An interacting network of T-box genes directs gene expression and fate in the zebrafish mesoderm Lisa M. Goering†‡, Kazuyuki Hoshijima†‡, Barbara Hug†‡, Brent Bisgrove†§, Andreas Kispert¶, and David Jonah Grunwald†ʈ †Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112; and ¶Max-Planck-Institut fu¨r Immunbiologie, Stu¨beweg 51, D-79108 Freiburg, Germany Communicated by Mario R. Capecchi, University of Utah, Salt Lake City, UT, June 10, 2003 (received for review April 10, 2003) T-box genes encode transcription factors that play critical roles in common developmental pathways, and competitive antagonism generating the vertebrate body plan. In many developmental governing downstream gene expression. Our findings demon- fields, multiple T-box genes are expressed in overlapping domains, strate that T-box genes can perform multiple region-specific establishing broad regions in which different combinations of functions within a developmental field and indicate how loss of T-box genes are coexpressed. Here we demonstrate that three function of one T-box gene can alter the function of other T-box T-box genes expressed in the zebrafish mesoderm, no tail, spade- genes expressed in the same field. tail, and tbx6, operate as a network of interacting genes to regulate region-specific gene expression and developmental fate. Materials and Methods Loss-of-function and gain-of-function genetic analyses reveal Isolation of ntl-Regulated Genes. A mesendoderm-specific cDNA three kinds of interactions among the T-box genes: combinatorial library was constructed by subtractive hybridization (26, 27): interactions that generate new regulatory functions, additive con- cDNA from midgastrula embryos was depleted for sequences tributions to common developmental pathways, and competitive expressed in isolated animal cap tissue (see Supporting Materials antagonism governing downstream gene expression. We propose and Methods, which is published as supporting information on that T-box genes, like Hox genes, often function within gene the PNAS web site, www.pnas.org). Independent transformants networks comprised of related family members. (1,728 in all) were arrayed and analyzed by differential hybrid- ization screening. cDNA from WT and individually genotyped b195 -box genes encode related transcription factors that regulate ntl mutant 70% epiboly embryos was used to generate Ttissue specification, morphogenesis, and cell proliferation WT-ntl, ntl-WT-subtracted, and ntl-unsubtracted cDNA probes. (1–3). In addition to tissue-specific roles, T-box genes govern Candidate ntl-dependent sequences hybridized only with WT-ntl regional identities within developmental fields (4–6). One puz- probe. Whole-mount in situ hybridization was performed on WT zling aspect of T-box gene function is the recurrent finding that and mutant sibling embryos to identify genes whose expression the primary cellular focus of the defect seen in a T-box mutant depended on ntl function. corresponds to only a limited portion of the expression domain of the mutated T-box gene (5). For example, whereas haploin- Genetics and Genotype Analysis. Embryos from natural spawnings were raised at 28.5°C (28). WT embryos were from the AB* line. sufficiency of TBX3 results in posterior forelimb deficiencies, the b104 b160 b195 gene is expressed in the anterior and posterior margin of the The spt , ntl , and ntl alleles were used (11, 20, 24, 29). developing hind limb and forelimb (7, 8). Similarly, although To analyze additive effects of mutant alleles on MyoD expres- Brachyury orthologues are expressed throughout the nascent sion, genotyping with allele-specific primers was performed on mesoderm in vertebrate embryos, loss of Brachyury function tissue removed before in situ hybridization (see Supporting blocks differentiation of the notochord, a dorsal mesoderm Materials and Methods). tissue, but has only limited effects on the morphogenesis of the Ectopic Gene Expression Experiments. ventrolateral mesoderm, allowing differentiation of the full Sequences encoding No Tail, range of mesoderm cell types derived from this tissue (9–11). the No Tail DNA-binding domain (DBD) (amino acids 1–229), These findings implicate factors that modify T-box gene function Tbx6, the Tbx6 DBD (amino acids 1–240), the repressor domain so that individual T-box genes carry out different functions in of the Drosophila Engrailed protein [amino acids 2–299 (30)], or the activation domain of VP16 (amino acids 19–101) were cloned different regions of their expression domain (12–15). Because ϩ ϩ multiple T-box genes are expressed in overlapping patterns in into the CS2 and CS2 MT plasmids (31). Proteins were in vitro Ј many developmental fields (7, 16–19), we hypothesized that expressed ectopically after injection of -generated 5 - capped mRNA (mMESSAGE mMACHINE kit, Ambion, Aus- T-box gene interactions contribute to regionalization of T-box tin, TX) along with fluorescent lineage tracer dye into one- to gene function. two-cell embryos. Normally cleaving embryos with widespread We examined individual and combined functions of three dye were selected for analysis. Myc-epitope-tagged protein was T-box genes, no tail (ntl), spadetail (spt), and tbx6, which are detected with the 9E10 antibody (Santa Cruz Biotechnology). To expressed exclusively in the developing zebrafish mesoderm measure gene expression in animal caps, caps were removed at (20–24). The three genes are expressed in broad domains that 3.5 h from injected embryos and incubated for5hat28°C (21), overlap and together mark all of the mesoderm (Fig. 1A). We and RNA expression was detected by RT-PCR and quantified demonstrate that ntl, the zebrafish orthologue of Brachyury, regulates different downstream targets in different portions of its uninterrupted expression domain. Because spt and tbx6 are Abbreviation: DBD, DNA binding domain. coexpressed with ntl in selected regions of the mesoderm, we Data deposition: The sequences reported in this article have been deposited in the GenBank analyzed whether these genes interact with ntl to modify its database [accession nos. AY150226 (mesogenin) and AY150227 (ntd5)]. function in a region-specific manner. Amacher et al. (25) recently ‡L.M.G., K.H., and B.H. contributed equally to this work. showed that ntl and spt provide some overlapping functions in the §Present address: Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112. mesoderm. In this report, we document three kinds of interac- ʈTo whom correspondence should be addressed at: Department of Human Genetics, tions among the three T-box genes: combinatorial interactions University of Utah, 15 North 2030 East, Salt Lake City, UT 84112. E-mail: grunwald@ that generate new regulatory functions, additive contributions to genetics.utah.edu. 9410–9415 ͉ PNAS ͉ August 5, 2003 ͉ vol. 100 ͉ no. 16 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1633548100 Downloaded by guest on October 1, 2021 reporter plasmids contained three T-sites 55 bp upstream of the promoter of Ϫ36 PRL-luc (32), a pGL3-derived plasmid (Pro- mega) containing 73 bp of the rat prolactin promoter TATA box region. Plasmid expressing Renilla luciferase constitutively was used as an internal reference to standardize transfection effi- ciency. Experiments were performed in triplicate and repeated at least twice. Results Region-Specific Functions of ntl in the Mesoderm. ntl, spt, and tbx6 are expressed exclusively in the developing zebrafish mesoderm (20, 21, 23). Overlapping expression of the three genes demar- cates distinct regions of mesoderm identities (Fig. 1A). Before gastrulation, at the time of mesoderm specification, the com- bined expression marks two domains: dorsal mesoderm, where ntl and spt are expressed without tbx6, and ventrolateral meso- derm, where all three genes are present. During gastrulation, the expression patterns of the genes are refined to generate four domains: anterior axial mesoderm, or prechordal plate, ex- presses only spt; posterior axial mesoderm, or presumptive notochord, expresses only ntl; nonaxial mesoderm generated by involution of the ventrolateral mesoderm expresses spt and tbx6 together; and noninvoluted ventrolateral mesoderm maintains expression of all three genes. We hypothesized that individual T-box genes have distinct functions in different portions of their expression domains and that the function of each T-box gene is defined in part by the combination of T-box genes with which it is coexpressed. To define cellular regions of ntl function, we identified genes whose expression depends on ntl by hybridizing a mesendoderm- specific target cDNA library with a probe enriched for sequences expressed in WT, but not ntl mutant, gastrulae (see Materials and Methods). Two classes of genes were recovered (Fig. 1B). One class, exemplified by ntl-dependent gene 5 (ntd5), was expressed solely in the presumptive notochord. A second class, exemplified Fig. 1. T-box genes have region-specific functions in the zebrafish meso- by mesogenin and wnt8 (33), was expressed in the noninvoluted derm. (A) Schematic representation of ntl, spt, and tbx6 expression domains ventrolateral mesoderm. Loss of expression of these genes was during gastrulation. (B) ntl regulates different target genes in the dorsal and ͞ not secondary to loss of mesoderm tissue: other markers of axial ventrolateral mesoderm. ntd5, which encodes a product with Sushi CCP do- and ventrolateral mesoderm continued to be expressed through mains found in adhesion and complement proteins (54), is expressed in the presumptive notochord of WT (Upper) but not of ntl mutant (Lower) embryos. midgastrula stages (data not shown and refs. 21, 34, and 35). wnt8 and mesogenin are expressed in the ventrolateral mesoderm in a ntl- Furthermore, mesogenin appears to be a proximal target of ntl, dependent manner. (C) mesogenin expression is first detected at 30% epiboly because its expression is detectable within 30 min of the onset of (Upper), but it is not initiated in ntl embryos (Lower). (D) mesogenin is ntl transcription in 30% epiboly WT embryos and fails to be expressed in WT, but not spt mutant, embryos; wnt8 is expressed in both types initiated in ntl mutants (Fig. 1C).