Trachealess (Trh) regulates all tracheal genes during Drosophila embryogenesis

Abstract

The Drosophila trachea is a branched tubular epithelia that transports oxygen and other gases. trachealess (trh), which encodes a bHLH-PAS transcription factor, is among the first genes to be expressed in the cells that will form the trachea. In the absence of trh, tracheal cells fail to invaginate to form tubes and remain on the embryo surface. Expression of many tracheal-specific genes depends on trh, but all of the known targets have relatively minor phenotypes compared to loss of trh, suggesting that there are additional targets. To identify uncharacterized transcriptional targets of Trh and to further understand the role of Trh in embryonic tracheal formation, we performed an in situ hybridization screen using a library of ~100 tracheal-expressed genes identified by the Berkeley Drosophila Genome Project (BDGP). Surprisingly, expression of every tracheal gene we tested was dependent on Trh, suggesting a major role for Trh in activation and maintenance of tracheal gene expression. A re-examination of the interdependence of the known early-expressed transcription factors, including trh, ventral veinless (vvl) and knirps/knirps-related (kni/knrl), suggests a new model for how gene expression is controlled in the trachea, with trh regulating expression of vvl and kni, but not vice versa. A pilot screen for the targets of Vvl and Kni/Knrl revealed that Vvl and Kni have only minor roles compared to Trh. Finally, genome-wide microarray experiments identified additional Trh targets and revealed that a variety of biological processes are affected by the loss of trh.

Fig.1

Trh autoregulates in the trachea but not in other tissues. (A) trh has three known alternative transcripts (RA, RB and RC), which encode nearly identical proteins that share the bHLH domain and two PAS domains (FlyBase). trh¹ and trh² are EMS-induced mutations. trh⁸ is an excision allele derived from a P element inserted 14 bp into the 5′ UTR of the trh-RA and trh-RB isoforms. (B) The amino acid sequences of the C-terminal region of PAS-A domain are aligned. The Ile (I) to Asn (N) mutation in trh¹ and trh² is conserved in Trh orthologues from other species (either the same or a similar residue is always found in this position). The amino acids completely conserved in all species are in green, the identical residues are in yellow, the similar residues are in cyan, and the different residues are in white. (C) A comparison of trh mRNA levels in wild type and trh mutant alleles reveals a loss of trh expression in the trachea (arrows) at later stages but no changes in the levels of expression in the salivary duct, filzkörper or CNS in trh mutants. (D) Trh protein levels are similar to trh mRNA levels in WT, trh¹ and trh² mutants, whereas no Trh protein is detected in trh⁸ mutants. Arrows indicate the trachea, black arrowheads indicate the early salivary gland, open arrowheads indicate the salivary duct, red arrowheads indicate the filzkörper, and green arrowheads indicate the CNS. The asterisk indicates cross-reactivity of the Trh antiserum with the secretory portion of the salivary gland, which does not express trh in late embryos.

Fig.2

trh regulates all tested tracheal genes. (A-H) Shown are in situs of several tracheal-expressed genes in wild-type and trh mutant embryos. Embryos in A-D are stage 11 and those in E-H are stage 15. At later stages, some genes are expressed in the entire trachea (E), whereas other genes are expressed in a subset of tracheal branches (F, H) or in specific tracheal cell-types (G). Regardless of the expression pattern of each gene, the tracheal expression in wild type (black arrows) is completely absent in the trh mutants (open arrows). Note that only tracheal expression is dependent on trh; expression in the other tissues, such as the salivary gland (D) or head/tail region (H), is not affected by the absence of trh (arrowheads in D, H). (I-K) Fluorescent in situ hybridization of tkv (red) with α-Trh staining (green) was done with wild type (I), trh² (J) and vvl^6A3 (K) mutants. tkv mRNA expression is still present although slightly reduced in the dorsal tracheal cells at stage 11 in both trh and vvl mutant embryos. (J, K) Fluorescent in situ hybridization reveals reduced expression of the early-expressed tracheal gene CG8312 in trh mutants (M) compared to wild type (L). White arrowheads in I-M indicate tkv or CG8312 mRNA expression in WT or trh mutant embryos. Scale bars: 50μm.

Fig.3

trh expression is largely unaffected by loss of vvl or kni. trh mRNA (A-C, G-I) and Trh protein (D-F, J-L) are expressed in the trachea of both vvl mutants and embryos deficiency for both kni and the related knrl gene (Df(3L)ri-79C) at both early (A-F) and late (G-L) stages. Asterisks in J-L indicate nonspecific background staining with the α-Trh antisera in the late embryonic salivary gland at late stages. Scale bars: 50μm.

Fig. 4

Trh is required for the expression/maintenance of Vvl and Kni. (A-F) α-Vvl staining (green) in wild type, trh² and Df(3L)ri-79C embryos. Wild type expression of Vvl is shown in A and D (arrows). The early expression of Vvl is relatively normal in early trh mutants (B, closed arrow), but subsequently disappears (E, open arrow). Asterisk indicates non-tracheal Vvl expression. Vvl protein is expressed to WT levels in Df(3L)ri-79C embryos at all embryonic stages (C, F, arrows). (G-L) Kni (green) expression requires trh but not vvl. Higher magnifications of the boxed region of G-L are shown in G’-L’. α-Trh or α-Tgo (red) signals mark the tracheal cells. Arrows indicate the Kni- and Trh (or Tgo)- doubly positive tracheal cells. Kni expression is completely absent in the trh mutant tracheal cells at all stages (H, K). Trh-positive cells in H’ do not express Kni in the trh² mutants (arrowheads). In contrast, Kni is normally expressed in the vvl mutants (I, L). Askterisks in G’-I’, K, L and L’ are non-tracheal cells expressing Kni. Embryos in A-C, G-I, and G’-I’ are stage11, and those in D-F, J-L, and J’-L’ are stage15. Scale bars: 50μm in A-L; 20μm in G’-L’.

Fig. 5

Vvl regulates a subset of tracheal genes whereas Kni does not affect expression of any of the tracheal genes tested. (A-F) Shown are examples of in situ hybridizations with tracheal-expressed genes in vvl and Df(3L)ri-79C. Embryos in A-C are stage 11 and those in D-F are stage 15. (A, B) Compared to wild-type tracheal expression levels (black arrows), sano and CG33006 levels are significantly reduced in vvl^6A3 mutants (open arrows). Note that salivary gland expression of sano is not affected by vvl loss (arrowheads in A). (C) CG18549 is expressed to wild-type levels in vvl mutants and in Df(3L)ri-79C embryos. (D) CG32499 is expressed in vvl mutants (open arrow), but in fewer cells compared to wild type (black arrow). (E, F). Two additional examples of tracheal genes (CG1157 and CG2016) whose expression is unaffected by loss of vvl or kni are shown.

Fig. 6

Microarray experiments identify additional genes regulated by Trh. (A) Volcano plot showing changes in expression levels and statistical significance of Trh target genes. Transcripts elevated 1.4 fold or more in trh mutants are labeled red. Transcripts reduced 1.4 fold or more are labeled blue. Trh downstream genes identified in the in situ screen are marked in green. Closed circles indicate significance with a p-value ≤0.05. Open circles indicate non-statistically significant changes. (B) Examples of the expression patterns of novel Trh targets identified by microarray in both wild type and trh mutant embryos. Tracheal-specific genes (Spn43Aa, Osi19, and CG12009, black arrows) as well as those expressed in other Trh-expressing tissues, such as the filzkörper (CG13640, black arrowhead), require trh for expression. Open arrows and arrowheads indicate the absence of expression in the trh mutant trachea and filzkörper, respectively. (C, D) RT-qPCR with downregulated genes (C) and upregulated genes (D). The fold changes were normalized to a housekeeping gene (actin5c) and the relative levels of normalized expression were shown compared to WT (1.0). Error bars represent standard error of the mean. *, p<0.05; **, p<10⁻³; ***, p<10⁻⁶.

Fig. 7

A model for gene regulation during tracheal development. The global patterning genes including Dpp, Wg and Sal specify the region where tracheal primordia form. Stat92E directly and independently regulates the expression of trh and vvl, the two earliest-expressed tracheal transcription factors. Trh is required for expression of all tracheal genes whereas Vvl is required for the expression of only 25-30% of tracheal genes. Trh also maintains its own expression and is required for the maintenance of vvl and the few other tracheal genes whose initial expression is independent of Trh (dotted lines).