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First published online 15 December 2008
doi: 10.1242/dev.027854

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Ancestry-independent fate specification and plasticity in the developmental timing of a typical Drosophila neuronal lineage

Department of Neuroscience and Cell Biology, University of Texas Medical Branch School of Medicine, Galveston, TX 77555, USA.

View larger version (123K): [in this window] [in a new window]   Fig. 1. The extra RP2 neuron in Drosophila mid mutants. Wild-type and mid mutant Drosophila embryos, 13 to 14 hours old, were stained with antibodies to Eve (A,B), Eve+Zfh1 (C-F) or Eve+22C10 (G-L). Anterior is up, the midline is marked by vertical lines. Arrow, RP2; arrow with asterisk, the extra RP2 (eRP2); small arrow, axon projection from an RP2 or eRP2. ISN, intersegmental nerve bundle; fp1, focal plane 1; fp2, focal plane 2.

View larger version (86K): [in this window] [in a new window]   Fig. 2. The elaboration of the extra RP2 lineage in mid mutants. (A-N) Wild-type and mid mutant Drosophila embryos of the indicated ages were stained with antibodies to Eve (A-D,J,M), Zfh1 (K,N), or Eve+Zfh1 (E-H,I,L). Only one hemisegment is shown in each panel; anterior is up. Arrowhead, GMC-1; arrowhead with asterisk, the extra GMC-1; arrow, RP2; arrow with asterisk, eRP2; thin arrow, sib; thin arrow with asterisk, esib. (O) The developmental timing of the bona fide RP2/sib lineage in wild-type Drosophila (WT) and the development of the eRP2 lineage in mid mutants.

View larger version (82K): [in this window] [in a new window]   Fig. 3. Double-mutant analysis between mid and insc, numb, pdm and wg. Drosophila embryos of the indicated genotypes and ages were stained with antibodies to Eve (A,B,E-J) or Eve+Zfh1 (C,D). Anterior is up; vertical lines indicate the midline. Arrow, RP2; arrow with asterisk, eRP2; smaller arrow, sib; arrowhead indicates missing RP2 lineage; arrowhead with asterisk indicates missing eRP2 lineage.

View larger version (62K): [in this window] [in a new window]   Fig. 4. Characterization of various mid alleles and the Mid antibody. (A) Mid protein in wild-type Drosophila and in three different mid alleles. The double-headed arrow indicates the region of Mid used to raise the antibody. (B) Western analysis of the Mid protein in the wild type and in various mid alleles and a mid, H15 deficiency. The large arrow indicates the Mid-specific band in the wild type; the small arrow indicates the truncated Mid protein in los1. (C) There is no maternal deposition of mid transcript to embryos. RT-PCR for mid transcript from unfertilized eggs (Unf) and embryos at 0-4, 4-8, 8-12 and 12-24 hours of development. rp49 was used as control. (D) There is no maternal deposition of Mid protein to embryos. Western blotting of embryos at 0-4, 4-6, 6-8 and 8-10 hours of development. The Mid band (arrow) is separated from a non-specific band in this blot. (E) Mid antibody is specific to Mid protein. Embryos are stained with Mid and Eve antibodies. Anterior is up; vertical lines mark the midline. In the wild type, Mid is expressed in a large number of neurons. The neuron that appears to change into an extra RP2 is indicated by the arrow. In mid1 embryos, no detectable Mid was present. This was also the case in mid and H15 deficiency (middf) embryos.

View larger version (89K): [in this window] [in a new window]   Fig. 5. Mid expression in NBs, GMCs and neurons. Wild-type (A-I) and los1 mutant (J-L) Drosophila embryos of the indicated ages stained with Mid (red) and Wg (green) antibodies. Anterior is up; vertical lines mark the midline. M, M-neuron; X?, this Mid-positive cell appears to be the X-cell; arrow with asterisk, the extra RP2. Rows of NBs are indicated by numbers at the midline. The schematics illustrate NB maps corresponding to A-C and D-F.

View larger version (109K): [in this window] [in a new window]   Fig. 6. Development of the M-lineage. (A-T) Wild-type (WT) and los mutant Drosophila embryos of the indicated ages were stained with antibodies to Mid and En (A-H), Mid and Eve (I-P), or Mid+Zfh1 (Q-T). Anterior is up; midline is marked by vertical line(s). Arrowhead, M-GMC; large arrow and M, M-neuron; small arrow and s, M-sib; white asterisks in N indicate another Mid-positive neuron and its smaller sibling cell. (U) The developmental timing of the M-lineage.

View larger version (84K): [in this window] [in a new window]   Fig. 7. Loss of Wingless activity affects the formation of row 5 NBs and expression of Gsb and Wg is unaffected in mid mutants but the expression of Gsb-n is lost from the eRP2. Wild-type (WT) and mid or wg mutant Drosophila embryos stained with antibodies to Gsb (Gsb-d) (A-C), Wg (D,E) or Gsb-n (F-H). Anterior is up; midline is marked by vertical line(s). Numbers in the midline indicate NB rows; NBs are indicated by row number followed by NB number (e.g. 5-6 = NB5-6 = NB6 on row 5).

View larger version (29K): [in this window] [in a new window]   Fig. 8. Transcriptional activation by Mid, H15 and Org from the consensus T-box-binding element (TBE) and the gsb-n promoter. (A) Consensus TBE and the degenerate TBE (highlighted in red) present in the Drosophila gsb-n promoter. (B) Expression and reporter constructs used for the transcriptional activation assays. (C) Activation of the luciferase gene linked to the consensus TBE. (D) Activation of the luciferase gene linked to the gsb-n promoter.

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