.............................................................. cdx4 mutants fail to specify blood progenitors and can be rescued by multiple hox genes Alan J. Davidson 1 , Patricia Ernst 2 , Yuan Wang 3 , Marcus P. S. Dekens 4 , Paul D. Kingsley 5 , James Palis 5 , Stanley J. Korsmeyer 2 , George Q. Daley 3 & Leonard I. Zon 1 1 Department of Medicine, Division of Hematology/Oncology, Children’s Hospital and Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA 2 Departments of Pathology and Medicine, Dana-Farber Cancer Institute and Harvard Medical School, Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA 3 Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA 4 Max-Planck-Institut fu ¨r Entwicklungsbiologie, Abteilung Genetik, Spemannstrasse 35, 72076 Tu ¨bingen, Germany 5 Department of Pediatrics, Center for Human Genetics and Molecular Pediatric Diseases, Universityof Rochester, Rochester, New York 14642, USA ............................................................................................................................................................................. Organogenesis is dependent on the formation of distinct cell types within the embryo. Important to this process are the hox genes, which are believed to confer positional identities to cells along the anteroposterior axis 1–3 . Here, we have identified the caudal-related gene cdx4 as the locus mutated in kugelig (kgg), a zebrafish mutant with an early defect in haematopoiesis that is associated with abnormal anteroposterior patterning and aber- rant hox gene expression. The blood deficiency in kgg embryos can be rescued by overexpressing hoxb7a or hoxa9a but not hoxb8a, indicating that the haematopoietic defect results from perturbations in specific hox genes. Furthermore, the haemato- poietic defect in kgg mutants is not rescued by scl overexpression, suggesting that cdx4 and hox genes act to make the posterior mesoderm competent for blood development. Overexpression of cdx4 during zebrafish development or in mouse embryonic stem cells induces blood formation and alters hox gene expression. Taken together, these findings demonstrate that cdx4 regulates hox genes and is necessary for the specification of haematopoietic cell fate during vertebrate embryogenesis. The yolk sac blood islands of amniotes develop from posterior mesoderm and form embryonic erythroid cells and endothelial cells. The equivalent site in zebrafish, known as the intermediate cell mass (ICM), arises from bilateral stripes of haematopoietic and vascular precursors in the posterior mesoderm. We found that embryos homozygous for kgg, an autosomal recessive mutation that was initially identified because of tail defects 4 , exhibit severe anaemia within the first day of development. Although blood cell numbers begin to recover by 5 days post-fertilization (d.p.f.), all mutants die between 7 and 10 d.p.f. To investigate the haematopoietic defect in kgg, we examined the expression of scl, gata1 and runx1 genes. At the 5-somite stage, the bilateral stripes of scl þ cells are thinner in kgg tv205 embryos than in wild-type controls (Fig. 1b). In addition, kgg tv205 mutants show a decreased number of gata1 þ erythroid precursors and a complete absence of runx1 expression in blood and neuronal cells. By 24 h post-fertilization (h.p.f.), kgg tv205 mutants have a severe reduction in the number of haemoglobin-expressing erythroid cells compared with wild-type siblings (Fig. 1b). By contrast, normal numbers of pu.1 þ myeloid cells are formed from the cephalic mesoderm in kgg tv205 embryos (data not shown). To study the development of the vasculature in the mutant, we examined the expression of the VEGF receptor flk1. At the 10- and 15-somite stages, kgg tv205 embryos have relatively normal numbers of angioblasts, although their conver- gence to the midline is delayed (Fig. 1c). By 24 h.p.f., the vasculature appears well formed in the mutants and the few blood cells that develop circulate normally. The pronephric kidney arises from mesoderm adjacent to the ICM precursors. In kgg tv205 mutants, the expression domains of the pronephric duct markers pax2.1 and cxcr4b are shortened (arrowheads in Fig. 1d), although unlike the scl stripes, the width of the pax2.1 stripe is unaffected. Transcripts for the glomerulus marker wt1, which are normally expressed in mesoderm adjacent to somites one to four, extend from somites one to six in kgg tv205 embryos (brackets in Fig. 1d), suggesting that the kgg tv205 mutation leads to an expansion of anterior kidney fates at the expense of more posterior fates. Other structures such as the head, notochord and somites appear grossly normal in kgg tv205 embryos, although the length of the embryo is shortened compared with wild-type embryos. The kgg mutation maps to linkage group 14 (Fig. 2a) near candidate genes including cdx4, smad5 and wnt8. An analysis of the complementary DNA sequence of wnt8 and smad5 from kgg mutants did not identify any mutations. cdx4 belongs to the caudal family of homeobox genes, which have been implicated in antero- posterior patterning 5–7 . Three caudal paralogues exist in mammals (Cdx1, Cdx2 and Cdx4), and mouse gene-targeting studies of Cdx1 and Cdx2 (Cdx4 has yet to be targeted) have demonstrated a role for these genes in the anteroposterior patterning of the axial skeleton 8–10 . In addition, Cdx2 þ/2 mice develop hamartomatous polyps in the colon that result from a transformation of the intestinal epithelium to a more anterior (gastric) fate 9,11,12 . Sequence analysis of the cdx4 gene from kgg tl240 mutants revealed a T to A transversion in nucleotide þ510, changing a conserved F170 residue in the homeodomain to a leucine (Fig. 2b). This mutation prevents the protein from binding to a Cdx4 consensus binding site in gel- shift experiments (see Supplementary Fig. 1). A partial deletion of the cdx4 gene, and at least one other neighbouring gene (chic1), was found in kgg tv205 mutants (Fig. 2a, b; see also Supplementary Fig. 2). We isolated the cdx4 transcript in kgg tv205 mutants by 3 0 rapid amplification of cloned ends (RACE) and found that exon two had become spliced onto downstream sequence that extended the cdx4 open reading frame by 11 amino acids (GFSSVFQSQSD-stop). Radiation hybrid mapping of this foreign sequence placed it 20 cR away from the cdx4 locus. To provide further evidence that the kgg mutant phenotype is caused by defects in cdx4, we injected wild- type embryos with cdx4 antisense morpholino oligonucleotides and found that the resulting morpholino mutants (known as morphants) phenotypically resembled kgg embryos (Figs 1b and 2c). Transcripts for cdx4 are first detected in the early gastrula but become restricted to the posterior-most cells during gastrulation and early somitogenesis (Fig. 2d). Double whole-mount in situ hybridization and sectioning at the 3-somite stage revealed that the cdx4 expression domain initially includes cells in the posterior mesoderm that express scl (Fig. 2d and data not shown). However, from the 5-somite stage onwards the expression domains of cdx4 and scl are largely non-overlapping. Similar expression profiles were found for the mouse orthologues of cdx4 and scl during early embryogenesis (Fig. 2e). At the late primitive streak stage (E7.25), Cdx4 transcripts are confined to mesodermal cells of the posterior embryo, the allantois and the forming yolk sac wall. Although Cdx4 is not expressed in the nascent blood islands, its expression domain does partially overlap with Scl in mesodermal cells of the posterior primitive streak and the posterior yolk sac. To further explore the function of cdx4 during embryonic haematopoiesis, we examined the effect of cdx4 overexpression in wild-type embryos. Embryos injected with cdx4 messenger RNA (7, 15 or 30 pg) display a range of ‘posteriorized’ phenotypes (see Supplementary Fig. 3a, b). By contrast, embryos injected with 15 pg of F170L mutant mRNA all exhibit a wild-type morphology (n ¼ 60 of 60 embryos injected; data not shown). The effect of cdx4 over- expression (15 pg) on blood development was examined at the 5- to 12-somite stages. Surprisingly, 12–20% of the injected embryos letters to nature NATURE | VOL 425 | 18 SEPTEMBER 2003 | www.nature.com/nature 300 © 2003 Nature Publishing Group