DEVELOPMENTAL DYNAMICS 238:835–852, 2009 RESEARCH ARTICLE Overlapping Functions of Cdx1, Cdx2, and Cdx4 in the Development of the Amphibian Xenopus tropicalis Laura Faas and Harry V. Isaacs* Using Xenopus tropicalis, we present the first analysis of the developmental effects that result from knocking down the function of the three Cdx genes present in the typical vertebrate genome. Knockdowns of individual Cdx genes lead to a similar range of posterior defects; compound Cdx knockdowns result in increasingly severe posterior truncations, accompanied by posterior shifts and reduction of 5؅ Hox gene expression. We provide evidence that Cdx and Wnt3A genes are components of a positive feedback loop operating in the posterior axis. We show that Cdx function is required during later, but not early stages of development, for correct regional specification of the endoderm and morphogenesis of the gut. Our results support the hypothesis that during amphibian development the overall landscape of Cdx activity in the embryo is more important than the specific function of individual Cdx proteins. Developmental Dynamics 238:835–852, 2009. © 2009 Wiley-Liss, Inc. Key words: Cdx; Xenopus; Hox; caudal; mesoderm; endoderm; morphogenesis; wnt Accepted 14 January 2009 INTRODUCTION (Brooke et al., 1998). In the vertebrate three germ layers in dynamic patterns lineage sequential genome duplication in the posterior of the developing body The Cdx family of homedomain tran- has led to cluster expansion and sub- axis (Gamer and Wright, 1993; Meyer scription factors has conserved func- sequent degeneration, such that the and Gruss, 1993; Marom et al., 1997; tions in the development of several typical vertebrate genome contains Pillemer et al., 1998; Reece-Hoyes et animal groups. The prototype of the Cdx family is the caudal gene, which the remains of four ParaHox gene al., 2002; Gaunt et al., 2003; Lohnes, is required for normal posterior devel- clusters but only three Cdx genes 2003). The expression patterns of the opment of Drosophila (MacDonald (Cdx1, Cdx2, and Cdx4). Only Cdx2 Cdx genes resemble that of the Hox and Struhl, 1986; Mlodzik and Ge- remains clustered with members of genes, in that all Cdx genes are typi- hring, 1987; Mlodzik et al., 1987). The the Pdx and Gsx classes (Ferrier et al., cally expressed in the posterior of the Cdx class, together with the Gsx and 2005; Illes et al., 2009). embryo but each individual Cdx gene Pdx classes, comprise the ParaHox Analysis of Cdx gene expression in exhibits a different anterior boundary family of homeobox genes. In the several vertebrate species, including of expression. This produces a nested cephalochordate amphioxus, the sin- mouse, chick, fish, and frog shows that set of Cdx gene expression, establish- gle representatives of each ParaHox Cdx genes are initially expressed dur- ing a gradient of Cdx activity along class are clustered, having a similar ing gastrula stages in overlapping do- the posterior axis (Marom et al., 1997; genomic organization to that of the mains in the mesoderm; subse- Pillemer et al., 1998). closely related Hox gene family quently, they are expressed in all Gene inhibition and overexpression Additional Supporting information may be found in the online version of this article. Department of Biology, University of York, York, United Kingdom Grant sponsor: Wellcome Trust. *Correspondence to: Harry V. Isaacs, Area 11, Department of Biology, University of York, York, YO10 5YW, UK. E-mail: [email protected] DOI 10.1002/dvdy.21901 Published online 13 March 2009 in Wiley InterScience (www.interscience.wiley.com). © 2009 Wiley-Liss, Inc. 836 FAAS AND ISAACS studies in several chordates including ascidians, fish, frogs, chick, and mouse have shown that the role of Cdx genes in posterior development has been conserved during evolution (Sub- ramanian et al., 1995; Pownall et al., 1996; Epstein et al., 1997; Katsuyama et al., 1999; Bel-Vialar et al., 2002; van den Akker et al., 2002; Davidson et al., 2003; Lohnes, 2003; Chaweng- saksophak et al., 2004; Shimizu et al., Fig. 1. Inhibition of Cdx protein translation by morpholino oligos (MOs). A–C: Western blots 2005; van Nes et al., 2006). Cumula- showing that the Cdx1, Cdx2, and Cdx2 MOs but not the standard control MO or the corresponding tive evidence suggests that Cdx fac- five base mismatch control MOs block the translation of myc-epitope tagged Cdx target proteins tors act as transducers of positional in embryos. Early cleavage stage embryos were injected with 10 pg of Cdx-myc mRNA or ϩ information by regulating the bound- co-injected with 10 pg of Cdx-myc mRNA 10 ng of standard control morpholino (cMO) or 10 ng of the corresponding translation blocking MOs (Cdx1 MO, Cdx2 MO, or Cdx4) or 10 ng of the aries of Hox gene expression domains corresponding five mismatch MOs (Cdx1-mmMO, Cdx2-mmMO, or Cdx4-mmMO). Data are pre- (Charite et al., 1998; van den Akker et sented for the set-1 morpholinos. al., 2002; Gaunt et al., 2004, 2008). In keeping with their regulatory role during axis development, Cdx logue of amniote Cdx2, and Xcad2, stages of development. Data in this proteins are crucial factors involved in and Xcad3 are the orthologues of Cdx1 study support the hypothesis that, anteroposterior patterning of the di- and Cdx4, respectively. during early amphibian development, gestive tract. The regulation of gene In the present study, we have un- individual Cdx genes have overlap- expression in the digestive tract by dertaken a systematic analysis of the ping function and that in a given re- Cdx proteins in vertebrates is well developmental effects resulting from gion of the embryo, it is the overall documented (Beck, 2004; Guo et al., single and compound knockdown of level of Cdx activity that is relevant, 2004). Furthermore, Cdx2 misexpres- the three Cdx family members in the rather than the specific function of in- sion has been implicated in homeotic frog Xenopus tropicalis using transla- dividual Cdx proteins. anterior to posterior transformation tion blocking antisense morpholino in the gut epithelium (Mutoh et al., oligos (MOs). Our study is the first to RESULTS 2002; Silberg et al., 2002), indicating a present data on the developmental ef- role for Cdx genes in establishing re- fects that result from knocking down Antisense Morpholino Oligos gional identity. the activity of the three Cdx genes That Block Cdx Translation Single and double gene knockout present in the typical vertebrate ge- Antisense morpholino oligos (AMOs) and knockdown experiments in vari- nome. targeted to the initiating AUG ous species support roles for Cdx gene Data in this investigation show that and/or the 5Ј UTR of the Xenopus function in anteroposterior patterning compound knockdown of the three Xe- tropicalis Cdx1, Cdx2, and Cdx4 mR- of the main body axis. However, estab- nopus Cdx genes gives rise to a highly NAs were tested for their ability to lishing a clear picture of the overall penetrant, severe truncation of the block translation of myc-epitope role of the Cdx family in these pro- posterior axis. Similar ranges of devel- tagged Cdx proteins (Fig. 1). A stan- cesses is difficult because individual opmental abnormalities are seen dard control MO (cMO) has little ef- Cdx genes exhibit significant overlap when each individual Cdx gene is fect on the efficiency of Cdx protein of expression during early develop- knocked down, indicating that the translation in whole embryos. In ment. It is, therefore, likely that there Cdx genes have overlapping functions contrast, the Cdx1, Cdx2, and Cdx4 is some functional redundancy among in posterior axial development. How- MOs (set-1 in the Experimental Pro- the Cdx genes and that some degree of ever, the increased severity of the phe- cedures section) efficiently block compensation may occur in the event notypes in the compound knockdowns translation from the corresponding of single and double gene deficiencies. argue in favor of a cooperative effect of target mRNA, whereas MOs with se- This underlines the importance of un- Cdx genes on posterior patterning. quences differing from the transla- dertaking studies involving inhibition We present data indicating that the tion blocking MOs by five bases of the activity of the full complement amphibian Cdx genes are components (mmMOs) have relatively little effect of Cdx genes present in model verte- of gene regulatory pathways, involv- on translation from the target mR- brate organisms. ing Wnt ligands and 5Ј Hox genes, NAs. A second set of translation In Xenopus the three Cdx genes that are required for morphogenesis blocking Cdx MOs (set-2 in the Ex- were originally designated Xcad1, and patterning in the posterior of the perimental Procedures section) also Xcad2, and Xcad3 (Pillemer et al., main body axis during postgastrula inhibit translation of their respec- 1998). However, for the sake of consis- stages. In addition, we find a require- tive targets but less efficiently (data tency, we have adopted the human ment for Cdx function in the normal not shown). Unless stated otherwise and mouse nomenclature for the frog morphogenesis and regional specifica- the set-1 Cdx morpholinos were used Cdx genes. Thus, Xcad1 is the ortho- tion of the amphibian gut during later in all subsequent experiments. Cdx KNOCKDOWN IN Xenopus tropicalis 837 Analysis of the Cdx similar profiles of phenotypes; how- Cdx activity plays a critical role in Knockdown Phenotype ever, the triple Cdx knockdown re- regulating axial development. sults in a much higher proportion of As another test of the specificity of Injection of the set-1 Cdx1, Cdx2, and the more severe type 3 phenotype. Tri- the knockdown effects, five base mis- Cdx4 MOs, individually or collec- ple Cdx knockdown using the set-2 match MOs were injected into em- tively, in the range of 10 ng to 20 MOs produced similar effects on axial bryos.