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Redundant function of Runt Domain binding partners, Big brother and Brother, during Drosophila development

¹ Department of Molecular, Cell and Developmental Biology,
² Molecular Biology Institute,
³ Department of Biological Chemistry and Department of Human Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA

View larger version (174K): [in a new window]   Fig. 1. A sensitized genetic screen for dosage-sensitive enhancers of lzts1. Scanning electron micrographs of adult eyes. Posterior is to the left and dorsal is up. All flies were reared at 25°C except for the one shown in C which was reared at 29°C. (A) The wild-type Drosophila eye has a regular array of ordered facets. (B) lzts1 flies, when reared at 25°C, have wild-type eyes. (C) When lzts1 flies are reared at 29°C, the eye appears rough and disorganized. (D) lzR1, which is a null allele of lz, gives rise to a more severe eye phenotype than lzts1 at any temperature. (E-I) Interacting mutations. When raised at 25°C, in a lzts1 background, loss of one copy of each enhancer locus causes a slight roughening of the posterior area of the adult eye. Under a light microscope the eyes also appear to be slightly glossy, suggesting a defect in lens secretion. (E) lzts1; en(lz)3C/+. (F) lzts1; en(lz)Y/+. (G) lzts1; en(lz)D/+. (H) lzts1; en(lz)4G/+. (I) lzts1; en(lz)4F/+.

View larger version (99K): [in a new window]   Fig. 2. Bgb and Run expression in the eye imaginal disc. Posterior is to the left. Arrow indicates the position of the furrow. (A-C) Wild-type eye disc stained with -Bgb antibody. (A) At the basal level, nuclear staining is seen in the undifferentiated pool of cells. This staining begins immediately posterior to the furrow. (B) At a slightly more apical level, Bgb protein is seen in the nuclei of the R8, R1 and R6 cells. The expression of Bgb in the R8 cell initiates within 1 column of the furrow, while the expression in R1 and R6 initiates about 4 columns posterior to the furrow. (C) At the most apical focal plane, cone cells (cc) and the R7 cell express Bgb beginning about 7 columns posterior to the furrow. The R7 cell expresses Bgb at a much higher level than in any of the other cells. (D,E) Wild-type eye disc stained with -Run antibody. (D) Run protein is detected in the nuclei of the R8 cell, initiating 1 column posterior to the furrow. Run expression was determined to be in R8 by the central location of this cell within an ommatidial cluster and through co-localization with the R8 specific marker, Boss (Kramer et al., 1991 and data not shown). (E) Run expression is seen in R7 about 7 columns posterior to the furrow. The timing of Run expression in R7 and R8 correlates well with the expression pattern of the Bgb protein in these cells. (F,G) Bgb expression in a lz77a7 background. lz77a7 is an eye-specific null allele of lz (Flores et al., 1998). (F) Bgb expression is limited to the R8 cell. (G) R7 expression of Bgb is absent. (H) Bgb expression in a lzsprite background. With this gain-of-function allele, lz is misexpressed in R3 and R4 (Daga et al., 1996). Arrowheads indicate a corresponding ectopic expression in R3 and R4 of the Bgb protein. (I-J) Run expression in a lz77a7 background. (I) Run expression in lz77a7 is seen only in the R8 cell. (J) At the slightly higher R7 focal plane, no Run staining is observed in the R7 cell. (K,L) S2 cells stained with -Bgb. (K) In cells transiently transfected with Bgb, the majority of the Bgb protein is found in the cytoplasm. (L) When Bgb and lz are co-transfected, the majority of Bgb protein moves to the nucleus. (M,N) S2 cells stained with -Lz. (M) In cells transiently transfected with lz cDNA, Lz protein is seen in the nucleus. (N) Cotransfection of Bgb and lz does not affect the localization of Lz protein.

View larger version (167K): [in a new window]   Fig. 3. Bgb phenotypes. (A-D) SEMs of adult eyes. Posterior is to the left and dorsal is up. All flies were reared at 25oC. (A) lzts1; BgbD/+. The BgbD mutant enhances lzts1 on the posterior side of the eye (compare with Fig. 1B). (B) lzts1; Df(3L)BgbK4/+. The BgbK4 deletion enhances the lzts1 eye phenotype. (C) lzR1. The lz null eye is very smooth and does not show any ommatidial organization. (D) lzts1; Df(3L)BgbK4/BgbD. Rare escapers of this genotype have a phenotype resembling a lz null eye. (E-M) Embryonic phenotype of Bgb and run mutants. Anterior is to the left and dorsal is up. Embryos were stained with mAb22C10 (E-J) or -Engrailed antibody (K-M). (E) Wild-type stage-15 embryo. mAb22C10 recognizes neurons of the chordotonal organs (box). These are easily identified on the basis of their stereotypical arrangement and position in abdominal segments A1-A7. (F) A schematic diagram of the lateral chordotonal neurons based on that by Campos-Ortega and Hartenstein (1997). Within each wild-type cluster (WT panel) the axon of the single lateral chordotonal neuron, lch1, pioneers the segmental nerve (SN) to its target in the midline. The remaining five lateral chordotonal neurons, lch5, follow the intersegmental nerve (ISN) to their target in the midline. In Bgb mutant embryos (Bgb panel), the axon of the lch1 neuron incorrectly follows the ISN to the midline. In this and subsequent panels an arrowhead indicates the absence of the lch1 axon in its normal position. This axon does not join the SN, but instead aberrantly turns toward the ISN and follows this incorrect path to the midline. The ventral sensilla, vp5, has an apical projection (asterisk in F and H to distinguish this structure from the axon of the lch1 neuron). (G,H) Lateral chordotonal neurons of (G) wild-type and (H) BgbD/BgbD stage-15 embryos. (G) The axons of the lch1 neurons correctly follow the SN to the midline (arrow). (H) In the mutants, axons of the lch1 neurons do not follow the SN (arrowheads), but incorrectly project towards the lch5 and follow the ISN to the midline. In this genetic background the phenotype is not fully expressed and only 2 of the 3 lateral chordotonal clusters shown have this defect. (I) Lateral chordotonal neurons of BgbD/Df(3L)BgbK4 stage-15 embryo. In these mutants, the axons of the lch1 neurons aberrantly project towards the lch5 and do not follow the SN to the midline (arrowheads). This phenotype is similar to, but stronger than, that seen in (H). (J) Lateral chordotonal neurons of runYP17/runYP17 stage-15 embryo. Only two sets of lateral chordotonal neurons are seen within the region corresponding to the previous panels because of a lack of segments in this mutant (Gergen and Wieschaus, 1986). The axon of one of the lch1 neurons shown misprojects and does not follow the SN to the midline (arrowhead). (K) Wild-type stage-11 embryo. Engrailed protein is expressed as a 14 stripe pattern in the posterior compartment of each segment. (L) BgbD/BgbD stage-11 embryo. Engrailed expression is as seen in wild type. (M) runYP17/runYP17 stage-11 embryo. These embryos show a segmentation phenotype, not seen in Bgb mutants.

View larger version (72K): [in a new window]   Fig. 4. Lack of Bgb eye phenotype in a lz+ background. (A) Somatic clone of BgbD/BgbD tissue in the eye. The mutant tissue was first identified at low magnification using the difference in pigmentation between the mutant and the wild-type tissue. This boundary was subsequently marked in the SEM with the black dotted line. No defects are evident in the external structure of the facets within the clone. (B,C) Plastic section through a somatic clone of BgbD/BgbD tissue. (B) In this clone, marked by the lack of pigment granules, the R1-6 and R7 cells (1-7) appear completely normal. (C) In this deeper section of the same clone, R8 cells (8) are seen to develop normally.

View larger version (104K): [in a new window]   Fig. 5. ds-RNA-mediated genetic interference. Cuticle preparations showing denticle belts corresponding to abdominal segments A1-A8 and thoracic segment T3. Anterior is to the left. (A) Wild-type injected with buffer only. T3 and A1-A8 denticle belts are clearly visible. (B) runYP17. This hypomorphic allele produces a weak segmentation phenotype. A deletion of the lateral edges between the A2/A3 denticle belts results in the curved appearance of these belts. Deletion of the naked cuticle between the A4/A5 and the A6/A7 denticles is also evident. (C) runXD106. This null allele produces a severe segmentation phenotype. Large deletions between the A2/A3, A4/A5, and A6/A7 denticle belts are apparent. These deletions result in mirror image duplications of the corresponding denticles. (D-F) Three independent examples of wild-type embryos injected with double stranded RNA corresponding to the run gene (ds-run). In each example there is a complete deletion of the naked cuticle between A2/A3, A4/A5, and A6/A7 which results in a mirror image duplication of the remaining denticles. This is the same phenotype as that seen in null alleles of run (compare with C). (G-I) Three independent examples of wild-type embryos injected with ds-Bgb. No perceptible alteration of the segmentation pattern is evident. These animals are indistinguishable from those injected with buffer (compare with A). (J-L) Three independent examples of wild-type embryos injected with ds-Bro. Defects in the segmentation pattern are similar to those seen in hypomorphic run mutants (compare with B). (M-O) Three independent examples of wild-type embryos injected with a mixture of ds-Bgb and ds-Bro. An extremely strong segmentation phenotype identical to that seen with null alleles of run (compare with C) and wild-type embryos injected with ds-run (compare with D-E) can be seen. Thus, although injection of ds-Bgb did not affect segmentation, and injection of ds-Bro produced a mild segmentation defect, injection of a mixture of both ds-Bgb and ds-Bro had a synergistic effect on the pattern of segmentation.

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