Regulation of gene expression downstream of a novel Fgf/Erk pathway during Xenopus development.

Activation of Map kinase/Erk signalling downstream of fibroblast growth factor (Fgf) tyrosine kinase receptors regulates gene expression required for mesoderm induction and patterning of the anteroposterior axis during Xenopus development. We have proposed that a subset of Fgf target genes are activated in the embyo in response to inhibition of a transcriptional repressor. Here we investigate the hypothesis that Cic (Capicua), which was originally identified as a transcriptional repressor negatively regulated by receptor tyrosine kinase/Erk signalling in Drosophila, is involved in regulating Fgf target gene expression in Xenopus. We characterise Xenopus Cic and show that it is widely expressed in the embryo. Fgf overexpression or ectodermal wounding, both of which potently activate Erk, reduce Cic protein levels in embryonic cells. In keeping with our hypothesis, we show that Cic knockdown and Fgf overexpression have overlapping effects on embryo development and gene expression. Transcriptomic analysis identifies a cohort of genes that are up-regulated by Fgf overexpression and Cic knockdown. We investigate two of these genes as putative targets of the proposed Fgf/Erk/Cic axis: fos and rasl11b, which encode a leucine zipper transcription factor and a ras family GTPase, respectively. We identify Cic consensus binding sites in a highly conserved region of intron 1 in the fos gene and Cic sites in the upstream regions of several other Fgf/Cic co-regulated genes, including rasl11b. We show that expression of fos and rasl11b is blocked in the early mesoderm when Fgf and Erk signalling is inhibited. In addition, we show that fos and rasl11b expression is associated with the Fgf independent activation of Erk at the site of ectodermal wounding. Our data support a role for a Fgf/Erk/Cic axis in regulating a subset of Fgf target genes during gastrulation and is suggestive that Erk signalling is involved in regulating Cic target genes at the site of ectodermal wounding.

A comparative analysis of fibroblast growth factor receptor signalling during Xenopus development.

BACKGROUND INFORMATION: The fibroblast growth factor (FGF) signalling system of vertebrates is complex. In common with other vertebrates, secreted FGF ligands of the amphibian Xenopus signal through a family of four FGF receptor tyrosine kinases (fgfr1, 2, 3 and 4). A wealth of previous studies has demonstrated important roles for FGF signalling in regulating gene expression during cell lineage specification in amphibian development. In particular, FGFs have well-established roles in regulating mesoderm formation, neural induction and patterning of the anteroposterior axis. However, relatively little is known regarding the role of individual FGFRs in regulating FGF-dependent processes in amphibian development. In this study we make use of synthetic drug inducible versions of Xenopus Fgfr1, 2 and 4 (iFgfr1, 2 and 4) to undertake a comparative analysis of their activities in the tissues of the developing embryo. RESULTS: We find that Xenopus Fgfr1 and 2 have very similar activities. Both Fgfr1 and Fgfr2 are potent activators of MAP kinase ERK signalling, and when activated in the embryo during gastrula stages regulate similar cohorts of transcriptional targets. In contrast, Fgfr4 signalling in naïve ectoderm and neuralised ectoderm activates ERK signalling only weakly compared to Fgfr1/2. Furthermore, our analyses indicate that in Xenopus neural tissue the Fgfr4 regulated transcriptome is very different from that of Fgfr1. CONCLUSION AND SIGNIFICANCE: We conclude that signalling downstream of Fgfr1 and 2 regulates similar processes in amphibian development. Interestingly, many of the previously identified canonical transcriptional targets of FGF regulation associated with germ layer specification and patterning are regulated by Fgfr1/Fgfr2 signalling. In contrast, the downstream consequences of Fgfr4 signalling are different, although roles for Fgfr4 signalling in lineage specification and anteroposterior patterning are also indicated.

Xenopus laevis FGF16 activates the expression of genes coding for the transcription factors Sp5 and Sp5l.

Fibroblast growth factors (FGFs) comprise a family of signalling molecules with essential roles in early embryonic development across animal species. The role of FGFs in mesoderm formation and patterning in Xenopus has been particularly well studied. However, little is known about FGF16 in Xenopus. Using in situ hybridisation, we uncover the expression pattern of FGF16 during early Xenopus laevis development, which has not been previously described. We show that the zygotic expression of FGF16 is activated in the mesoderm of the early gastrula as a ring around the blastopore, with its first accumulation at the dorsal side of the embryo. Later, FGF16 expression is found in the otic vesicle, the branchial arches and the anterior pituitary, as well as in the chordal neural hinge region of the tailbud. In addition, we show that FGF16 can activate the MAPK pathway and expression of sp5 and sp5l. Like FGF16, sp5 is expressed in the otic vesicle and the branchial arches, with all three of these genes being expressed in the tailbud. These data provide evidence that FGF16 is present in the early mesoderm and can activate the expression of developmentally important transcription factors.

N1-Src Kinase Is Required for Primary Neurogenesis in Xenopus tropicalis.

The presence of the neuronal-specific N1-Src splice variant of the C-Src tyrosine kinase is conserved through vertebrate evolution, suggesting an important role in complex nervous systems. Alternative splicing involving an N1-Src-specific microexon leads to a 5 or 6 aa insertion into the SH3 domain of Src. A prevailing model suggests that N1-Src regulates neuronal differentiation via cytoskeletal dynamics in the growth cone. Here we investigated the role of n1-src in the early development of the amphibian Xenopus tropicalis, and found that n1-src expression is regulated in embryogenesis, with highest levels detected during the phases of primary and secondary neurogenesis. In situ hybridization analysis, using locked nucleic acid oligo probes complementary to the n1-src microexon, indicates that n1-src expression is highly enriched in the open neural plate during neurula stages and in the neural tissue of adult frogs. Given the n1-src expression pattern, we investigated a possible role for n1-src in neurogenesis. Using splice site-specific antisense morpholino oligos, we inhibited n1-src splicing, while preserving c-src expression. Differentiation of neurons in the primary nervous system is reduced in n1-src-knockdown embryos, accompanied by a severely impaired touch response in later development. These data reveal an essential role for n1-src in amphibian neural development and suggest that alternative splicing of C-Src in the developing vertebrate nervous system evolved to regulate neurogenesis.SIGNIFICANCE STATEMENT The Src family of nonreceptor tyrosine kinases acts in signaling pathways that regulate cell migration, cell adhesion, and proliferation. Srcs are also enriched in the brain, where they play key roles in neuronal development and neurotransmission. Vertebrates have evolved a neuron-specific splice variant of C-Src, N1-Src, which differs from C-Src by just 5 or 6 aa. N1-Src is poorly understood and its high similarity to C-Src has made it difficult to delineate its function. Using antisense knockdown of the n1-src microexon, we have studied neuronal development in the Xenopus embryo in the absence of n1-src, while preserving c-src Loss of n1-src causes a striking absence of primary neurogenesis, implicating n1-src in the specification of neurons early in neural development.

Human Lin28 Forms a High-Affinity 1:1 Complex with the 106~363 Cluster miRNA miR-363.

Lin28A is a post-transcriptional regulator of gene expression that interacts with and negatively regulates the biogenesis of let-7 family miRNAs. Recent data suggested that Lin28A also binds the putative tumor suppressor miR-363, a member of the 106~363 cluster of miRNAs. Affinity for this miRNA and the stoichiometry of the protein-RNA complex are unknown. Characterization of human Lin28's interaction with RNA has been complicated by difficulties in producing stable RNA-free protein. We have engineered a maltose binding protein fusion with Lin28, which binds let-7 miRNA with a Kd of 54.1 ± 4.2 nM, in agreement with previous data on a murine homologue. We show that human Lin28A binds miR-363 with a 1:1 stoichiometry and with a similar, if not higher, affinity (Kd = 16.6 ± 1.9 nM). Further analysis suggests that the interaction of the N-terminal cold shock domain of Lin28A with RNA is salt-dependent, supporting a model in which the cold shock domain allows the protein to sample RNA substrates through transient electrostatic interactions.

lin28 proteins promote expression of 17∼92 family miRNAs during amphibian development.

BACKGROUND: Lin28 proteins are post-transcriptional regulators of gene expression with multiple roles in development and the regulation of pluripotency in stem cells. Much attention has focussed on Lin28 proteins as negative regulators of let-7 miRNA biogenesis; a function that is conserved in several animal groups and in multiple processes. However, there is increasing evidence that Lin28 proteins have additional roles, distinct from regulation of let-7 abundance. We have previously demonstrated that lin28 proteins have functions associated with the regulation of early cell lineage specification in Xenopus embryos, independent of a lin28/let-7 regulatory axis. However, the nature of lin28 targets in Xenopus development remains obscure. RESULTS: Here, we show that mir-17∼92 and mir-106∼363 cluster miRNAs are down-regulated in response to lin28 knockdown, and RNAs from these clusters are co-expressed with lin28 genes during germ layer specification. Mature miRNAs derived from pre-mir-363 are most sensitive to lin28 inhibition. We demonstrate that lin28a binds to the terminal loop of pre-mir-363 with an affinity similar to that of let-7, and that this high affinity interaction requires to conserved a GGAG motif. CONCLUSIONS: Our data suggest a novel function for amphibian lin28 proteins as positive regulators of mir-17∼92 family miRNAs.

Cdx ParaHox genes acquired distinct developmental roles after gene duplication in vertebrate evolution.

BACKGROUND: The functional consequences of whole genome duplications in vertebrate evolution are not fully understood. It remains unclear, for instance, why paralogues were retained in some gene families but extensively lost in others. Cdx homeobox genes encode conserved transcription factors controlling posterior development across diverse bilaterians. These genes are part of the ParaHox gene cluster. Multiple Cdx copies were retained after genome duplication, raising questions about how functional divergence, overlap, and redundancy respectively contributed to their retention and evolutionary fate. RESULTS: We examined the degree of regulatory and functional overlap between the three vertebrate Cdx genes using single and triple morpholino knock-down in Xenopus tropicalis followed by RNA-seq. We found that one paralogue, Cdx4, has a much stronger effect on gene expression than the others, including a strong regulatory effect on FGF and Wnt genes. Functional annotation revealed distinct and overlapping roles and subtly different temporal windows of action for each gene. The data also reveal a colinear-like effect of Cdx genes on Hox genes, with repression of Hox paralogy groups 1 and 2, and activation increasing from Hox group 5 to 11. We also highlight cases in which duplicated genes regulate distinct paralogous targets revealing pathway elaboration after whole genome duplication. CONCLUSIONS: Despite shared core pathways, Cdx paralogues have acquired distinct regulatory roles during development. This implies that the degree of functional overlap between paralogues is relatively low and that gene expression pattern alone should be used with caution when investigating the functional evolution of duplicated genes. We therefore suggest that developmental programmes were extensively rewired after whole genome duplication in the early evolution of vertebrates.

An essential role for LPA signalling in telencephalon development.

Lysophosphatidic acid (LPA) has wide-ranging effects on many different cell types, acting through G-protein-coupled receptors such as LPAR6. We show that Xenopus lpar6 is expressed from late blastulae and is enriched in the mesoderm and dorsal ectoderm of early gastrulae. Expression in gastrulae is an early response to FGF signalling. Transcripts for lpar6 are enriched in the neural plate of Xenopus neurulae and loss of function caused forebrain defects, with reduced expression of telencephalic markers (foxg1, emx1 and nkx2-1). Midbrain (en2) and hindbrain (egr2) markers were unaffected. Foxg1 expression requires LPAR6 within ectoderm and not mesoderm. Head defects caused by LPAR6 loss of function were enhanced by co-inhibiting FGF signalling, with defects extending into the hindbrain (en2 and egr2 expression reduced). This is more severe than expected from simple summation of individual defects, suggesting that LPAR6 and FGF have overlapping or partially redundant functions in the anterior neural plate. We observed similar defects in forebrain development in loss-of-function experiments for ENPP2, an enzyme involved in the synthesis of extracellular LPA. Our study demonstrates a role for LPA in early forebrain development.

Lin28 proteins are required for germ layer specification in Xenopus.

Lin28 family proteins share a unique structure, with both zinc knuckle and cold shock RNA-binding domains, and were originally identified as regulators of developmental timing in Caenorhabditis elegans. They have since been implicated as regulators of pluripotency in mammalian stem cells in culture. Using Xenopus tropicalis, we have undertaken the first analysis of the effects on the early development of a vertebrate embryo resulting from global inhibition of the Lin28 family. The Xenopus genome contains two Lin28-related genes, lin28a and lin28b. lin28a is expressed zygotically, whereas lin28b is expressed both zygotically and maternally. Both lin28a and lin28b are expressed in pluripotent cells of the Xenopus embryo and are enriched in cells that respond to mesoderm-inducing signals. The development of axial and paraxial mesoderm is severely abnormal in lin28 knockdown (morphant) embryos. In culture, the ability of pluripotent cells from the embryo to respond to the FGF and activin/nodal-like mesoderm-inducing pathways is compromised following inhibition of lin28 function. Furthermore, there are complex effects on the temporal regulation of, and the responses to, mesoderm-inducing signals in lin28 morphant embryos. We provide evidence that Xenopus lin28 proteins play a key role in choreographing the responses of pluripotent cells in the early embryo to the signals that regulate germ layer specification, and that this early function is probably independent of the recognised role of Lin28 proteins in negatively regulating let-7 miRNA biogenesis.

Early transcriptional targets of MyoD link myogenesis and somitogenesis.

In order to identify early transcriptional targets of MyoD prior to skeletal muscle differentiation, we have undertaken a transcriptomic analysis on gastrula stage Xenopus embryos in which MyoD has been knocked-down. Our validated list of genes transcriptionally regulated by MyoD includes Esr1 and Esr2, which are known targets of Notch signalling, and Tbx6, mesogenin, and FoxC1; these genes are all are known to be essential for normal somitogenesis but are expressed surprisingly early in the mesoderm. In addition we found that MyoD is required for the expression of myf5 in the early mesoderm, in contrast to the reverse relationship of these two regulators in amniote somites. These data highlight a role for MyoD in the early mesoderm in regulating a set of genes that are essential for both myogenesis and somitogenesis.

Gsx transcription factors repress Iroquois gene expression.

We have previously shown that the Gsx family homeobox gene Gsh2 is part of the regulatory network specifying dorsoventral pattern of primary neurons in the developing amphibian embryo. Here, we investigate the role of Gsx transcription factors in regulating the transcription of Iroquois family homeobox genes in the amphibian neural plate. Iroquois genes are key regulators of neural patterning and their expression is coincident with that of the Gsx genes during open neural plate stages. We show that Gsx proteins repress Iroquois expression in the embryo and conversely, inhibition of Gsx activity with either antisense morpholino oligos or an anti-morphic Gsx protein up-regulates Iroquois expression. These data indicate that Gsx factors act as negative regulators of Iroquois gene expression in the amphibian neural plate and support a model in which the Gsx proteins promote neuronal differentiation by repressing the expression of known inhibitors of neuronal differentiation such as Iro3.

Conserved and novel roles for the Gsh2 transcription factor in primary neurogenesis.

The Gsx genes encode members of the ParaHox family of homeodomain transcription factors, which are expressed in the developing central nervous system in members of all major groups of bilaterians. The Gsx genes in Xenopus show similar patterns of expression to their mammalian homologues during late development. However, they are also expressed from early neurula stages in an intermediate region of the open neural plate where primary interneurons form. The Gsx homologue in the protostome Drosophila is expressed in a corresponding intermediate region of the embryonic neuroectoderm, and is essential for the correct specification of the neuroblasts that arise from it, suggesting that Gsx genes may have played a role in intermediate neural specification in the last common bilaterian ancestor. Here, we show that manipulation of Gsx function disrupts the differentiation of primary interneurons. We demonstrate that, despite their similar expression patterns, the uni-directional system of interactions between homeodomain transcription factors from the Msx, Nkx and Gsx families in the Drosophila neuroectoderm is not conserved between their homologues in the Xenopus open neural plate. Finally, we report the identification of Dbx1 as a direct target of Gsh2-mediated transcriptional repression, and show that a series of cross-repressive interactions, reminiscent of those that exist in the amniote neural tube, act between Gsx, Dbx and Nkx transcription factors to pattern the medial aspect of the central nervous system at open neural plate stages in Xenopus.

Characterisation of the fibroblast growth factor dependent transcriptome in early development.

BACKGROUND: FGF signaling has multiple roles in regulating processes in animal development, including the specification and patterning of the mesoderm. In addition, FGF signaling supports self renewal of human embryonic stem cells and is required for differentiation of murine embryonic stem cells into a number of lineages. METHODOLOGY/PRINCIPAL FINDINGS: Given the importance of FGF signaling in regulating development and stem cell behaviour, we aimed to identify the transcriptional targets of FGF signalling during early development in the vertebrate model Xenopus laevis. We analysed the effects on gene expression in embryos in which FGF signaling was inhibited by dominant negative FGF receptors. 67 genes positively regulated by FGF signaling and 16 genes negatively regulated by FGF signaling were identified. FGF target genes are expressed in distinct waves during the late blastula to early gastrula phase. Many of these genes are expressed in the early mesoderm and dorsal ectoderm. A widespread requirement for FGF in regulating genes expressed in the Spemann organizer is revealed. The FGF targets MKP1 and DUSP5 are shown to be negative regulators of FGF signaling in early Xenopus tissues. FoxD3 and Lin28, which are involved in regulating pluripotency in ES cells are shown to be down regulated when FGF signaling is blocked. CONCLUSIONS: We have undertaken a detailed analysis of FGF target genes which has generated a robust, well validated data set. We have found a widespread role for FGF signaling in regulating the expression of genes mediating the function of the Spemann organizer. In addition, we have found that the FGF targets MKP1 and DUSP5 are likely to contribute to the complex feedback loops involved in modulating responses to FGF signaling. We also find a link between FGF signaling and the expression of known regulators of pluripotency.

Overlapping functions of Cdx1, Cdx2, and Cdx4 in the development of the amphibian Xenopus tropicalis.

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.

FGF signalling modulates transcriptional repression by Xenopus groucho-related-4.

BACKGROUND INFORMATION: Developmental cell signals co-operate in the processes of cell specification and tissue patterning during embryogenesis. Interactions between the FGF (fibroblast growth factor) and Wnt signalling pathways have been demonstrated in a number of developmental processes, including mesoderm formation in amphibian embryos. However, the mechanism underlying the interactions between these key signalling pathways remains unclear. RESULTS: In the present study, we find that the ability of TLE4/Xgrg4 (transducin-like enhancer of split 4/Xenopus groucho-related gene 4) to inhibit a transcriptional target of canonical Wnt signalling is reduced in the presence of FGF and that this is partially dependent on a consensus site for MAPK (mitogen-activated protein kinase) phosphorylation in TLE4/Xgrg4. CONCLUSIONS: These data suggest to us a novel molecular mechanism where FGF and Wnt signalling pathways interact at the level of the co-repressor TLE4/Xgrg4: the weakening of TLE4/Xgrg4 repression by FGF signalling, combined with the stabilization of beta-catenin by Wnt signals, enhances expression of Wnt target genes.

Cloning and expression analysis of the anterior parahox genes, Gsh1 and Gsh2 from Xenopus tropicalis.

Gsx class proteins are members of the ParaHox homeodomain transcription factor family with conserved roles in specification and patterning of the nervous system. We report the cloning of two Gsx genes, Gsh1 and Gsh2, from the frog Xenopus tropicalis. We demonstrate the existence of a single, intact Xenopus ParaHox cluster, containing Gsh1, Pdx, and Cdx2, plus three degenerate clusters containing Gsh2, Cdx1, and Cdx4. Anterior expression boundaries of genes from the intact ParaHox cluster are co-linear with respect to their genomic organization. We show that Gsh1 and Gsh2 exhibit complex, overlapping patterns of expression within the anterior nervous system from open neural plate stages. We also find that expression of Gsh2, Nkx6, and Msx1 across the medio-lateral axis of the amphibian neural plate is strikingly similar to that of related genes in the Drosophila neuroectoderm. These findings provide further evidence for a conserved pathway regulating dorso-ventral patterning in the Bilateria.

FGF4 regulates blood and muscle specification in Xenopus laevis.

BACKGROUND INFORMATION: FGF (fibroblast growth factor) signalling is known to be required for many aspects of mesoderm formation and patterning during Xenopus development and has been implicated in regulating genes required for the specification of both blood and skeletal muscle lineages. RESULTS: In the present study, we have specifically knocked down the expression of FGF4 using AMO (antisense morpholino oligonucleotide)-mediated inhibition and demonstrate that FGF4 acts in the dorsal marginal zone to restrict blood development and promote the development of skeletal muscle. In addition, we used a drug inhibitor of FGF signalling and an inducible form of FGFR1 (FGF receptor 1) to identify a period of competence during late blastula and gastrula stages when FGF signalling acts to regulate blood versus muscle specification. Notably, we found that it is the dorsal activity of FGF that is required to restrict the expression of SCL (stem cell leukaemia) to the ventral blood island. CONCLUSIONS: Our data indicate that FGF4 is a key organizer-derived signal involved in the process of dorsoventral patterning of the mesoderm.

Expression of enzymes involved in thyroid hormone metabolism during the early development of Xenopus tropicalis.

BACKGROUND INFORMATION: There are significant indications that amphibians require TH (thyroid hormones) prior to their involvement in the regulation of metamorphosis and before the development of a functional thyroid. RESULTS: In order to investigate the potential role for TH in pre-metamorphic Xenopus tropicalis we have cloned cDNAs for, and analysed the expression of, TPO (thyroid peroxidase), 5'DII (type II iodothyronine deiodinase) and 5DIII (type III iodothyronine deiodinase), enzymes involved in TH metabolism. Zygotic expression of TPO was detected in neurula stage embryos. Expression was observed in the notochord and later in the thyroid. The notochord was also a common site of expression for 5'DII and 5DIII. Other sites of 5'DII expression are the otic vesicles, retina, liver, blood-forming region, branchial arches and brain. 5DIII is also expressed in the brain, retina, liver, developing pro-nephros, blood-forming region and branchial arches. Embryos exposed to the TPO inhibitor methimazole showed a distinctive dose-dependent phenotype of a crimped notochord and shortened axis, together with alterations in (125)I(-) uptake. CONCLUSIONS: These data suggest a novel extrathyroidal role for TH during early development, and support the proposal that embryos require thyroid signalling for normal development prior to metamorphosis.

FGF signal transduction and the regulation of Cdx gene expression.

Cdx homeodomain transcription factors play important roles in the development of the vertebrate body axis and gut epithelium. Signaling involving FGF, wnt and retinoic acid ligands has been implicated in the regulation of individual Cdx genes. In this study we examine the requirement for FGF-dependent signal transduction pathways in the regulation of Cdx gene expression. In the amphibian Xenopus laevis the earliest expression of Cdx1, Cdx2 and Cdx4 is within the developing mesoderm. We show that a functional FGF signaling pathway is required for the normal expression of all three amphibian Cdx genes during gastrula stages. We show that FGF stimulation activates signaling through both the MAP kinase pathway and the PI-3 kinase pathway in Xenopus tissue explants. However, our analysis of these pathways in gastrula stage embryos indicates that the MAP kinase pathway is required for Cdx gene expression, whereas the PI-3 kinase pathway is not. We show that FGF and wnt signaling can interact in the regulation of Cdx genes and during gastrula stages the normal expression of the Cdx genes requires the activity of both pathways. Furthermore, we show that wnt mediated Cdx regulation is independent of the MAP kinase pathway.

A consensus Oct1 binding site is required for the activity of the Xenopus Cdx4 promoter.

Cdx homeodomain transcription factors have multiple roles in early vertebrate development. Furthermore, mis-regulation of Cdx expression has been demonstrated in metaplasias and cancers of the gut epithelium. Given the importance of Cdx genes in development and disease, the mechanisms underlying their expression are of considerable interest. We report an analysis of the upstream regulatory regions from the amphibian Xenopus laevis Cdx4 gene. We show that a GFP reporter containing 2.8 kb upstream of the transcription start site is expressed in the posterior of transgenic embryos. Deletion analysis of the upstream sequence reveals that a 247-bp proximal promoter fragment will drive posterior expression in transgenic embryos. We show that 63 bp of upstream sequence, that includes a consensus site for POU-domain octamer-binding proteins, retains significant promoter activity. Co-expression of the octamer-binding protein Oct1 induces expression from a Cdx4 reporter and mutation of the octamer site abolishes activity of the same reporter. We show that the octamer site is highly conserved in the promoters of the human, mouse, chicken, and zebrafish Cdx4 genes and within the promoters of amphibian Cdx1 and Cdx2. These data suggest a conserved function for octamer-binding proteins in the regulation of Cdx family members.