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Elbow and Noc define a family of zinc finger proteins controlling morphogenesis of specific tracheal branches

¹ Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel
² EMBL, Heidelberg, Germany
³ Department of Biology, New York University, New York, USA

View larger version (73K): [in a new window]   Fig. 1. Tracheal phenotype following elB overexpression. (A) Wild-type stage 14 embryo stained with anti-lumen antibody. Note the visceral branch (VB, arrow). (B) In btl-Gal4/EP-elB the VB is not formed in most metameres (arrow). (C) Wild-type stage 14 embryo stained with anti-Trh antibody, marking the tracheal nuclei. Note the presence of five or six nuclei in the dorsal branch formed by tracheal pit 3. (D,D') After tracheal misexpression of ElB, embryos were stained with anti-ElB antibody. Note the nuclear staining and the presence of seven nuclei in the dorsal branch (DB) formed by tracheal pit 3 (arrow). The stalled visceral branch cells remain in the transverse connective (arrowhead). (E) The number of nuclei in the dorsal branch was quantitated in wild type (red bars, n=30), ElB overexpression (green, n=52) and elB mutant embryos (blue, n=54). (F,G) btl-Gal4/UAS-elB embryos were stained with anti-ElB antibodies. Note the reduced visceral branches (arrowhead) and the extended dorsal branches (arrow).

View larger version (122K): [in a new window]   Fig. 2. Effects of elB overexpression on tracheal gene expression. (A) The enhancer trap line l(2)01351 is expressed in a pair of cells in the dorsal trunk of each metamere (arrowhead) and in the visceral branch (arrow). The tracheal nuclei are marked by anti-Trh staining (red), and the enhancer trap by anti-ß-gal (green). (B) After elB tracheal misexpression, the enhancer trap line (green) continues to be expressed in the dorsal trunk (arrowhead), but is not observed in the residual visceral branch cells. Tracheal nuclei (red) are marked by anti-ElB. (C) kni-lacZ (green) is a target of Dpp signaling in the trachea in the dorsal branch and lateral trunks. It is also expressed weakly in the visceral branch. Red: anti-Trh. (D) After elB tracheal misexpression, kni expression (green) is retained in the reduced dorsal branches and the residual visceral branch (arrow). Red: anti-ElB. kni expression in the visceral branch that is independent of Dpp signaling was previously reported in wild-type embryos (Vincent et al., 1997; Chen et al., 1998). (E) Sal is expressed in the dorsal trunk (arrow) and oenocytes (arrowhead). Red, anti-Trh; green, anti-Sal. (F) After elB tracheal misexpression, Sal protein is retained in the oenocytes (arrowhead), but is not detected in the trachea (arrow). Red, anti-Trh; green, anti-Sal.

View larger version (80K): [in a new window]   Fig. 3. Sequence of ElB and Noc and gene structure. (A) Alignment of the protein sequence of ElB, Noc and the two human homologs. The following domains are marked: activation domain (red), Groucho binding sequence (purple), cysteine-rich domain (green), Zn finger (blue) and proline-tyrosine rich domain (orange). (B) Gene structures of elB and noc, and the position of the EP and Gal4 elements. elB and noc are positioned in opposite transcriptional orientations, and the ATG codon of each positioned within the first exon. The imprecise excisions generated in elB and noc are shown.

View larger version (116K): [in a new window]   Fig. 4. Expression of ElB. (A) At stage 11, elB RNA is detected in all tracheal pits. (B) By stage 12, expression is reduced in the central part of the pit, and retained in the dorsal branch and lateral trunks. (C) At stage 13, expression is detected only in the lateral trunk, most prominently in the first metamere. (D) Insertion of a Gal4 element upstream to elB induced (at stage 14) expression of UAS-elB in the lateral branches (arrowhead), but not in the visceral branch or dorsal trunk. Red, anti-ElB; green, 2A12 (which marks the tracheal lumen). (E) At stage 16 the expression of UAS-elB in the spiracular branch, transverse connective, lateral trunk and ganglionic branch was observed. (F) At the same stage, the dorsal branch fusion cells also expressed ElB (arrows).

View larger version (85K): [in a new window]   Fig. 5. elB mutant tracheal phenotypes. (A) Lumen staining of wild-type embryo at stage 14. (B) In elB47 mutant embryos, the lateral trunk anterior branch is not detected (arrow), and fusion between lateral trunks in adjacent metameres is not observed. (C) Anti-Trh staining of a wild-type embryo at stage 12 shows the initial stages of branching. (D) In elB mutants at the same stage, the lateral trunk anterior branch is stalled. (E) Wild-type embryo at stage 14. (F) In elB mutants, the lack of lateral trunk anterior migration is evident by stage 14. (G,I) Lumen and anti-Trh staining of the dorsal branches of a wild-type embryo. (H,J) In elB mutants, fusion between adjacent dorsal branches is observed (arrows). (K,L) Rescue of the tracheal phenotype of elB47 mutant embryos by btl-Gal4/UAS-elB was examined. Embryos misexpressing ElB were identified by anti-ElB staining, and stalled visceral branch migration (upper arrowhead in K). Over 120 such embryos were examined. elB47 embryos were scored by subtle defects in dorsal and ganglionic branch migration (arrowheads). Complete rescue of LTa migration (arrow in K) and partial rescue of dorsal branch migration (arrow in L) were observed.

View larger version (110K): [in a new window]   Fig. 6. Tracheal pMad patterns in wild-type and elB mutant embryos. (A) In wild-type embryos pMad (green) is induced at stage 11 by dorsal and lateral stripes of Dpp expression. pMad is observed in approx. five dorsal tracheal cells and 10-15 ventrolateral tracheal cells. (B) At stage 13 pMad is still detected in the same cells. (C,D) In elB mutants at stages 11 and 12, normal pMad is detected in the trachea. All tracheal nuclei are marked by anti-Trh (red).

View larger version (96K): [in a new window]   Fig. 7. Parallel activities of ElB and Dpp pathway. (A,B) In wild-type embryos, at stage 14 Sal expression (red) is restricted to the dorsal trunk, and kni-lacZ (green), a Dpp-target gene, is expressed in several tracheal branches including the dorsal branches shown here. (C,D) In elB mutant embryos, expression of kni was retained. Note, however, that the number of kni-expressing cells in the dorsal branches was reduced. The remaining kni cells were located within the dorsal trunk (arrows in D). In addition, in contrast to wild-type embryos, some of the dorsal branch cells continued to express Sal (arrows in C). Thus, while ElB is not necessary for kni expression, it is required in conjunction with Kni for repression of Sal expression in the dorsal branches, and for the capacity of all putative dorsal branch cells to detach from the dorsal trunk. (E) Additional experiments demonstrated that ElB and Dpp function in parallel. Expression of elB RNA is not elevated when activated Tkv is induced in all tracheal cells by btl-Gal4. (F) The same driver can induce uniform tracheal expression of elB when crossed to the EP2039 element upstream to elB. (G) In btl-Gal4/EP2039 embryos, defects in visceral branch were observed (arrowhead) while the LTa appeared normal. (H) In btl-Gal4/UAS-tkv* embryos, an excess of dorsal branch cells was detected (arrowhead) while again the LTa was normal. (I) In embryos misexpressing both constructs in the trachea, an excess of LTa cells was observed (arrow).

View larger version (120K): [in a new window]   Fig. 8. Expression and mutant phenotype of noc. (A) noc RNA is first seen at stage 5 in the anterior region of the embryo. (B) At stage 6 a striped pattern is observed. (C) At stage 11, prominent expression is seen in the tracheal pits. (D) Expression is broad and uniform from stage 13. (E) Uniform tracheal expression of a noc EP2000 element does not give rise to tracheal phenotypes or the elimination of expression of the visceral enhancer trap marker l(2)01351 (arrow, green). (F) noc mutant embryos show tracheal defects that are similar to but weaker than the defects seen in elB mutants. Note the reduced lateral trunk anterior (arrow) and the shortened ganglionic branch (arrowhead). Tracheal nuclei are stained with anti-Trh (red). (G) In wild-type embryos, only the terminal cell of each dorsal branch expresses SRF (green, arrow). (H,I) In noc mutant embryos, the fusion cell also expressed SRF (arrows). This may account for the lack of dorsal trunk fusion in noc and elB mutants. It correlates with the specific late expression of ElB in the fusion cells, and implies a role for ElB/Noc in repressing expression of terminal cell markers in the fusion cell. (J) Fusion of lateral branches was also disrupted in noc mutants (arrows).

View larger version (41K): [in a new window]   Fig. 9. Interactions between ElB, Noc and Groucho proteins. GST pull-down experiments were carried out, using GST fusions of ElB constructs, Noc, or an unrelated human 60 kDa protein. The scheme shows the fragments of ElB and Noc used for GST fusion. Domains are marked by the same color code as Fig. 3A. In vitro translated, labeled ElB, Noc or Groucho proteins were incubated with the GST constructs. ElB can associate with itself, with Noc and with Groucho.

View larger version (43K): [in a new window]   Fig. 10. Model for the activity of ElB in the trachea. Noc is an essential partner in the dimer. It is broadly expressed and thus does not give rise to a phenotype upon overexpression. Conversely, elB expression in the trachea is restricted to the dorsal branch and lateral trunks from stage 12 (red). This restricted expression is crucial for the correct determination of branch-specific cell fates. Overexpression of ElB represses the expression of genes in the visceral branch (VB) and dorsal trunk (arrows), while elB mutant embryos exhibit defects in the migration of the lateral trunks and dorsal branch (arrows). We suggest that normally ElB/Noc repress transcription of visceral-branch genes in the lateral trunks, and dorsal-trunk genes in the dorsal branch, thus contributing to the definition of branch-specific cell fates. Subsequently, ElB expression is refined to the fusion cells of the dorsal branch, where ElB/Noc are required for repression of terminal cell markers.