clone, as occurs with Vangstbm-6/Vangstbm-6 single mutant clones in the pupal wing.
Unlike embryos from dsh3 germline clones, embryos from dsh3; sggM11 germ-line clones have no dorsal hole in their cuticles (although their dorsal cuticle is severely puckered), and have a dorso-ventrally polarised and extended leading edge at stage 13.
dshmut6.T:Avic\GFP-EGFP rescues the embryonic lethality of dsh3 animals. The resulting adults have largely wild-type wings and both leg bristles and wing hairs show normal planar polarisation. The rescued adults show a strong ectopic leg joint phenotype.
dsh3 animals rescued to adulthood by dsh1.T:Avic\GFP-EGFP show a strong ectopic leg joint phenotype.
Some dsh3 animals rescued to adulthood by dshT:Avic\GFP-EGFP show an ectopic leg joint phenotype.
dshΔQ rescues the lethality of dsh3 flies, but a small fraction of the survivors have mild polarity and patterning defects.
dshQ108 shows only 24% rescue of the lethality of dsh3 flies. The surviving adults have polarity and patterning defects including notched wings, disordered bristles and bifurcated legs.
dsh3 flies carrying dsh1.tAa are viable and have a dsh1 phenotype. Mutant embryos show shortening of the cuticle, fusion of denticle bands and absence the head skeleton and filzkorper. This phenotype is completely rescued by injection of dshcAa or dshΔbPDZ mRNA, partially rescued by injection of dshΔDIX or dshΔEP+ mRNA, and is not rescued by injection of dshΔDEP+, dshDIX, dshbPDZ or dshDEP+ mRNA.