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First published online 16 August 2006
doi: 10.1242/dev.02525

Development 133, 3517-3527 (2006)
Published by The Company of Biologists 2006
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Fork head and Sage maintain a uniform and patent salivary gland lumen through regulation of two downstream target genes, PH4{alpha}SG1 and PH4{alpha}SG2

Elliott W. Abrams*, Whitney K. Mihoulides and Deborah J. Andrew{dagger}

Department of Cell Biology, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205-2196, USA.


Figure 1
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  		Fig. 1. Fkh regulates expression of SG1 and SG2. (A,B) Expression of SG1 and SG2 RNA and (D) SG1 protein was absent in fkh mutants. (C) Expression of pipe was unaffected by loss of fkh. (E) SG2 (red) colocalized with the ER marker KDEL (green) and SG1 (green) colocalized with SG2 (red) (top and bottom panels, respectively). We examined expression of genes with a fkh null mutation in the background of the H99 deficiency, which removes the pro-apoptotic genes; this background allows salivary gland cell survival in a fkh mutant (Myat and Andrew, 2000Go). Expression of pipe, SG1 and SG2 were the same in fkh mutants that also did not carry the H99 deficiency.

 

Figure 2
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  		Fig. 2. Regulation of SG1 by Fkh is indirect. (A) 972 nucleotides of sequence upstream and spanning the translation start site (green letters) of SG1 were sufficient to direct Fkh-dependent salivary gland expression of the lacZ reporter gene. Only two of the four putative Fkh-binding sites in the 972 nucleotide sequence (b and d, blue sequence and filled blue ovals) were bound by Fkh protein in vitro and competed for binding (Aa, Ab; arrowheads indicate P32-labelled oligos, the migration of which in the gel has been slowed owing to Fkh binding; the triangles on the top of each gel indicate relative concentrations of cold competitor). Whereas unlabelled wild-type d oligos competed well for binding with labeled wild-type d, the mut d oligo did not compete (compare left and right gels in Aa, arrowheads). Wild-type b oligos competed less well than did wild-type d for binding but better than mut d (compare gel in Ab with gels in Aa, arrowheads). Three consensus bHLH binding sites (CAXXTG) are present in the 972 nucleotide SG1 enhancer (red sequences and red filled circles). (B-D) Wild-type versions of the SG1 972-lacZ construct gave high level ßgal expression in the salivary gland (arrows) and variable expression in other tissues (non-salivary gland staining in B-I corresponds to hemocytes). (E-G) Mutations in the two in vitro Fkh-binding sites (b and d) did not affect the salivary gland expression of the reporter constructs. (H,I) Expression of the SG1 972-lacZ construct was absent in salivary glands (arrows) of fkh mutants, although expression in other tissues (hemocytes) was unaffected (here, embryos were stained in the presence of NiCl, causing the reaction substrate to turn purple/black instead of red/brown, as in the embryos in B-G).

 

Figure 3
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  		Fig. 3. Regulation of SG2 by Fkh is direct. (A) 885 nucleotides of sequence upstream and spanning the translation start site of SG2 (green letters) was sufficient to direct Fkh-dependent salivary gland expression of the lacZ reporter gene. Although the 885 nucleotide sequence contains four putative Fkh-binding sites (A, blue ovals and blue sequences), only three of the sites (f,g,h, blue filled ovals) were bound by Fkh protein in vitro or competed for binding (Aa, Ab). Whereas both unlabelled wild-type h and wild-type f compete well for binding with P32-labelled wild-type h, neither mut h nor mut f compete for binding (compare left to right gels in Aa and Ab). Adding cold competitor in all cases reduced the amount of labeled oligo that failed to enter the gel, making it appear as if the addition of mutant oligos increased the specific binding of Fkh to the labeled oligo, especially on the gel on the right with the mut site f competitor. Six consensus bHLH binding sites (CAXXTG) are present in the 885 nucleotide SG2 enhancer (A, red circles and red sequences). (B-D) Embryos carrying a wild-type SG2 885-lacZ construct had ßgal expression in the salivary glands (arrows) beginning at stage 11 and continuing through embryogenesis. (E-G) Embryos carrying the SG2 885 fkh-lacZ constructs with mutations in the three Fkh in vitro binding sites (f,g,h) had reduced salivary gland expression at early stages (arrow indicates the area outlined in black, which is the unstained salivary gland) and variable expression at later stages (arrows in F,G). (H,I) The salivary gland expression (arrows) of the SG2 885-lacZ construct visible in wild type (H) was absent in the salivary glands of fkh mutants (I). Embryos in H,I were stained in the presence of NiCl.

 

Figure 4
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  		Fig. 4. sage is expressed specifically in the salivary gland and its later expression requires Fkh. (A) sage was detected in the salivary glands of wild-type embryos beginning at the placode stage through the end of embryogenesis. (B) sage expression after stage 12 was absent in fkh mutants. Compare staining indicated by arrows in left panels in the fkh heterozygotes, which also show lacZ expression from the TM3 Ubx-lacZ balancer chromosome (a-c), with staining indicated by arrows in the fkh mutants in the right panels (d-f). Salivary glands do not invaginate in fkh mutants (d-f).

 

Figure 5
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  		Fig. 5. sage is directly regulated by Fkh and Sage. (A) 1.2 kb of sequence upstream and spanning the translation start site (green letters) of sage is sufficient to direct Fkh-dependent salivary gland expression of the lacZ reporter gene. This sequence contains five consensus bHLH-binding sites (red circles and sequences) and one Fkh-binding site (blue oval and sequence). (B,C) The wild-type Sage 1.2-lacZ construct gave robust salivary gland expression both early and late. (D,E) Expression of the wild-type Sage 1.2-lacZ was diminished in fkh mutant salivary glands, which do not invaginate. (F,G) Mutations disrupting either the Fkh site or (H,I) the bHLH sites resulted in reduced salivary gland expression of ßgal. (J,K) Loss of both Fkh and bHLH sites resulted in reduced expression of the reporter gene to levels observed in fkh mutants (compare salivary gland expression of ßgal in J and K with D and E).

 

Figure 6
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  		Fig. 6. SG2 is directly regulated by Fkh and Sage, whereas SG1 is not. (A) Global expression of sage, achieved by driving UAS-sage with a tub-GAL4 driver, resulted in ectopic expression of both SG1 and SG2 in tissues outside the salivary gland, notably in the foregut (fg), hindgut (hg) and Malphigian tubules (mt) (Aa-a'', Ab-b''). (Aa) SG1 was also expressed in two rows of cells in each segment during stage 11 (arrows). (Ac-c'') The tissues that expressed ectopic SG1 and SG2 also express Fkh protein, including faint expression in two rows of cells in each segment during stage 11 (Ac, arrows). (B) Mutations in the bHLH binding sites (Ba-a'') or the bHLH and Fkh-binding sites (Bb-b'') in the SG1 972 enhancer did not affect expression of the lacZ reporter. (C) Mutations in the bHLH binding sites (Ca-a'') or the bHLH and Fkh-binding sites (Cb-b'') in the SG2 885 enhancer resulted in either reduction or complete loss of salivary gland expression of the lacZ reporter, respectively.

 

Figure 7
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  		Fig. 7. Loss of SG1 and SG2 results in regions of salivary tube closure. (A) Df(3R)Exel6216 deletes only 16 genes, eight of which are the previously characterized PH4{alpha} genes (asterisks), including SG1 and SG2 (Abrams and Andrew, 2002Go). (B) Df(3R)Exel6216 salivary glands have abnormal lumenal morphology. (C) Confocal images of salivary glands stained with CrebA (blue), ßHSpectrin (red) and {alpha}Spectrin (green) revealed regions of tube dilation, constriction and apparent closure (asterisks) in the Df(3R)Exel6216 embryos (the images -1 to -4 are different focal planes from the same glands). (D) Composite images of ~50-80 0.3 µm sections of salivary glands stained with CrebA (blue) and Crb (red) revealed uniform lumen diameters wild-type embryos (left panel) but variable lumenal diameters in the Df(3R)Exel6216 embryos (right panel). (E) Expression of either SG1 (left panels) or SG2 (right panels) driven by a fkh-GAL4 driver rescues the salivary gland lumenal irregularities of Df(3R)Exel6216 embryos. Although all of the embryos of the genotypes shown in the lower two sets of panels in E were expected to express either SG1 or SG2, expression of both proteins was variable, with undetectable expression in some embryos (middle panels). Detectable SG1 and SG2 expression correlated with rescue of the lumenal defects (lower panels). Df(3R)Exel6216 is balanced over TM6B, Ubx-lacZ, allowing us to distinguish the heterozygous from homozygous deficiency embryos by also staining with an {alpha}-ßgal antiserum. ßgal staining (red) can be seen in the heterozygous embryos in the top panels in E near the distal end of the salivary gland.

 

Figure 8
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  		Fig. 8. Loss of SG1 and SG2 affects the quality and quantity of lumenal secretory content. (A-C) TEM analysis of wild-type (Oregon R) and Df(3R)Exel6216 salivary glands revealed differences in both the volume and morphology of apical secretions. (A-A''') Thin sections of wild-type salivary gland cells revealed uniform apical cell surfaces surrounding a relatively large lumenal space filled with fibrillar matrix material (asterisks). (B-B''') Thin sections of Df(3R)Exel6216 salivary glands revealed irregular and small lumenal spaces containing matrix of increased density (asterisks) and large regions where the apical cell surfaces appear to meet. (C,C') New adherens junctions (AJs) appear to have formed at the contact site of cells arising from opposite sides of the lumen of Df(3R)Exel6216 salivary glands (arrowheads indicate AJs). Secretory vesicle number and morphology were similar in wild-type and Df(3R)Exel6216 salivary glands, as were other aspects of salivary gland cell morphology. A-A''', B-B''' and C-C' are images from the same gland showing increased magnification from left to right; in some cases the angle of the image is changed slightly from panel to panel. Boxed regions in A'', B'' and C correspond to magnified images shown in A''', B''' and C', respectively. Df(3R)Exel6216 was balanced over TM3, twi-GFP, allowing us to select homozygous deficiency embryos prior to fixation.

 

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© The Company of Biologists Ltd 2006