Requirement of the LIM homeodomain transcription factor tailup for normal heart and hematopoietic organ formation in Drosophila melanogaster

Abstract

Dorsal vessel morphogenesis in Drosophila melanogaster serves as a superb system with which to study the cellular and genetic bases of heart tube formation. We used a cardioblast-expressed Toll-GFP transgene to screen for additional genes involved in heart development and identified tailup as a locus essential for normal dorsal vessel formation. tailup, related to vertebrate islet1, encodes a LIM homeodomain transcription factor expressed in all cardioblasts and pericardial cells of the heart tube as well as in associated lymph gland hematopoietic organs and alary muscles that attach the dorsal vessel to the epidermis. A transcriptional enhancer regulating expression in these four cell types was identified and used as a tailup-GFP transgene with additional markers to characterize dorsal vessel defects resulting from gene mutations. Two reproducible phenotypes were observed in mutant embryos: hypoplastic heart tubes with misaligned cardioblasts and the absence of most lymph gland and pericardial cells. Conversely, a significant expansion of the lymph glands and abnormal morphology of the heart were observed when tailup was overexpressed in the mesoderm. Tailup was shown to bind to two DNA recognition sequences in the dorsal vessel enhancer of the Hand basic helix-loop-helix transcription factor gene, with one site proven to be essential for the lymph gland, pericardial cell, and Svp/Doc cardioblast expression of Hand. Together, these results establish Tailup as being a critical new transcription factor in dorsal vessel morphogenesis and lymph gland formation and place this regulator directly upstream of Hand in these developmental processes.

FIG. 1.

Dorsal vessel phenotypes observed in tup mutant embryos. (A to D) Toll-nGFP transgene expression in (A) wild-type (wt), (B) homozygous Df(2L)OD15, (C) homozygous tup¹, and (D) homozygous tup^isl-1 embryos. Embryos are at stage 16 of development. (E and F) H15-lacZ transgene expression in (E) wild-type and (F) homozygous tup¹ embryos. Embryos are at stage 14 of development. (G and H) D-MEF2 protein expression in (G) wild-type and (H) homozygous tup¹ embryos. Embryos are at stage 15 of development. Open arrowheads point to missing cardioblasts and pronounced gaps in dorsal vessels of mutant embryos. All embryos are oriented with the anterior to the left. Abbreviations: as, amnioserosa cells; cb, cardioblasts; sm, somatic body wall muscles.

FIG. 2.

tup expression in cells within and associated with the dorsal vessel. (A) Low-magnification (×20) image of a wild-type (wt) embryo immunostained for the Tup protein. Tup expression in the dorsal vessel (dv) is indicated. (B) High-magnification (×40) image of a wild-type embryo immunostained for Tup protein. Tup expression is observed (from anterior to posterior) in lymph glands (lg), pericardial cells (pc), cardioblasts (cb), and alary muscles (am). (C) Mapping the location of regulatory modules controlling tup dorsal vessel expression. Boxes within the tup gene correspond to exon sequences. (D) Expression of the tup-F4-GFP transgene in all four types of Tup-positive cells of the dorsal vessel. All embryos are at stage 16 of development and oriented with the anterior to the left.

FIG. 3.

Determination of the molecular mutation present in the tup¹ allele. (A) Two very similar forms of Tup are generated by the alternative splicing of pre-mRNA. The LIM and homeobox domains of the proteins are indicated. A single nucleotide mutation present in the tup¹ allele results in the change of an amino acid crucial for zinc finger formation within the first LIM domain; specifically, the codon for Cys-57 is mutated to one encoding Tyr-57. (B and C) Double staining of (B) wild-type (wt) and (C) homozygous tup¹ embryos for Tup and D-MEF2 reveals an absence (open arrowhead) of the Tup protein in mutant animals. The embryos are at stage 13 of development and oriented with the anterior to the left. Abbreviations: as, aminoserosa cells; cb, cardioblasts; sm, somatic body wall muscles.

FIG. 4.

High-resolution analyses of tup dorsal vessel and lymph gland phenotypes. (A and B) tup-F4-GFP transgene expression in (A) wild-type (wt) and (B) homozygous tup¹ embryos. Lymph glands (lg), pericardial cells (pc), and cardioblasts (cb) of the normal dorsal vessel are indicated in the wild-type embryo. (C and D) Col protein and tup-F4-GFP transgene expression in (C) wild-type and (D) homozygous tup¹ embryos. Arrows point to Col expression in the posterior signaling centers of the lymph glands associated with the normal dorsal vessel, and comparable Col staining is observed along the defective heart tube of the mutant embryo. Col expression is also observed in bilateral posterior structures in the wild-type embryo, which are out of focus in the tup¹ embryo due to a defect in dorsal closure. (E and F) Hand-GFP transgene expression in (E) wild-type and (F) homozygous tup¹ embryos. Prominent GFP expression in the lymph glands, pericardial cells, and cardioblasts in the normal dorsal vessel is highlighted. (G and H) Srp protein and Hand-GFP transgene expression in (G) wild-type and (H) homozygous tup¹ embryos. Arrows point to the coincident expression of Srp and Hand in the lymph glands of the wild-type embryo. (I and J) Odd protein and Toll-nGFP transgene expression in (I) wt and (J) tup¹ embryos. Prominent Odd expression in the lymph glands and pericardial cells is highlighted, while Toll expression in cardioblasts is also indicated in the wild-type embryo. (B, D, F, H, and J) All five of the markers, or marker combinations, reveal the absence of most lymph gland (closed arrowheads) and pericardial (open arrowheads) cells in defective dorsal vessels found in tup¹ mutant embryos. Mutant embryos also exhibit defects in dorsal closure, with prolonged GFP expression in cells of the amnioserosa using the Toll-nuclear GFP (nGFP) marker (J). All embryos are at stage 16 of development and oriented with the anterior to the left.

FIG. 5.

Two Tup DNA recognition sites are present in the Hand 513-bp HCH enhancer. The locations of Tup CTAATG binding elements, relative to previously defined Tin and GATA protein binding sites (15), are indicated in the Hand DNA. At the bottom, electromobility gel shift assays reveal that Tup can selectively bind to double-stranded oligonucleotides containing the Tup-1 or Tup-2 sequence. Abbreviations: MT, Tup-site-mutated oligonucleotide; WT, wild-type oligonucleotide.

FIG. 6.

Tup is a direct transcriptional regulator of the Hand dorsal vessel enhancer. (A, left) Schematic of the Hand 513-bp HCH enhancer and three Tup site mutant versions thereof. (A, right) Function of wild-type and mutated Hand-GFP DNAs in transgenic embryos. Activities of the various enhancers in Tin or Svp/Doc cardioblasts (cb), lymph glands (lg), or pericardial cells (pc) are indicated as positive (+), negative (−), or greatly reduced (+/−). (B and C) Normal cellular expression profile of GFP in embryos harboring the wt-Hand-GFP or mTup1-Hand-GFP transgenes. (D and E) Representative embryos expressing the mTup2-Hand-GFP or mTup1/2-Hand-GFP construct. With both mutated DNAs, GFP is observed in Tin cardioblasts but absent from the lymph glands (closed arrowheads) and pericardial cells (open arrowheads). Reporter expression is greatly diminished in Svp/Doc cardioblasts (arrows) with both mutations. (F) Twi-Gal4>UAS-tup embryo expressing the wt-Hand-GFP marker. This forced expression condition results in an expanded population of prohemocytes in the lymph glands (horizontal bar and asterisk) and a disorganized heart region (arrow). (G) Expression of the tup-F4-GFP transgene in a homozygous Hand^ko mutant embryo. tup expression appears to be normal in cardioblasts and pericardial cells but reduced in the lymph glands. All embryos are at stage 16 of development and oriented with the anterior to the left.

FIG. 7.

Proposed functions for Tup in regulating lymph gland, pericardial cell, and Svp/Doc cardioblast gene expression.