spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 15 March 2006
doi: 10.1242/dev.02321

Development 133, 1445-1455 (2006)
Published by The Company of Biologists 2006
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Related articles in Development
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Garces, A.
Right arrow Articles by Thor, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Garces, A.
Right arrow Articles by Thor, S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Specification of Drosophila aCC motoneuron identity by a genetic cascade involving even-skipped, grain and zfh1

Alain Garces1 and Stefan Thor2,*

1 INSERM U 583, INM-Hopital St Eloi, 80 rue Augustin Fliche, 34091 Montpellier Cedex 5, France.
2 Division of Molecular Genetics, Department of Physics, Chemistry and Biology, Linkoping University, S-581 83 Linkoping, Sweden.


Figure 1
View larger version (98K):

[in a new window]
  		Fig. 1. Grain is expressed in ISN motoneurons and in subsets of interneurons. Stage 14 (A-H) and stage 12 (I) embryos stained for Grn (A,E,I), Repo (I), ß-gal (A,C,E,G) and Myc (B,C,D,F,G,H). Dorsal (A-D,I), mid-dorsal (C') and intermediate (E-H) focal planes of the VNC. Anterior is upwards in all panels. Grn expression within one (C,C',I), two (A,E) or three (B,C,F-H) segments. grnlacZ is expressed in all Grn-positive neurons (A,E). We noticed consistently weaker expression of Grn and grnlacZ (or grnGAL4) in the RP2 motoneuron (arrowhead) compared with other grn-expressing neurons (A,D). Mutually exclusive expression patterns of grnGAL4/UAS-{tau}lacZ with islet-{tau}mycEGFP (B,F), and with Hb9-GAL4 (C,C',G) in subsets of motoneurons and interneurons. Overlap of Grn with Eve and Zfh1 in aCC, RP2 (D) and the U motoneurons (asterisks, H). The pCC interneuron, which is located posterior to aCC does not express grnlacZ (A) or Grn (D). In stage 12 embryos, we find overlap of Grn and Repo (I) in one glia cell (*). This glia cell rapidly becomes Grn negative at later stages (compare with A1) but maintains ß-gal expression when probed with grnlacZ (A2) probably owing to the stability of the ß-gal protein. (J) Schematic showing grn-expressing cells in the VNC and the grn-expressing ISN motor axon projections in the periphery. The five U motoneurons are depicted in dark brown.

 

Figure 2
View larger version (84K):

[in a new window]
  		Fig. 2. grain is required for ISN motor axon projections. Stage 16 embryos stained with {alpha}-Fas2 (green in A-C,G), RN2-GAL4 driving UAS-mEGFPF (green in D-H), CQ2-GAL4 driving UAS-mEGFPF (green in I,J) and Phalloidin-TX (magenta in A-F,H-J). Arrows and arrowheads indicate axons terminals contacting dorsal (2 and 10) and dorsal-most (1 and 9) muscles, respectively. (A) In wild type, the ISN nerve innervates muscles 2/10 and 1/9. (B) In grn mutants, ISN fails to innervate muscles 1/9, but axonal projections are seen contacting muscles 2/10. Bracket denotes a partially penetrant muscle patterning phenotype, evident as an imprecise insertion of muscles 21-24 into the body wall. (C) In grn rescue (RN2-GAL4/UAS-grn; grn-/-) ISN innervates muscles 2/10 and 1/9 as in wild type. (D) In control, RN2-GAL4/UAS-EGFPF reveals muscle 1 innervation by aCC and muscle 2 innervation by RP2. (E) In a grn mutant background, RN2-GAL4/UAS-EGFPF reveals that although muscle 1/9 is not innervated by aCC, axon terminals from aCC and/or RP2 contact muscles 2/10. (F) zfh1 can partially rescue grn mutants (RN2-GAL4/UAS-zfh1; grn-/-) and the lack of muscle 1 innervation (arrowheads) is less severe than in grn mutant. (G) Overlap between RN2-GAL4/UAS-EGFPF (green) and {alpha}-Fas2 (magenta) revealing axons terminals for aCC and RP2. This reporter allows for a precise analysis of aCC and RP2 terminals in the periphery. (H) In grn mutants, 36% of hemisegments (n=69) show ectopic innervation of muscle 8 together with defasciculation of aCC and RP2 motor axons (see also oblique arrow in E). (I) In control, CQ2-GAL4/UAS-EGFPF reveals muscle 9 innervation by U1 and muscle 10 innervation by other U motoneurons. (J) In a grn mutants, CQ2-GAL4/UAS-EGFPF reveals that muscle 9 is not innervated (by U1), while U axon terminals contact muscles field 2/10. (K) Quantification of muscles 1/9 innervation in different genetic backgrounds. (L,M) Schematic showing the grn mutant phenotypes (M) compared to wild type (L).

 

Figure 3
View larger version (87K):

[in a new window]
  		Fig. 3. eve is necessary for grain expression and for Hb9 repression in both aCC and RP2 motoneurons. Stage 12 (A-D) or stage 15 (F-I) eve{Delta}RP2A/+ heterozygote (eve mosaic/+) (A,C,F,H) and eve{Delta}RP2A homozygote mutant (eve mosaic) (B,D,G,I) embryos. Arrows and arrowheads indicate aCC and RP2, respectively (visualized using RN2-GAL4/UAS-{tau}lacZ). (A,C,F,H) eve mosaic/+ RN2-GAL4/UAS-{tau}lacZ showing that Grn is expressed in aCC and RP2 at stage 12 and stage 15, while Hb9 is not. (B,D) In stage 12 eve mosaic mutant, Hb9 is derepressed in aCC and RP2 while Grn expression is not detectable in aCC but maintained in RP2. (G,I) In stage 15 eve mosaic mutants, Hb9 remains derepressed in aCC and partly in RP2, while Grn expression is not detectable in aCC but maintained in RP2. At this stage, Grn expression in RP2 appears even stronger in eve mosaic compared with wild type. (E,J) Quantification of these phenotypes.

 

Figure 4
View larger version (38K):

[in a new window]
  		Fig. 4. Zfh1, pMad and Vnd expression is affected in eve mutants. (A-D,F-I) Stage 15 eve mosaic/+ (A,C,F,H) and eve mosaic (B,D,G,I). Arrows and arrowheads indicate aCC and RP2, respectively (visualized using RN2-GAL4/UAS-{tau}lacZ). (A,C) Zfh1 expression is robust in control aCC and RP2 motoneurons. (B,D) In eve mosaic mutants, Zfh1 is lost from aCC, but unaffected in RP2 motoneurons. (E) Quantification of these phenotypes. (F) In control, pMad staining is evident in both aCC and RP2, but lost from these neurons in eve mosaic mutants (G). (H) In control, Vnd is specifically expressed by the pCC interneuron (double arrowhead) but expression is lost in eve mosaic mutants (I).

 

Figure 5
View larger version (100K):

[in a new window]
  		Fig. 5. In grain mutants, loss of Zfh1 expression is restricted to the aCC motoneuron. Stage 15 wild-type (A,D,G), grn mutant (B,E,H), zfh1 mutant (C,F) and grn rescue (I) (using RN2-GAL4/UAS-grn; grn-/-) embryos stained for Eve and pMad (A-C), Eve and Zfh1 (D-F) or Grn and Zfh1 (I). (G,H) RN2-GAL4/UAS-mEGFPF embryo stained with {alpha}-Hb9. (A-C) pMad staining in grn and zfh1 mutants appears unaffected within aCC and RP2. (D-F) In grn mutants, Zfh1 expression is not detectable in the aCC motoneuron, but RP2 maintains Zfh1 expression. Grn expression is not affected in aCC or RP2 in zfh1 mutants. (G,H) Hb9 expression is unaffected in grn mutants. (I) In grn rescue experiments, Zfh1 expression is restored in aCC showing the cell autonomous effect of grn on Zfh1 expression in this motoneuron. Arrowheads and asterisks indicate aCC and RP2, respectively.

 

Figure 6
View larger version (123K):

[in a new window]
  		Fig. 6. eve and grain play additional roles outside of the eve->grn->zfh1 cascade. (A-E) Stage 15 embryo stained for Grn (A,B) ß-Gal (A-D) and Zfh1 (A,C,E). B-E are identical to A but with different combinations of color channels to facilitate the observation of Grn and Zfh1 expression in aCC (arrows) and RP2 (arrowheads). grn is unable to rescue eve mosaic mutants (UAS-grn, eve mosaic; RN2-GAL4, UAS-{tau}lacZ), evident as a failure of aCC and RP2 to project axons out of the VNC, and of aCC to express Zfh1. (F-H) Stage 15 embryo stained for Myc and Zfh1, expressing only UAS-EGFPF (F), UAS-eve (G) or co-misexpressing both eve and grn (H). (F) In the control, dMP2 axons project posteriorly in the longitudinal connective and never exit the VNC laterally (n=62). (G) Ectopic eve triggers lateral VNC exit, but only in 5% of hemisegments. (H) Ectopic eve and grn (UAS-eve, UAS-grn, dMP2-GAL4; UAS-EGFPF) triggers lateral VNC exit in 40% of hemisegment (n=84). There is no evidence of Zfh1 expression in dMP2 neurons (yellow circles), in the control (F) or in the misexpression backgrounds (G). Arrowheads indicate dMP2 axons exiting the VNC.

 

Figure 7
View larger version (98K):

[in a new window]
  		Fig. 7. grain expression is under the control of Notch signaling. (A) In wild type, Vnd is expressed in the pCC interneuron, but this expression is lost in spdoG104 mutant (B,B'). (C,G) Vnd is derepressed in the aCC motoneuron when Notch signaling is activated using RN2-GAL4/UAS-NotchICD (intracellular domain of a constitutive activated form of Notch). The arrow indicates aberrant axonal projection (probably from aCC and/or RP2). (D) In grn mutants, derepression of Vnd is not observed in aCC (or in RP2) suggesting that grn does not repress Vnd in this sibling neuron. (E) In wild type, Grn is not expressed in pCC. (F,H) In spdoG104 mutants, Grn (and grnGAL4) is derepressed in the pCC neuron. Grn is also derepressed in the RP2sib; 4 Eve-positive neurons (observed in B) are indicated by a vertical bar and an asterisk. (G,I) Activation of Notch (RN2- GAL4/UAS-NotchICD) led to a loss of Grn (and grnlacZ) expression in aCC and RP2. Arrowheads and asterisks indicate aCC and RP2, respectively.

 

Figure 8
View larger version (79K):

[in a new window]
  		Fig. 8. grain and vnd do not act in a cross-repressive manner in aCC, pCC and RP2. (A-F) Stage 15 embryos stained for Grn and Vnd. (A-C) Ectopic grn expression in pCC (double arrowhead; RN2-GAL4/UAS-grn) does not suppress Vnd expression in this cell. (D-F) Ectopic Vnd expression in aCC (arrow) and RP2 (arrowhead; RN2-GAL4/UAS-vnd) does not suppress Grn expression in these cells.

 

Figure 9
View larger version (24K):

[in a new window]
  		Fig. 9. An eve->grn->zfh1 genetic cascade specifies aCC motor axon identity. Within the VNC, GMC1-1a is one of the first GMCs to divide and produces two postmitotic neurons: the aCC pioneer motoneuron and its sibling the pCC interneuron. In aCC and pCC, eve expression is independent of the activity of Notch signaling, whereas grn and zfh1 are suppressed by Notch signaling, and vnd is activated by Notch. The GMC4-2a divides later and produces the RP2 motoneuron and the RP2sib. In contrast to aCC/pCC, in the RP2 neuron, eve, grn and zfh1 do not regulate each other and in addition expression of all three genes is dependant upon Notch signaling. Although pros function is essential for proper GMC1-1a fate, GMC4-2a specification is under control of concerted activities of pros, hkb, ftz and pdm1. The orange boxes indicate genes regulated and/or sensitive to Notch signaling.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2006