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Results: 8

1.
Figure 6

Figure 6. Either ‘loss’ or ‘gain’ of gγ1 function leads to germ cell migration defects.. From: G?1, a Downstream Target for the hmgcr-Isoprenoid Biosynthetic Pathway, Is Required for Releasing the Hedgehog Ligand and Directing Germ Cell Migration.

Embryos from either the gγ1/ Cy0, en:LacZ stock or from UAS-gγ1 X elav-GAL4 cross were collected and fixed using standard histochemical technique. Wild type embryos derived from Oregon R stock were used as control (Panels A and B). Embryos from the gγ1/ Cy0, en:LacZ stock were identified by simultaneously staining them with b-galactosidase antibody (not shown). Germ cell migration was assessed using anti-Vasa antibodies. A: Wild type stage 13 embryo. B: Wild type stage 15 embryo. C: gγ1N159 stage 13 embryo. D: gγ1N159 stage 15 embryo. E: UAS-gγ1/ elav-GAL4 stage 15 embryo. F: UAS-gγ1/ elav-GAL4 stage 15 embryo.

Girish Deshpande, et al. PLoS Genet. 2009 January;5(1):e1000333.
2.
Figure 2

Figure 2. Reduced wingless expression in embryos compromised for gγ1.. From: G?1, a Downstream Target for the hmgcr-Isoprenoid Biosynthetic Pathway, Is Required for Releasing the Hedgehog Ligand and Directing Germ Cell Migration.

Panels A and B: Embryos from gγ1N159/ Cy0, en:LacZ stock were collected and fixed using standard procedures. Embryos were genotyped by probing with β-galactosidase antibodies (imaged in red: not shown), while Wg accumulation was visualized by probing with Wg (imaged in green) antibodies. Embryos carrying en:LacZ express β-galactosidase whereas homozygous mutant embryos do not. The embryo in panel A was positive for β-galactosidase (not shown) and has at least one wild type copy of gγ1. Note the high level of Wg accumulation in the stripes. The embryo in panel B β-galactosidase negative, and is homozygous for the gγ1N159 mutation. Note the lower level of Wg expression. Panels C and D: Embryos from a gγ1k0817/Cyo, en:LacZ stock were collected and fixed using standard procedures. As in panels A and B embryos were genotyped by probing with β-galactosidase antibodies (imaged in red: not shown), while Wg accumulation was visualized by probing with Wg (imaged in green) antibodies. The embryo in C is positive for β-galactosidase, while the embryo in D is not. Note the difference in Wg accumulation in the blow-up of three Wg stripes.

Girish Deshpande, et al. PLoS Genet. 2009 January;5(1):e1000333.
3.
Figure 3

Figure 3. Localization of Smoothened receptor is altered in embryos compromised for gγ1.. From: G?1, a Downstream Target for the hmgcr-Isoprenoid Biosynthetic Pathway, Is Required for Releasing the Hedgehog Ligand and Directing Germ Cell Migration.

Embryos from gγ1N159/ Cy0; en:LacZ stock were collected, fixed using standard protocol and were subsequently identified by staining simultaneously with the β-galactosidase and Smo antibodies. Smo was imaged with the secondary antibodies coupled with Alexa 546. The figure shows stage 10–11 embryos. In the wild type control (gγ1N159/ Cy0; en:LacZ embryos) the intercellular distribution of Smo protein has a parasegmentally repeating pattern (see arrows in Panel A). In a ∼5 cell wide stripe across each parasegment Hh signaling leads to the relocalization of Smo protein to the membrane. In the remaining cells in each parasegment (∼5 cell wide stripe) Smo protein remains largely cytoplasmic. This can be seen in the magnified view in Panel B. In gγ1 embryos, this parasegmentally repeating pattern of Smo protein localization is largely lost (see Panel C). In most cells in each parasgement the Smo protein remains diffusely distributed through cytoplasm (see Panel D).

Girish Deshpande, et al. PLoS Genet. 2009 January;5(1):e1000333.
4.
Figure 8

Figure 8. Germ cell migration defects induced by partial loss of gγ1 are enhanced further by reducing either hmgcr or hh activity.. From: G?1, a Downstream Target for the hmgcr-Isoprenoid Biosynthetic Pathway, Is Required for Releasing the Hedgehog Ligand and Directing Germ Cell Migration.

Embryos between stages 12–15 of the indicated genotype were stained with anti-Vasa and β-galactosidase antibody and staining was visualized with standard immunohistochemcial techniques. Total number of germ cells that failed to associate with SGPs and remained scattered were counted per embryo. 25 embryos of each genotype were analyzed. Top panel shows that when embryos are heterozygous either for gγ1 or hmgcr, more than 80% of the embryos display 0–2 lost germ cells (blue and red bars respectively). But when embryos are simultaneously compromised for both gγ1 and hmgcr, more than 60% of the embryos have 7 or more lost germ cells (yellow bars). Bottom panel shows similar synergistic interaction between gγ1 or hmgcr. Although the enhancement in germ cell migration defects is less severe compared to that seen with hmgcr (30% of the total number of embryos of the genotype gγ1/+; hh/+ show more than 7 lost germ cells), the germ cell migration defects in embryos simultaneously compromised for gγ1 and hh are clearly more severe as opposed to either gγ1/+ or hh/+ embryos.

Girish Deshpande, et al. PLoS Genet. 2009 January;5(1):e1000333.
5.
Figure 1

Figure 1. gγ1 can dominantly suppress the wing abnormalities including pattern duplication induced by hh MRT.. From: G?1, a Downstream Target for the hmgcr-Isoprenoid Biosynthetic Pathway, Is Required for Releasing the Hedgehog Ligand and Directing Germ Cell Migration.

Panel A shows an example of Class III type of wing defects induced by ectopic expression of Hh in the anterior compartment in hh MRT animals. Panel B shows a wing from an animal of the genotype hh MRT/gγ1. Almost complete suppression (Classified as Class I or II) of the wing phenotype can be seen. Panel C shows a graphic representation of the suppression of the wing defects. Roughly 250 single wing blades of the indicated genotypes were analyzed and classified into 5 different categories depending on the severity of the phenotype as previously described in Felsenfeld and Kennison, [27]. All the experimental as well as control crosses testing hhMRT suppression were carried out at 18°C.

Girish Deshpande, et al. PLoS Genet. 2009 January;5(1):e1000333.
6.
Figure 5

Figure 5. Distribution of Hh ligand is altered in qm embryos.. From: G?1, a Downstream Target for the hmgcr-Isoprenoid Biosynthetic Pathway, Is Required for Releasing the Hedgehog Ligand and Directing Germ Cell Migration.

Embryos from qm/ Cy0, ftz-LacZ stock were collected, fixed using standard protocol and were subsequently identified by staining simultaneously with the β-galactosidase (not shown) and Hh antibodies (imaged in red). Panel A: Wild type control (qm/Cy0, ftz-LacZ). Panel B: qm embryo. In the control embryos, Hh protein synthesized in two rows of cells per parasegment is released and spreads through the segment. Within the Hh expressing cells, Hh protein is localized around the membrane in a grainy or punctate pattern (see arrowheads in Panel A). In qm the release/transmission of the Hh protein is abnormal. The level of Hh in the interstripe region is considerably diminished suggesting that like gγ1, the qm gene is required for the efficient release and/or transport of the Hh protein. Consistent with this suggestion, Hh protein appears to accumulate in the Hh expressing cells. Like gγ1, the distribution of Hh in the expressing cells is abnormal. Instead of the characteristic grainy or punctate pattern of Hh protein localized around the cell membranes, Hh accumulates in clumps or aggregates (see arrows).

Girish Deshpande, et al. PLoS Genet. 2009 January;5(1):e1000333.
7.
Figure 4

Figure 4. Spread of Hh ligand is restricted in gγ1− embryos.. From: G?1, a Downstream Target for the hmgcr-Isoprenoid Biosynthetic Pathway, Is Required for Releasing the Hedgehog Ligand and Directing Germ Cell Migration.

Embryos from gγ1N159/ Cy0; en:LacZ stock were collected, fixed using standard protocol and were subsequently identified by staining simultaneously with the β-galactosidase and anti-Hh antibodies. Hh specific signal was imaged with the secondary antibodies coupled to Alexa 546 (Red). The figure shows stage 10–11 wild type (panel A) and gγ1N159 (panels B and C) embryos. In wild type embryos, two rows of cells per segment express Hh protein. In these cells Hh protein is distributed around the membrane in a grainy or punctate pattern (see arrowheads). The Hh ligand is released and it spreads in a graded fashion through the parasegment. In the gγ1N159 embryos (panels B and C) Hh is not properly released from hh expressing cells and high levels of Hh protein accumulate in these cells. The grainy pattern of Hh protein around the cell membrane is less evident and instead Hh protein accumulates in larger clumps or aggregates that appear to be distributed in the cytoplasm rather than just at the membrane (see arrows). Panels D, E and F are enlargements of the Hh stripe in the embryos shown in panels A, B and C respectively. Arrows in panel D show the grainy punctate pattern of Hh protein in wild type embryos (the LPS), while the arrows in panels E and F show the larger clumps or aggregates of Hh protein that accumulate in cells of mutant embryos.

Girish Deshpande, et al. PLoS Genet. 2009 January;5(1):e1000333.
8.
Figure 7

Figure 7. Manipulations of gγ1 activity disrupt germ cell migration.. From: G?1, a Downstream Target for the hmgcr-Isoprenoid Biosynthetic Pathway, Is Required for Releasing the Hedgehog Ligand and Directing Germ Cell Migration.

(A) Ectopic expression of gγ1 in hh producing cells not in hh receiving cells can induce germ cell migration defects. Whole mount staining of stage 13–15 embryos with antibodies against Vasa protein. Females carrying two copies of UAS-gγ1 were mated independently either with the ptc-GAL4/ptc-GAL4 males (panels on left) or hh-GAL4/TM6 Ubx-LacZ males (panels on right). Embryos (10–14 hr old) were collected, fixed and then probed with β-galactosidase and Vasa antibodies. In the case of hh-GAL4 driver, embryos of the correct genotype were identified by the absence of β-galactosidase. The staining was visualized using standard immunohistochemical techniques. As can be seen by the comparison of the panels, gγ1 is able to induce germ cell migration defects only when it is overexpressed using hh-GAL4 whereas gγ1 overexpression using ptc-GAL4 leads to essentially wild type germ cell migration. As shown in the bar-diagram in the lower half of A, more than 90% of the ptc-GAL4/UAS-gγ1 embryos (represented with red colored bars) display less than 2 lost germ cells whereas close to 30% of the hh-GAL4/UAS-gγ1 embryos (represented with blue colored bars) have more than 5 lost germ cells. hh-GAL4/UAS-gγ1 (n = 74), ptc-GAL4/UAS-gγ1 (n = 53). (B) Ectopic expression of gγ1 and gγ1-ΔCAAX in the mesoderm induces germ cell migration defects. In this experiment embryos produced by females carrying the UAS:gγ1 or UAS gγ1-ΔCAAX mated to twist GAL4 males were stained with Vasa antibody and the number of lost or scattered germ cells in each embryo was counted. As shown in the bar graph, ectopic expression of either Gγ1 or Gγ1-ΔCAAX using the twist driver induced germ cell migration defects. As explained in the text, these defects likely arise for different reasons. Ore R (n =  20), UAS-gγ1 (N = 69), UAS- gγ1-ΔCAAX (n = 53).

Girish Deshpande, et al. PLoS Genet. 2009 January;5(1):e1000333.

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