Regulation of the Xenopus Xsox17α1 promoter by co-operating VegT and Sox17 sites

The gene encoding the Sox F-group transcription factor Xsox17α1 is specifically expressed throughout the entire region of the Xenopus blastula fated to become endoderm, and is important in controlling endodermal development. Xsox17α1 is a direct target of the maternal endodermal determinant VegT and of Sox17 itself. We have analysed the promoter of the Xenopus laevis Xsox17α1 gene by transgenesis, and have identified two important control elements which reside about 9 kb upstream at the start of transcription. These elements individually drive transgenic endodermal expression in the blastula and gastrula. One contains functional, cooperating VegT and Sox-binding consensus sites. The Sox sites in this region are occupied in vivo. The other responds to TGF-β signals like Activin or Nodals that act through Smad2/3. We propose that these two regions co-operate in regulating the early endodermal expression of the Xsox17α1 gene.
Introduction
The endoderm of Xenopus embryo arises in two successive phases, involving firstly cell-autonomous gene action, followed by dependence on cell signalling. The cell-autonomous phase is directly initiated by the maternal T-box transcription factor VegT, but then some key genes associated with endodermal differentiation become dependent on signalling by the group of TGF-β family members that signal through Smad2/3 These include the Nodal-related proteins or Xnrs, Derrière, Vg1 and Activin. In this second phase, cells are sensitised to TGF-β signalling by the maternal VegT that they inherit (Clements et al., 2001; Clements and Woodland, 2003; Engleka et al., 2001; Hudson et al., 1997; Yasuo and Lemaire, 1999). Since the essential TGF-β signalling molecules are themselves induced by VegT, and VegT is indispensable for endoderm development (Xanthos et al., 2001), the overall effect is that only a critical mass of VegT-containing cells can generate sufficient signalling to sustain expression of these key endodermal genes. Scattered VegT containing cells will fail to become endoderm and conform to their surroundings. In contrast, some other genes (e.g. Xnr4), which are directly induced by VegT do not become signal-dependent. Finally, in the gastrula, endodermal gene expression becomes independent of cell signalling (Yasuo and Lemaire, 1999). This interpretation of endoderm initiation, establishment and maintenance is heavily based on studying the expression of the VegT target Xsox17, an HMG-box transcription factor, although Mix.1 and Mixer behave in a similar fashion.
There are three Sox17 genes in Xenopus laevis, Xsox17α1, α2 and β, but since no differences in their activity have so far been detected, for most purposes we refer to them collectively as Xsox17. The transcription of these Sox F group genes is activated prior to the mid-blastula transition (MBT), when very low levels are found ubiquitously, but Xsox17 transcription in the vegetal pole is enormously upregulated at MBT, precisely marking out the territory of the future endoderm through late blastula, gastrula and neurula stages (Hudson et al., 1997; Zorn et al., 1999). The Xsox17 genes have a key role in endoderm formation. Their ectopic expression induces endodermal gene expression, as well as changing the fate of cells. Interfering with their expression with a dominant negative Engrailed Xsox17 fusion construct has the converse effect, inhibiting endodermal gene expression and shifting cells out of an endodermal fate in intact embryos (Clements and Woodland, 2000; Hudson et al., 1997). The use of antisense morpholino oligos shows that the individual genes have non-redundant late roles in the developing mid- and hindgut, but that together they are needed for the completion of gastrulation (Clements et al., 2003). This correlates well with the essential role of the single-murine Sox17 gene for early development of endoderm fated to become mid- and hindgut, although in the absence of Sox17 there is also later loss of foregut cells (Kanai-Azuma et al., 2002). In zebrafish, two related Sox genes, Casanova and Sox17, are important in forming the endoderm; the former is more upstream and its mutants indicate that it has a vital role in endoderm development (Aoki et al., 2002). While mutations in the zebrafish Sox17 gene have not been described, it is likely that in the zebrafish endodermal gene network the combined action of Casanova and Xsox17 are roughly equivalent to the overall action of Xsox17 in Xenopus, particularly allowing for the fact that Casanova expression in the yolk syncytial layer is Nodal-independent, allowing a parallel with the Xenopus cell autonomous phase to be drawn (Kikuchi et al., 2001; Woodland and Clements, 2003). In Xenopus, ablation of Xsox17 expression with morpholinos halts gastrulation at an early stage. However, the immediate effects on gene expression were initially reported to be modest and were restricted to the direct Xsox17 targets Endodermin and Hnf-1β (Clements et al., 2003). However, much wider effects have now been observed using microarrays (Sinner et al., 2006).
Since Xsox17 is a crucial gene in the early endoderm, and its expression defines the endodermal territory, we have analysed the regulatory elements in the Xsox17α1 promoter approximately 9 kb upstream of transcriptional initiation. We have identified two small elements, which can confer endodermal expression on a reporter gene in the early embryo. We have analysed one of these in detail and show that its activity depends on co-operating VegT and Sox17-binding sites, whereas the other regulatory region responds to Activin.
Materials and methods
Biological materials
Eggs of X. laevis were obtained, fertilised, dejellied and cultured by standard methods, as described previously (Wilson et al., 1986). Oocytes, complete with their follicle, were manually removed using watchmakers forceps in modified Barths' saline (MBS; 110 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 0.8 mM MgSO4, 15 mM Tris–HCl, pH 7.5).
Transgenic methods
These followed the procedure of Kroll and Amaya (1996), except that a reduced amount of egg extract was used in the incubation of stored frozen sperm nuclei with DNA (2 μl in a 25 μl reaction). The reaction typically contained 150–200 ng of linearised plasmid DNA and 0.5 μl of a 1:200 dilution of restriction enzyme (2 units/ml). Injections of dejellied unfertilised eggs were performed in 6% Ficoll, 0.4× Marc's modified Ringer (MMR) or 0.4× MBS in polyheme-coated plastic dishes. Correctly cleaving eggs were sorted at the 2- to 4-cell stage and incubated in 6% Ficoll, 0.1× modified Barth's medium. GFP fluorescence was monitored using a Leica MZFLIII dissecting microscope.
Transient transgenesis in embryos and oocytes
For transient expression in embryos, DNA constructs were linearised and purified with the Qiagen gel extraction kit. 50 pg DNA together with 5 pg control Renilla luciferase reporter in 10 nl water were injected bilaterally, with or without mRNA, into the animal or vegetal poles of 2-cell embryos, cultured in 6% Ficoll, 0.4× MBS. Embryos were analysed at gastrula or neurula stages using the Promega Dual Luciferase Reporter Assay System. Triplicate batches of 10 embryos were homogenised in 600 μl passive lysis buffer and incubated on ice for 10 min. Samples were centrifuged at 13,000 rpm for 2 min at 4 °C, and supernatants removed. They were equilibrated to room temperature for 30 min, and 60 μl assayed for bioluminescence after addition of 50 μl luciferin stock using the Luminoskan RS apparatus (Labsystems). Normalisation of the experimental reporter was achieved by quenching of the firefly luciferase reaction and measurement of Renilla luciferase luminescence.
Oocyte nuclei were microinjected with 18 nl water containing 300–500 pg circular firefly test plasmid, plus one third this amount of control Renilla luciferase plasmid, with or without transcription factor mRNA. After culturing overnight in MBS, they were processed for luciferase activity as detailed above.
Cloning and characterisation of the Xsox17α1 gene
The Xsox17α1 gene was isolated by screening a X. laevis gilli PAC library (RZPD) with an Xsox17α1 cDNA probe. One clone (BUMSP710J2012Q3) reacted strongly with this probe. A positive Not 1 fragment was subcloned into Bluescript and this sequence encompassed all 12 kb of 5′ upstream sequence present in the PAC, the transcribed region itself and 3 kb downstream of the 3′ UTR.
Transgenic constructs and mutagenesis
Mutant promoter constructs were created by hybrid overlap extension PCR, using a series of external and overlapping internal primers. Primer sequences were as follows: B1 ext up 5′CAACACTCACATTC 3′. B1 T-box ext down 5′CTTGAGAATGGGACTGTGTTAACAAACAATGATGATCAGAACTCTGG 3′, Sox A int up 5′ CTTGGGAACTAGTTGTGGATC 3′, B1 Sox A int down 5′GATCCACAACTAGTTCCCAAG 3′, B1 Sox B int down 5′ CTTGAGAATGGGACTGTGTTAACAACCATGGATGGTGTGAACTCTGG 3′, B1 Sox B + T-box ext down 5′CTTGAGAATGGGACTGTTTTAACAACCATGGATGATCAGAACTCTGG 3′. Underlined text denotes mutated sequence. External primers had 5′ Sac1 and 3′ Kpn1 extension sequences. Amplification was performed over 20 cycles with 55 °C annealing temperature. Products were Qiagen column purified, cut with restriction enzyme, and cloned into pGL3basic-act-luc (transient assay) or pGL3basic-act-mgfp5 (transgenic assay).
Electrophoretic band-shifts (EMSAs)
EMSAs were performed using VegT protein synthesised in vitro in the rabbit reticulocyte system. 10 pmol single-stranded DNA oligo was 5′ end-labelled with 20 μCi 32P γ-ATP using T4 polynucleotide kinase. Forward and unlabelled reverse strands were annealed by heating to 90 °C for 5 min, followed by slow cooling overnight. DNA was recovered by ethanol precipitation and resuspended in TE buffer. 15 μl binding reactions contained 1 μl in vitro translated protein, 2 μg [poly dI.dC].[poly dI.dC], 3 μl MDB buffer (20 mM HEPES, pH 7.9, 100 mM KCl, 12.5 mM MgCl2, 0.1 mM EDTA, 17% glycerol w/v, 2 mM DDT). The probe was added following 10-min incubation at 30 °C, followed by a further 10-min incubation. Control binding reactions included a 50-fold excess of unlabelled specific competitor probe. Samples were analysed on a 15% polyacrylamide gel at 4 °C (200 V, 2 h). Gels were dried and autoradiographed or analysed with a phosphoimager. Probe sequences used were: B1 5′-TGTCCAGAGTTCACACCATCATTGTTTGTTA-3′, T-box mutant 5′-TCTCCAGAGTACGCACATTCATTGTTTGTTA-3′, T-box consensus 5′-TCTCCAGAGTTCACACCTTCATTGTTTGTTA-3′. Underlined text denotes T-box half site and variations thereof.
Chromatin immunoprecipitation (ChIP)
30 gastrula stage embryos were fixed in 1% formaldehyde for 10 min at room temperature, and fixation reversed by addition of 125 mM glycine for 30 min. Embryos were washed in MMR (25 mM NaCl, 0.5 mM KCl, 0.25 mM MgCl2, 0.25 mM CaCl2, 1.25 mM HEPES, pH 7.5), and homogenised in 500 μl low salt extraction buffer (25 mM Tris–Cl, pH 7.5, 70 mM KCl, 1 mM EDTA, 20% glycerol, 5 mM DTT + protease inhibitors). Shearing was performed by sonication at full power for 6 × 10 s with 2-min breaks on ice. Shearing efficiency was assessed by agarose gel electrophoresis following reversal of cross links by the addition of 200 mM NaCl and incubation at 65 °C for 5 h, followed by proteinase K treatment, phenol extraction and ethanol precipitation. This preparation also yielded input DNA for quantification of PCR reactions.
100 μl sheared chromatin preparation was diluted with 100 μl IP buffer (50 mM Tris–Cl, pH 8, 100 mM NaCl, 2 mM EDTA, 1 mM DTT, 1% NP-40 + protease inhibitors) and pre-cleared with 40 μl protein A agarose (Sigma) for 2 hrs at 4 °C. Supernatant was incubated with 4 μl serum for 2 hrs at 4 °C. A no antibody control was also included. 40 μl 50% slurry protein A agarose pre-saturated with 1 mg/ml BSA and 0.3 mg/ml salmon sperm DNA in IP buffer was added, and reactions incubated overnight at 4 °C. Beads were washed successively in IP buffer plus 0.1% sodium deoxycholate, IP buffer with 500 mM NaCl, IP buffer with 250 mM LiCl, and TE (10 mM Tris, pH 8, 200 mM NaCl). Following a pulse spin, 250 μl elution buffer (1% SDS, 0.1 M NaHCO3) was added to the beads and elution repeated until a 500 μl volume was obtained. Cross-links were reversed and DNA recovered as described above.
PCR was performed as described previously. 32 cycles of amplification were used. Primer sequences are as detailed below:C3B1 F5′ GCCAATAGACACCTTTCTAG 3′C3B1 R5′ GAGAATGGGACTGTGTTAAC 3′Xsox17α ORF F5′ GGACGAGTGCCAGATGATG 3′Xsox17α ORF R5′ CTGGCAAGTACATGTGTCC 3′Xom Promoter F5′ TGTTGGCTGAGTAGGAATGAGAGG 3′Xom Promoter R5′ AGGCAGAGATCAGTACCACCT 3′
The Xom primers are from Messenger et al. (2005).
Results
Structure of the Xsox17α1 gene
The Xsox17α1 gene was isolated from a X. laevis gilli PAC library (RZPD) using an Xsox17α1 cDNA probe. A Not 1 fragment that encompassed all 12 kb of 5′ upstream sequence present in the PAC and 3 kb downstream of the 3′ UTR was subcloned into Bluescript. The transcribed sequence contains a single intron of 705 bp, starting in codon 119. The upstream region was sequenced up to − 9.5 kb, although sequence from approximately − 7.7 to − 9.2 kb was highly repetitive and proved unsequenceable. The transcriptional start site was determined by primer extension (data not shown).
In parallel, an Xsox17β phage λ clone with 6 kb of 5′ upstream sequence was isolated and all of its 5′ regions sequenced. Since the alpha and beta genes have identical early expression patterns, a comparison was made of the proximal 5′ promoter regions (Supplementary Fig. 1). There are many conserved features, including motifs for binding several transcription factors known to be relevant to endoderm development (Homeodomain, SMAD, T-box and Sox proteins), as well as a GA-rich region at about − 1860 bp. No other Xsox17β or Xsox17α2 clones were identified in any library.
Transgenic analysis of the promoter of the Xsox17α1 gene
Initially the entire 12 kb upstream of the Xsox17α1 gene was fused to a GFP expression cassette to give MR19. A second construct was made which also contained 3 kb downstream of the transcribed sequence. This was placed downstream of the GFP cassette (MR21) (Fig. 1, top panel). When the method of Kroll and Amaya (1996) was used to make transgenics containing these sequences, GFP expression was observed throughout the pre-endoderm of the gastrula, including the superficial region surrounding the blastopore lip, which is known as the involuting or extra-blastoporal pre-endoderm (Figs. 1A, B). Expression was screened by GFP fluorescence, which under-reports in the vegetal region (Ahmed et al., 2004), and it was confirmed by in situ hybridisation to GFP mRNA (C) which also under-reports in vegetal tissues, but to a much lesser degree if suitable protocols are used (Sasai et al., 1996). Bearing this in mind, Fig. 1 shows that the pattern at early and late gastrula was similar to the in situ hybridisation pattern of expression of the endogenous gene (E, F). Comparison of MR19 and MR21 transgenics indicated that they were similar in the gastrula (H), but that the 3′ region of MR21 reduced expression outside the endoderm at later stages. This sequence has not been investigated further. The 260 bp minimal promoter was negative in terms of endodermal expression (I), but was expressed elsewhere. Typical numerical results for the transgenics are shown in Supplementary Tables 1 and 2. Constructs either consistently gave endodermal expression in the gastrula, or consistently failed to do so.
There was later expression of GFP in the pharynx and hindgut of the tailbud tadpole (Supplementary Fig. 2). This corresponds to regions where the endogenous gene is expressed, as judged by RT–PCR (Clements et al., 2003), but there was no expression in the region of the developing gall bladder, where the gene is strongly expressed at this stage (Hudson et al., 1997; Zorn and Mason, 2001). Later there was robust expression in the foregut and proctodeum, as well as in the pancreas (Supplementary Fig. 2). Zorn and Mason (2001) did not report expression in the pancreas at these stages, so this may represent either incorrect regulation or persistent, stable GFP expression. There was also expression in lateral line organs and in the brain (fore/mid brain boundary, cranial nerves). It is not known if Xsox17α1 is expressed in all these regions. However there was GFP expression in the lens, and RT–PCR shows that Xsox17 is expressed there, as are other Sox genes (data not shown) (Schlosser and Ahrens, 2004; Zygar et al., 1998). There was also expression in the olfactory organs, which has been shown for the endogenous genes by in situ hybridisation (Zorn, personal communication), as well as in skeletal elements of the developing limbs.
We have focused on blastula to early gastrula stage expression of the promoter because this is the time that the endodermal domain, marked by Xsox17 expression, is mapped out. To define the regions responsible, we first showed that there were no sequences promoting endodermal expression in the single intron or the 3 kb downstream of the cDNA sequence (not shown). We then showed that although the 260 bp proximal region contains sequences conserved in the Xsox17β promoter, including T-box sites (Supplementary Fig. 1), it drove expression everywhere except the future endoderm. It is thus expressed only where Xsox17α1 is not. However, later it gave expression in the pharynx, duodenum, pancreas, stomach and proctodeum, as illustrated for longer promoters in Supplementary Fig. 2. The 6 kb upstream of Xsox17β also did not drive gastrula endodermal expression (not shown) and we could not identify any Xsox17α2 clone. Thus a comparison of endodermal elements in the three Xsox17 genes could not be made. While it is possible to compare sequences with those in Xenopus tropicalis, any interpretation would be dubious since the detailed regulation of X. tropicalis has not been studied in this species.
A wider screen for regulatory elements in Xsox17α1 was made with a deletion series (Fig. 2). Early endodermal expression required regions about 9 kb from the start of transcription. This defined a 1.1 kb “Endodermal element”, which alone drove strong GFP expression in the early vegetal/endoderm region. Internal deletions show that the E-element can drive strong endodermal expression when placed on the first 1.7 kb 5′ to the start of transcription, which itself only gave low GFP expression in non-endodermal regions of gastrulae and later stages (Fig. 2J). Later experiments, described below, investigated sub-fragments of this region fused to a basal promoter from a Xenopus cytoskeletal actin gene that is expressed in embryonic striated muscle (Ahmed et al., 2004; Latinkic et al., 2002), showing that the E-element does not require sequences in the proximal region of the promoter for endoderm-specific expression. Thus, in the context of the sequences studied, the E-element is the only region both necessary and sufficient to direct expression in the progenitor of the endoderm and in the early gastrula.
Dissection of the E-element
The E-element was divided into three sub-fragments (A–C), which were coupled to the basal cytoskeletal actin promoter and tested in transgenics (Fig. 3A). Only B and C gave vegetal expression. Each was further divided into two approximately equal fragments. For B, only B1 drove vegetal/endodermal expression, but neither C sub-fragment did. However a sub-fragment from the central region of C gave vegetal expression, albeit with relatively high animal expression (Fig. 3A). Since the C1 and C2 fragments were made with an overlapping 18 bp region to avoid disrupting a regulatory sequence, there clearly are sequences within C1 and C2 which must co-operate to give the endodermal expression of C3.
Thus two independent Xsox17α1 control regions, B1 and C3, are capable of driving expression in the presumptive endoderm of the gastrula. The expression of B1 (Figs. 3A, C) is notably similar to the endogenous Xsox17 genes (Figs. 1E, F). The GFP expression in the vegetal regions is detectable by early stage 10, the onset of gastrulation. Given that GFP requires an hour or more to mature and fluoresce, vegetal expression of the transgene must begin in the blastula, during the initiation and/or establishment phases of Xsox17 regulation (Clements et al., 1999; Yasuo and Lemaire, 1999). As discussed earlier, GFP under-reports in the vegetal pole (Ahmed et al., 2004), so any detectable vegetal fluorescence indicates strong actual expression.
Transgenic analysis of the T-box and Sox sites in element B1
B1 contains several consensus binding sites for known endodermal regulators (Fig. 3B; Supplementary Fig. 3), including a variant T-box binding site (core CACCA rather than CACCT) and two canonical Sox sites (core CATTG). To test if the variant T-box binds VegT, we performed electrophoretic gel mobility shift assays (EMSA), comparing the binding of VegT to this sequence and the consensus sequence identified in the Derrière promoter (White et al., 2002). Fig. 3D shows that the binding of the two is similar. As a control, no binding was observed to a sequence containing a mutant site (core CACAT).
The T-box and the two Sox sites were mutated and their efficiency in transgenics assessed (Fig. 3C). The numbers of transgenic embryos in a typical experiment are shown in Supplementary Table 2. The double Sox site mutant gave vegetal pole expression which was somewhat less intense than that produced by the wild-type B1. There was also raised relative expression in the animal pole, suggesting that these sites may play a possible repressive role in the non-endodermal region. The T-box mutant also gave vegetal expression, although again it appeared to be at level lower than from an unmutated B1 element, although this is difficult to quantify. When the T-box site was mutated in conjunction with the Sox sites (Fig. 3C, triple mutant), expression of GFP was excluded from the endodermal domain.
Transient transgenic analysis of B1 and C3
Although in transgenics the constructs either gave endodermal expression, or they did not, the results are not quantitative and the variability in the absolute level of expression from embryo to embryo makes it difficult to compare constructs objectively. Transient transgenesis using the quantifiable reporter luciferase circumvents this problem. DNA may be introduced into different parts of the embryo, or injected it into a region where Xsox17 is not expressed (i.e. the animal hemisphere) with or without mRNAs encoding specific regulatory molecules, like VegT. To enable this, promoter elements, together with the cytoskeletal actin basal promoter, were cloned into pGL3basic (Promega), which lacks eukaryotic promoter and enhancer sequences. The DNA was linearised for injection because sometimes this is necessary for expression (Wilson et al., 1986).
First constructs containing B1 and C3 elements were injected into vegetal and animal hemispheres, to establish whether the transient expression mirrors the natural high expression of Xsox17 in the vegetal compared to the animal pole. Fig. 4A shows that B1 is much more active in the vegetal than the animal hemisphere, in agreement with the transgenics. This is also true, to a reduced degree, of the C3B1 combined fragment. Surprisingly, C3 showed reduced expression in the vegetal relative to the animal hemispheres. This may partly be because these results are expressed as a ratio of animal to vegetal expression. High expression in the animal hemisphere was also seen in GFP transgenics where we simply scored for vegetal expression. Presumably inhibitory elements are lacking.
B1 contains T-box and Sox-binding sites that were essential for high endodermal expression in transgenics. We tested the functionality of the T-box site by co-injecting B1 DNA plus VegT mRNA into the animal pole, where VegT mRNA is normally present only at a low level. The basal promoter did not respond to VegT in these assays (not shown). VegT stimulates B1 activity by an amount comparable to that produced by vegetal compared to animal hemisphere injections (Fig. 4B). In contrast, C3 is inhibited slightly, even though it contains a consensus T-box site core sequence (CACCT). Flanking nucleotides of this site are divergent (AGACACCT; consensus TCACACCT), and White et al. found that this sequence bound VegT only weakly in EMSAs (White et al., 2002). The C3B1 fragment produced an intermediate result. Thus B1, but not C3, responds positively to VegT and the responses of B1 and C3 to VegT mirror their activity in vegetal versus animal poles.
Since VegT, directly or indirectly, induces expression of many transcription factors which are part of the mesendodermal gene network, including other T-box proteins (Eomes and Xbra), we sought to confirm that VegT directly interacts with B1 in two ways. It is of course known that the endogenous Xsox17 genes are direct VegT targets from experiments with inducible VegT fusions (Clements et al., 1999; Clements and Woodland, 2003; Yasuo and Lemaire, 1999). Firstly we mutated the T-box site, which greatly reduced, but did not eliminate the stimulation by VegT (Fig. 4C, lane 3). This implicates a T-box protein in the regulation. Secondly, we examined expression in oocytes in response to co-injected VegT (White et al., 2002). The amount of transcription seen when DNA is injected into an oocyte nucleus is comparable to an embryo, this being supported by stores of chromosomal and transcriptional proteins. However there is only one nucleus in an oocyte, so the amount of downstream nuclear gene expression that the oocyte can support over the time course of our experiments is negligible, even over an 18-h time course (White et al., 2002). One hour of transcription in the late blastula would be equivalent to 10,000 h for the oocyte, equating to well over a year. This suggests that any effects of VegT on the co-injected DNA are directly dependent on VegT itself. Fig. 4C (last three lanes) shows that VegT stimulates B1 transcription in oocytes and that this is absent for the T-box mutant. The stimulation is less in oocytes than in embryos, presumably because cooperating genes are not significantly induced by VegT in oocytes. For example endogenous Xsox17 expression is dependent both on VegT itself and on the TGF-β signalling that VegT induces (Clements et al., 1999; Clements and Woodland, 2003; Xanthos et al., 2001). To test if the B1 element responds to TGF-βs downstream of VegT (directly or indirectly), we blocked TGF-β family signalling by co-expressing VegT and a truncated Activin receptor (tXAR) in animal hemispheres (Hemmati-Brivanlou and Melton, 1992). Fig. 4C, lane 4, shows that the stimulation of B1 by VegT is reduced, but not eliminated. These results are consistent with the proposal that B1 responds to VegT directly via its T-box site, but that co-operating molecules downstream of VegT-induced TGF-β signalling are also necessary for the full VegT induction in an embryo. Consistent with this interpretation, in oocytes there is no VegT stimulation of the B1 element with a mutated T-box because VegT cannot induce downstream genes such as TGF-β signals (Fig. 4C, last lane).
Transgenesis also showed that B1 contained functional Sox-binding sites and it was already known that the endogenous Xsox17α genes are direct Xsox17 targets (Sinner et al., 2004). Fig. 4D shows that in the animal hemisphere B1 responds strongly to Xsox17 and expression in the oocyte supports the view that this is a direct effect. Xsox17α and β induce themselves and each other (Sinner et al., 2004), but the autoinduction is quite slow, as shown in Fig. 4E. The luciferase inductions fitted this because there was no significant induction by Xsox17 at stage 10.5 (not shown), but it was clear by stage 17 (8 h later at 23 °C). Xsox17 probably normally co-operates with other factors that are maternal or downstream of VegT on certain promoters. However, this only applies to a subset of promoters, whereas other genes like Hnf-1β do not show this delay, presumably because any co-operating factors are already present in the animal hemisphere. It is likely that when Xsox17 alone is expressed ectopically, it slowly induces the co-operating factors needed for auto-induction, eventually establishing a partial endodermal gene network. Normally the cooperating molecules are maternal or are induced by VegT. In contrast to B1, C3 did not respond to Xsox17, as expected from its lack of consensus Sox sites.
While C3 was activated by neither VegT nor Xsox17, it contains several consensus Smad-binding sites coupled to those for co-operating FoxH1 (Fast1), suggesting that it might respond to TGF-βs like Activin. Fig. 4F shows that this is indeed the case, suggesting that it might mediate the TGF-β response of Xsox17α1. In oocytes, C3 is induced by constituently active Smad2, indicating that the induction by Activin is direct (Fig. 4G). In contrast, B1 shows very little response to Activin in embryos. This is surprising since many gene products that induce Xsox17α1 are downstream of Activin, including the Xsox17s themselves. While in this experiment the luciferase was assayed before Xsox17 would have exerted its slow effect in animal caps (see above), there is little effect even at later time points (not shown), which suggests that the level of Xsox17 induced by Activin is too low to produce a strong induction without co-operating VegT. We show below that VegT and Xsox17 co-operate in regulating B1, and synergy between VegT and Activin was previously noted (Clements and Woodland, 2003).
Mutational analysis of B1 shows that one Sox site is most important and it co-operates with the VegT site
To assess the roles of the two Sox sites in B1, the response of a series of single, double and triple mutants of Sox and T-box sites was assessed by injection into animal hemispheres. Most of the response to VegT was dependent on the T-box site and the neighbouring B Sox site, whereas removal of the more distant A Sox site had much less effect (Fig. 5B). When all three sites were removed, there was no induction by VegT, and no expression in the vegetal pole. Since removal individually of either the B Sox or the T-box sites, but not the A Sox site, removed much of the response of B1, maximal induction clearly requires the presence of both B Sox and T-box sites. This suggests that there is synergy between the VegT and Xsox17 bound to these sites. The role of the two Sox sites in responses to Sox17 is examined in Fig. 5C. This confirms that Sox site B has the main activity and the T-box site is unimportant in induction by Sox17 alone.
It is possible that in the endoderm itself, the B1 Sox sites respond to Sox proteins other than Xsox17. To test this B1 DNA was injected into the vegetal pole, followed by injection of the three Xsox17 antisense morpholino oligos (MOs) that block translation of the three X. laevis Xsox17 mRNAs (Clements et al., 2003). The enhanced activity of B1 in vegetal poles, as compared to animal, was eliminated, whereas a control MO had no effect (Fig. 5D). This confirms that the endogenous Xsox17s are responsible for the high activity of the B1 element in the developing endoderm.
The B1 element is bound to Xsox17 in vivo
To confirm that the Sox sites in B1 are functional we needed to show that they are occupied by Xsox17 in the living embryo. We therefore performed a chromatin immunoprecipitation (ChIP) on mid-gastrulae stage embryos. Chromatin was isolated, sheared to DNA fragments of about 500 bp and immunoprecipitated with an Xsox17β anti-serum (Clements et al., 2003). Fig. 6A shows PCRs from several regions in the resulting genomic DNA. The Xsox17 B1 plus C3 element was clearly precipitated by the anti-serum, but not pre-immune serum. Conversely, the Xsox17α1 ORF was not precipitated, as expected since it is about 9 kb distant from the E-element. A second control was to probe for an enhancer previously used for immunoprecipitation with an Xbra anti-serum (Messenger et al., 2005). This ventrally expressed gene should not be regulated by Xsox17 and indeed the promoter element fails to precipitate with Xsox17β antiserum. Therefore, as expected from the transgenic data, the E-element is bound to Xsox17β in vivo.
Discussion
Expression of the Xenopus Xsox17 genes defines the endodermal territory in the mid-blastula, when cells start rapid transcription and make the initial decisions to become endoderm. For this reason, we have focused on the regulation of one of these genes, Xsox17α1, in the blastula and early gastrula. We have been able to identify two regulatory elements at approximately minus 9 kb that drive transcription in the future endoderm at blastula and gastrula stages. Study of the endogenous genes has suggested that the regulation of the Xsox17 genes is very dynamic, passing rapidly through initiation, establishment and maintenance phases (see Introduction). In the initiation phase, Xsox17α1 is a direct target of the localised maternal transcription factor VegT, in the establishment phase it becomes signal-dependent and eventually maintenance is cell-autonomous. One would hope to find the basis of these regulatory processes in the Xsox17α1 promoter.
Activity of the C3 endodermal element
Of the two adjacent elements capable of driving endodermal expression, B1 responds strongly to VegT and Xsox17, and C3 to Activin, but not to VegT. We have principally focused on B1, but we have made a preliminary analysis of C3. This 89-bp region must contain cooperating elements since two fragments, each containing half of C3 plus an 18-bp overlapping region, were negative in terms of endodermal expression. The Activin responsiveness of C3 correlates with its possession of three separate pairs of closely associated FoxH1/Fast1 and Smad sites, which are known direct effectors of Activin/Nodal signalling (Chen et al., 1997; Germain et al., 2000). However, Activin induces the expression of many downstream molecules, which could then be responsible for indirect induction of this element. To prove the effect was direct we showed that C3 is stimulated by activated Smad2 in the oocyte. This indicates a direct effect, since the single nucleus of this cell has 10,000-fold less transcriptional capacity to induce downstream effectors of Activin/Smad action than does the early gastrula.
Maternal depletion of FoxH1/Fast1 by Kofron et al. (2004) showed that it was not essential for Xsox17α expression. However, as they point out, Xenopus embryos also express XFast-3, which binds to the same target sequence (Howell et al., 2002), and could therefore redundantly regulate Xsox17 through the same promoter elements.
C3 responds strongly to Activin, but with respect to vegetal expression and response to VegT, its behaviour is paradoxical. It is surprising that it does not respond to VegT in the embryo, both because C3 contains a consensus T-box core sequence and because VegT induces the expression of TGF-βs that, like Activin, act through Smads 2/3; indeed VegT depends on this signalling for its overall biological effect. The T-box site in C3 entirely overlaps a Fast1/Smad pair of sites, which may have a bearing on the fact that VegT actually inhibits C3 basal expression, just as its expression is inhibited in the vegetal pole compared to the animal. This is consistent with the observation that removal of the T-box site removes the inhibition (data not shown). C3 was identified by its ability to direct expression in the vegetal pole of transgenics, however here we simply scored expression in the vegetal pole itself, disregarding animal regions. Conversely, the luciferase measurements are of the ratio either of vegetal to animal expression or of VegT stimulated to control expression in the animal region. Therefore a high level of animal expression would mask vegetal activity of the promoter. However, it is important to note that both the VegT induction and vegetal expression are consistent. We believe that the response to TGF-βs is the important property of C3 and other problems are introduced by looking at small regulatory regions in isolation, where synergising and inhibitory effects are absent.
Previous work has shown that Xsox17 expression switches from direct VegT response to TGF-β signal dependence. C3 is a therefore a good candidate for controlling the signal-dependent process. It is conceivable that the inhibitory effects we notice bear on the hypothetical inhibitor that make Xsox17 signal dependent in the establishment phase of endoderm development.
The activity of the B1 endodermal element
The activity of B1 is more straightforward. It is highly expressed in the presumptive endoderm in transgenics and it is much more highly expressed in the vegetal than the animal hemisphere in transient assays. It is also strongly stimulated by VegT. It contains a divergent T-box site which binds VegT in vitro and which mutation shows is partially responsible for the VegT stimulation in the embryo. Expression in oocytes shows that B1 responds directly to VegT through this site, although the stimulation is less than in embryos. This correlates with the fact that blocking TGF-β signalling in embryos with a truncated Activin receptor reduces VegT stimulation to about the oocyte level. Thus B1 responds directly to VegT and synergistically to other molecules that are downstream of the VegT-induced TGF-βs, which would principally be Nodal-related signals. These synergistic molecules are the Xsox17 proteins themselves in an autoregulatory loop.
B1 responds directly to Xsox17. Other molecules, such as Gata 4–6, are downstream of Xsox17 and could in principle regulate B1. Although B1 contains a possible variant Gata-binding site, mutation of the two consensus Sox sites removes much of the activity of B1, and the triple Sox/VegT mutant is unresponsive both to VegT and vegetal pole expression. Further, mutation of the possible Gata site has no effect on the response of B1 to VegT (not shown). The direct action of Xsox17 on B1 was confirmed by transcriptional assays in oocytes. These observations fit with the fact that Xsox17 auto-induces itself (Sinner et al., 2004), but endogenous Xsox17 genes are very slow to be induced when Xsox17 is expressed ectopically in the animal cap, and this is equally true of the B1 regulatory region. Previously we have found the same for a direct Sox inductive site in the Endodermin promoter (Ahmed et al., 2004). In fact, we believe that the Sox sites in B1 are already needed at blastula stages because they are required for full vegetal expression of the GFP transgene as early as the onset to gastrulation, judged by GFP fluorescence. Since GFP takes several hours to mature (Davis et al., 1995), there must have been an earlier requirement for the Sox factors. We have argued previously, in connection with the Endodermin promoter, that Xsox17 must normally co-operate with other molecules found in the vegetal pole. In the animal cap Xsox17 presumably establishes the endodermal gene network more slowly than its normal establishment by multiple maternal inputs (Sinner et al., 2006). Since B1 is rapidly induced by VegT, and this depends largely on the Sox sites and Xsox17 expression (as shown by Sox site mutants and blocking Xsox17 action with combined Xsox17 group morpholinos), it seems that VegT is able to co-operate with Xsox17 to produce more rapid Sox action.
The kinetics of reporter expression suggest that the VegT and the T-box site are not simply needed at the very onset of endodermal gene expression in the initiation phase, but that this continues into gastrulation during the establishment phase. It is provocative that the Sox site of principal importance in B1 is very near to the VegT site, suggesting a direct interaction between VegT and Xsox17, although we have been unable to detect this by immunological co-precipitation.
The role of Xsox17 auto-induction in the endodermal network
The idea that a key endodermal gene, Xsox17, induces itself is attractive because it would give stability to Xsox17 expression and hence to the endodermal network. The requirement for signalling also adds a fail-safe for cells that become misplaced from the endodermal domain, but still initiate direct VegT-dependent endodermal gene expression. These conform to the fate of their neighbours (Clements et al., 1999; Clements and Woodland, 2003; Wardle and Smith, 2004; Wylie et al., 1987; Yasuo and Lemaire, 1999). However, blocking Xsox17 action with morpholino oligos has no effect on Xsox17 expression, even though other Xsox17 targets, including HNF1-β, Endodermin and Gata5, are down-regulated (Clements et al., 2003; Sinner et al., 2006). The most obvious reason for this paradox is redundancy. For example Xsox17 can be induced by Gata4, 5 and 6 and the first two are only partially and the latter not at all affected by Xsox17 morpholinos (Clements et al., 2003, and unpublished data). Xsox17 is a direct Gata6 target (Afouda et al., 2005), but while B1 and C3 elements contain possible divergent Gata sites, mutating these in B1 does not prevent its response to Gata4–6. Furthermore, only the Sox and T-box sites of B1 are essential for expression in the endoderm. While other Xsox17-inducing factors, like Mix/Bix proteins, could be relevant, another candidate is maternal Xsox7, which is localised in the vegetal pole (Zhang et al., 2005). Indeed, we have shown that overexpression of Xsox7 in the animal hemisphere results in the induction of Xsox17, although again this is slow, as with Xsox17 itself (data not shown).
Nevertheless, the case for Xsox17 auto-regulation is compelling, now being based on three independent lines of evidence. Firstly, Xsox17 induces itself in ectopic expression experiments, and this is direct (Sinner et al., 2004); secondly, there are essential Sox sites in a promoter element that drives endodermal gene expression in transient and true transgenics; thirdly ChIP analysis shows that the endodermal element is bound to Xsox17 in vivo. Based on the activity of a glucocorticoid derivative of VegT in the absence of protein synthesis, Xsox17 is a direct target of VegT (Clements and Woodland, 2003), and our results show that it co-operates with Xsox17 (and possibly other transcription factors) to establish and amplify Xsox17 gene expression. The fact that Xsox17 expression is ultimately dependent on TGF-β signalling makes this amplification and stabilisation of its own expression signal-sensitive and hence subject to a community effect (Gurdon et al., 1993a,b). We propose that this involves the C3 element. A simplified version of this network is shown in Fig. 6. An important point to note is that other members of the endodermal network, like the Mix/Bix genes, are similar to Xsox17 with respect to VegT induction and signal dependence.
There are still many issues to be resolved relating to the regulatory elements. A VegT-induced inhibitor of its own action in inducing Xsox17 was postulated to explain the switch to signal dependence of VegT action, and this is still unknown. It may relate to the repressive effect of VegT and vegetal position on C3, a repression that is lifted by TGF-β signalling. There also appear to be inhibitors of expression in other parts of the embryo. For example, the loss of the Sox sites appears to increase the background expression in the animal pole. This could involve the action of the inhibitor Xsox3 (Zhang et al., 2003, 2004). At later stages. there is also expression of most transgenes in regions like the axis, where Xsox17 is not expressed. This was not seen in a construct including 3 kb of 3′ sequence, suggesting that late inhibitory sites are present in this region.
The regulatory interactions described here are elements within the core endodermal network (Sinner et al., 2006). The form of the regulation, where VegT induces several factors, such as TGF-βs and Xsox17, and co-operates with them to regulate an important control gene, is an important recurring regulatory motif, the feed-forward motif (Mangan and Alon, 2003). In this case, it also brings about autoregulation. Autoregulatory effects of proteins in transcription factor networks are thought to have special importance. Mochizuki (2005) found that the number of possible stable states of gene networks depended only on the number of autoregulated components. Since it is reasonable to equate stable states with differentiated states, the autoregulation of a key endodermal transcription factor would be most significant in endodermal differentiation.

xenopus
sox17
promoter
vegt
endoderm
tgf-β
transgenic embryo


The Role of User Input in Shaping Online Information From the National Cancer Institute

The National Cancer Institute (NCI) was among the first federal agencies to recognize the potential of the Internet for disseminating health-related information. The evolution and refinement of NCI's online cancer information has been substantially “user driven”—from the launch of CancerNet in 1995 to the recent redesign of its award-winning successor, the NCI website. This article presents an overview of NCI's multi-pronged approach to gathering input about its online information products, including stakeholder meetings, focus groups, standard and customized online user surveys, usability testing, heuristic reviews, and search log analysis. Also highlighted are some of the many enhancements that have been made to NCI's online cancer information products based on user input.
Introduction
The National Cancer Institute (NCI) was among the first federal agencies to recognize the potential of the Internet for disseminating health-related information, and it launched its CancerNet website in 1995. This site was a natural extension of NCI's information dissemination efforts, which have been carried out in response to mandates from Congress in the National Cancer Act of 1971 [1] and subsequent legislation. Table 1 outlines major milestones in the development of NCI's Web presence.
Table 1
Milestones in the development of NCI's website
Year
Milestone
1995
CancerNet website is launched.
1999
cancerTrials website is launched.
1999
CancerNet website is redesigned.
2002
NCI's overarching website [2] is redesigned; CancerNet and cancerTrials websites are subsumed into the redesigned site.
2004
NCI website is redesigned.
A large part of NCI's pre-1995 information dissemination efforts was targeted at health professionals through the Physician Data Query (PDQ®) cancer information database, which contains information summaries on numerous cancer-related topics and a cancer clinical trials registry. PDQ was available to medical librarians, physicians, oncology nurses, and other professionals through the National Library of Medicine's online information system [3-6].
There was, however, a new dynamic in the development of the Web. Cancer patients were coming online in large numbers, seeking to be informed decision makers in their own care. Simultaneously, the patient advocacy community was becoming more vocal in requesting that NCI provide products geared to patients. NCI responded to this growing audience by organizing the CancerNet website by audience type, with entry points for patients, health professionals, and researchers, and with information categorized accordingly.
The evolution and refinement of NCI's online cancer information has been notably “user driven.” NCI has adopted a multifaceted approach to gathering feedback and other information about how its information products are used. This has included pre- and post-design tests in usability labs, heuristic or expert review, informal user feedback, standard online user surveys, focus groups, analysis of site usage and search logs, and special user survey projects. Each generation of NCI's Web presence has been informed by user feedback. NCI staff members were crucial leaders in developing usability guidelines and standards that are now widely accepted in the industry, and NCI was one of the first federal agencies to conduct systematic usability testing with its CancerNet website.
This article presents an overview of the methods NCI has used to gather input about its online information products and services. It is not the result of research projects that set out to test specific hypotheses about the impact of specific user-driven enhancements. Rather, it presents an approach to information architecture and design of a website that uses a variety of methods to gauge user behaviors and preferences. It highlights some of the many enhancements that have been implemented in response to user data and feedback. While NCI's website contains a wealth of additional information about cancer research opportunities, funding, NCI programs/initiatives, cancer statistics, and information for the news media, space limitations prevent a discussion of the role of user input in the design and implementation of these areas. The focus of this article will be enhancements to patient-oriented cancer information and information about clinical trials.
How NCI Gathers User Input and Feedback
Stakeholder Input
NCI solicits user input prior to any major online system design or redesign. For example, in response to a growing need for clinical trials information, and prior to a major redesign of the backend database and the user interface of its CancerNet website, NCI organized the Clinical Trials Information System meeting in Chantilly, Virginia, USA, in 1998.
Approximately 200 patients, advocates, clinicians, oncology nurses, clinical investigators, and health information providers representing the core users of NCI's online information resources came together to brainstorm the design of a clinical trials information system.
Figure 1Before and after screen shots of the CancerNet home page, showing a shift in focus from audience to topic with the 1999 redesign
Some of the key recommendations of the meeting were the following: (1) that the NCI website avoid segmenting information pathways according to type of user (patient, physician, researcher); (2) that information be customized to provide varying degrees of technical detail, complexity, and reading level; and (3) that users be able to easily move between these levels. It was also recommended that the NCI website integrate clinical trials information with the full spectrum of cancer information; include information about clinical trials, patient rights, and the informed consent process; and include a feature covering news topics related to clinical trials.
One of the outcomes of this meeting was the development of a new NCI website, cancerTrials, to provide an educational context for the PDQ clinical trials registry that was offered on CancerNet. The cancerTrials website was launched in 1999. In addition to guidance on how to search the PDQ registry, visitors to the new site were offered original articles explaining what cancer clinical trials were, how they worked, and where to find them. They also were offered brief summaries of recently announced cancer trial results and other timely news related to the US clinical trials system.
The subsequent redesign of CancerNet in 1999 [7] carried out the Chantilly recommendation to abandon the partition of the site by audience (Figure 1). Now, the site gave all users information organized around a standard set of topics. Information was presented at varying levels for most of the common cancers—including the “What You Need To Know” series for the most basic introduction, patient and health professional versions of the PDQ cancer information summaries, and abstracts (summaries) of clinical trial protocols written for patients and health professionals. The new design also made it easy for users to switch between the different information levels. Input obtained at the Chantilly meeting continues to influence the development of NCI's cancer information products and their presentation to users.
Ongoing Feedback from CIS Information Specialists
Information specialists at NCI's Cancer Information Service (CIS) are the front line of NCI's interactions with the cancer community, particularly the public [8]. Through the CIS toll-free telephone service (1-800-4-CANCER) and “LiveHelp” online chat sessions, information specialists help individuals who are seeking cancer information. As needed, they can assist callers and website visitors with NCI online tools and resources. As “power users” of the NCI website, they often help test new features. Regular feedback from the CIS to website staff helps drive website improvements.
User Surveys
A critical factor in achieving continuous improvement of NCI's Web resources is soliciting user feedback to learn what works, what doesn't, and where gaps in information or functionality exist. In preparation for the 1999 redesign of CancerNet, an online survey asked users to identify the information they were seeking (Table 2), difficulties they encountered on the site, features they found useful, and additional information or features that were needed. Users were also asked about their general Web usage and basic demographics.
Table 2
Type of information users were seeking on CancerNet (1999)
Percentage
of Respondents
(N = 780)
Information Sought
22.8
Information on a specific type of cancer
18.6
Treatment information (general and specific)
11.3
Clinical trial information (specific trials, general information, trial results)
8.1
Symptoms of cancer, causes, risk factors, detection, diagnosis, prevention
6.8
Specific term (type of tumor or other term—not by name of cancer)
4.7
Cancer literature/articles
4.5
New treatments, news, recent findings, current research projects
3.5
Patient support (pain relief, diet/nutrition, survivorship, exercise, follow-up, questions to ask doctor)
3.1
Side effects
2.9
Statistics (incidence rates, survival rates, mortality rates)
2.8
Drug information
2.8
Access to other cancer resources (treatment facilities, physician names/specialties, national tumor registry, cost information, insurance coverage, patient support group)
2.2
Caregiver information (how to help patient, what to expect as disease progresses, how to talk to patient, etc)
2.1
History of cancer research, information for reports/projects
1.8
NCI publications (ordering information)
0.8
Alternative treatments
0.6
Genetic information (general and specific)
0.4
Search engine for the site
0.3
Information about oncology professions
Figure 2The “Types of Cancer” page on CancerNet
Feedback from the online survey, along with input from the Chantilly meeting, guided the redesign of CancerNet in 1999. On the redesigned site, users could start with the “Types of Cancer” page (Figure 2), which enabled users to quickly find information about specific cancers. They could then choose a cancer-specific home page (Figure 3), where information related to the cancer was organized by topics such as “Introductory Overview,” “Statistics,” “Treatment,” and “Clinical Trials.”
Figure 3The “Breast Cancer” page on CancerNet, an example of a cancer-specific home page
In 2002, NCI's overarching website was redesigned, and the CancerNet and cancerTrials websites became the Cancer Information and Clinical Trials portal areas of the redesigned site. In 2004, the NCI site underwent another redesign, once again guided by extensive evaluation and user input.
NCI's early decision to provide information tailored for patients and their families continues to be supported by surveys conducted during the past five years. Data from 1999 showed that 44% of visitors to the site described themselves as cancer patients or family members or friends of a cancer patient. Data from the American Customer Satisfaction Index (ACSI) survey posted on the NCI website in 2004 showed that more than 50% of respondents identified themselves as cancer patients or family or friends. The next largest audience in 2004 was health care providers, about 13%. NCI continues to keep the patient at the center of many of its online resources—PDQ's cancer information summaries and clinical trial abstracts, clinical trial results summaries, fact sheets and other information products, and the website's dictionary are all written for lay audiences.
American Customer Satisfaction Index (ACSI Survey)
Both before and after the 2004 redesign, the website displayed the ACSI survey [9]. This survey gathers input from users at points within the website. The ACSI survey can be utilized site-wide or for a certain URL. One version of the survey can be posted to appear randomly on all pages of the site (Appendix 1), and another can be set to appear on a group of related pages to collect in-depth data on a particular subject.
The ACSI methodology provides continuous online feedback and is a uniform, national, cross-industry measure of customer satisfaction. A core set of ACSI questions measures overall satisfaction, and customized questions can be added regarding individual websites or pages.
Data from the survey are helpful in supporting or dispelling impressions of who uses a site and what their information needs are. For example, data from the 2004 ACSI survey showed that approximately 57% of NCI's website visitors are first-time users of the site. This underscores the need for intuitive site structure and navigation tools that can be easily grasped by users with no prior knowledge of the site. Multiple paths to core information, such as cancer-specific home pages and clinical trial search tools, were created in 2004 to help new users easily find the most sought-after information. While we cannot make a direct correlation between these enhancements and increased customer satisfaction, the ACSI survey results published in December 2004 named the NCI website the “best in customer satisfaction” in the portal/department main site category [10]. Overall satisfaction among visitors to major government online portals was 72.1, on a scale of 0 to 100. The NCI website led the category for government sites with an overall satisfaction score of 80. In the first quarter of 2005, the NCI website was again the highest scoring government portal site, with a score of 80. NCI expects to further analyze ACSI data to inform additional improvements to the website.
Usability Testing and Expert Review
Usability testing helps ensure that products and services address the needs and interests of website visitors [11,12]. In lab sessions with representative users, testers pose scenarios (see Appendix 2) and solicit comments to gauge the effectiveness of page designs, functions, navigation paths, labels and terminology, and other elements. Data from iterative testing inform the refinement of key pages and the development of new features. NCI also consults with experts on user-centered design to help ensure that its information products keep pace with current standards and trends. For example, prior to the launch of the redesigned NCI website in 2004, a panel of experts was involved in heuristic reviews, and their recommendations led to additional refinements prior to the launch.
Search Log Analysis
Search log analysis played an important role in the 2004 redesign of the NCI website. Each year, users enter approximately 2.5 million free-text searches in the basic search box on the site. More than 50% of searches are for types of cancer or specific body systems or locations. To give visitors immediate access to information on the most common cancers, prominent links for each of these cancers were added to the site's home page, along with multiple links to an A to Z list of cancers to enable easy information retrieval (Figure 4). The same selection of links to common cancers and the A to Z list was also placed on the site's Cancer Topics portal page (which replaced the Cancer Information portal page introduced in the 2002 redesign). (For more information about search log analysis, see “Best Bets on the Website” below.)
Figure 4The home page of the current NCI website, prominently featuring “Types of Cancer” and links to “Common Cancer Types”
Selected User-Driven Enhancements
Best Bets on the Website
When the NCI website was redesigned in 2002, the site's search tool was supplemented with a “Best Bets” feature that gives users a concise list of editorially selected NCI sites and pages that are displayed above the full set of search results. Whereas the full set of search results, which are generated by a free-text search of NCI's Web content, can number thousands of documents for a given search term, the Best Bets offer an average of two links, with a range of one to 18. There are currently 677 Best Bets categories (eg, lung cancer, mammography, cancer diagnosis program) with selected Spanish-language categories included.
To populate Best Bets initially, a team of information experts identified cancer-related information categories, selected the most relevant NCI sites and pages for each, and created a table of related terms for each category name. When a search term is entered in the search box on the site and a category name or related term matches the term or any part of the term, the associated list of Best Bets is displayed.
Search log analysis after the launch of the Best Bets feature in 2002 validated the choice of category names and related terms, the majority of which proved to be among the more popular search terms. Since 2002, the Best Bets database has been edited by NCI staff as needed, on the basis of periodic analysis of search logs and knowledge of new and changing NCI Web content. Log analysis has prompted a considerable expansion of the Best Bets database by suggesting new category names and related terms. In addition, there have been a few instances in which the large number of searches on a topic indicated the need for new content. These findings have already led to the creation of two important pieces of content (which, in turn, were classified as Best Bets), namely a fact sheet about cancer staging and a substantial resource on the NCI website called the “Tobacco and Cancer” home page. Best Bets categories, related terms, and links have also been added in response to comments from users.
Clinical Trials Portal Redesign
In the summer and fall of 2002, NCI initiated a multi-pronged review of the Clinical Trials portal of its website (Figure 5) to determine whether the portal was meeting the needs of its users. Particular attention was given to the Clinical Trial Results section of the portal; articles in the section are also referred to as “news summaries” [13].
Figure 5The Clinical Trials portal home page on NCI's website (2002)
Evaluation Methods
The 2002 evaluation used six qualitative and quantitative methods:
The initial phase of the evaluation involved key informant interviews with NCI staff integral to the development of the portal.
A diary activity was conducted to capture feedback from users who were representative of three of the portal's target audiences, including patient advocates, oncology nurses, and CIS information specialists. Participants were asked to complete a written, formatted diary entry for each visit they made to the portal in the course of their regular activities over a period of one month.
Focus groups and in-depth interviews were later conducted to gain more feedback.
Two online surveys were posted in the Clinical Trials portal of NCI's website. A general survey was presented to each user who visited any page of the portal except for news summaries. A news summary survey was presented to users who visited news summaries. Session cookies were used to recognize possible repeat visitors and to serve up the survey once per visitor during a 30-minute time period (to minimize both the burden on the public and duplicate responses). Once duplicate responses were eliminated, the adjusted survey sample contained 1589 general survey responses and 207 news summary survey responses.
Usability testing was conducted to determine whether users could easily find and understand the news summaries. Perceived usefulness of the news summaries was also explored in usability testing with six participants.
Server log file entries were analyzed using WebTrends log analysis software to collect the following usage statistics: unique visitors, visitors who visited once, visitors who visited more than once, sessions, median visit length, page views, and visits from referring sites.
Key Findings
Several key findings emerged from these evaluation methods [14]. The top three categories of information that visitors were looking for were (1) specific cancer clinical trials (ie, they wanted to search the PDQ registry); (2) recent research results about a specific cancer treatment, test, or prevention; and (3) recent research about a specific type of cancer.
With regard to the Clinical Trials portal, most users found the information they needed, were able to understand it, and found it useful. However, they had difficulty finding their way to the Clinical Trial Results news summaries, even though this type of information was among the top three categories of information desired. When directed there (or when identified as having been there via the pop-up exit survey), users found the summaries to be useful, understandable, and well organized.
Informed Changes
These findings were used to inform changes to the content and design of the Clinical Trials portal over the course of 2003 and again during the 2004 redesign of the NCI website (Figure 6). For example, to make it quicker and easier for users to search the PDQ clinical trials registry, the website's basic search form for clinical trials was added to the Clinical Trials portal home page [15]. Links to this form are also located throughout the pages of the Clinical Trials portal and elsewhere on the site.
Figure 6The Clinical Trials portal home page on the 2004 redesigned NCI website
To further help visitors locate specific trials in which they might be interested, a new section was created called Featured Clinical Trials [16]. This section is updated on a weekly basis and includes brief profiles of key NCI-sponsored clinical trials, with links to more detailed information about the trial. Both the Featured Clinical Trials and the Clinical Trial Results sections were redesigned to allow users to browse by type of cancer and to search the collections by keyword from anywhere in the sections.
In addition, links to the Clinical Trial Results pages organized by type of cancer are more prominently displayed on the Clinical Trials portal home page, and teasers (brief description and link) for the two most recently posted Results articles are prominently displayed on the Clinical Trials portal home page.
Improved Searching for Clinical Trials
The PDQ clinical trials registry has been a key component of NCI's online cancer information services from its inception in the 1980s [3]. Originally designed for health professionals, the registry is now also widely used by patients and is one of the most popular features of NCI's website. Since January 2003, more than 50000 visitors per month, on average, have searched for clinical trials. Designing a search application that works equally well for patients, caregivers, health professionals, and researchers has been a major challenge, and NCI has relied on feedback from users as well as insight from experts to guide each version of the clinical trials search form.
Since its appearance on the Web on CancerNet, the complexity of the clinical trials search form has been a topic of discussion within NCI. The PDQ clinical trials registry began as part of a DOS-based, menu-driven system used almost exclusively by health professionals, medical librarians, and cancer information specialists. Developers were wary of transplanting the sophisticated search functionality of this system to NCI's website because many of the site's visitors had little familiarity with clinical trials, cancer staging, treatment choices, and other elements in the original system. Initial methods of clinical trial searching on NCI's CancerNet website included a form with limited search options and clinical trial descriptions written in technical language, a legacy from the original system. Simply written, patient-friendly descriptions of clinical trials were introduced in 1997.
Two-Step Search Form
The second-generation search form that was launched in 1999 was based on recommendations from the Chantilly meeting, data from an online feedback form on the website, analysis of the search form, and personal interviews. In addition, a prototype of the form was developed through iterative rounds of usability testing. A two-step search form was designed to allow users to search by common search parameters, such as type of cancer, type of trial, and geographic location. Users could then review their search results or choose to narrow their search with other parameters, such as stage of cancer, drug (including brand and generic names), type of treatment, and trial sponsor. Users were also given the option of viewing two descriptions of each clinical trial, one for patients and one for health professionals.
Other changes to the search form based on usability testing included a user's guide for less experienced users, annotated labels for search parameters with links to more detailed explanations, and explanations of how to select multiple items per field (eg, selecting several stages of breast cancer).
Audience-Focused Search Form
In the 2002 redesign of the NCI website, the clinical trials search form was included in the Clinical Trials portal of the site, giving users a more integrated information pathway that grouped information such as patient safety, informed consent, and insurance issues with the listing of clinical trials.
A major consideration in this redesign was the addition of a specific new group of users—information specialists from the CIS, whose duties include assisting patients, their families, and health professionals in identifying clinical trials of interest. Information specialists had previously used the DOS-based, menu-driven PDQ search system that allowed them to perform complex searches, review results, refine as needed, and then prepare an “information packet” that could be emailed or mailed to callers. Web designers visited a CIS regional office to understand the needs of this group of users and did extensive usability testing with them prior to launching the revised form.
Given the diversity of users, it became clear that a single search form was not ideal. Some users found the detailed choices on the form confusing and beyond what they needed. An interactive search form that guided users through the search process was considered, but such a form would require JavaScript, which does not meet Web accessibility requirements for federal government websites. It was determined that the best approach was to develop two search forms with different levels of complexity. Web accessibility requirements could be satisfied by creating one form without JavaScript, and a more complex, interactive form could be created with JavaScript.
The basic search form, designed for the patient, caregiver, or busy health professional, provided three search options—cancer type, type of trial, and zip code proximity. The results were also presented in a format more suited to the casual Web user, who was accustomed to clicking on a search result link to go to a page that contained more information. Usability testing had also indicated that users did not normally click on the check boxes that were provided with the search results in order to prepare a “package” for viewing or printing as a batch.
The advanced search form [17] was JavaScript enabled with key enhancements that included (1) dynamic population of the cancer subtype/stage search options based on cancer type selection, and (2) expanded trial site and location searching, including searches by zip code proximity and hospital. In addition, browse lists for drugs, hospitals, and investigators were added to support more precise searching. Users could search for a character string and find appropriate values to add to the search form, or they could browse data-generated pick-lists alphabetically for drug, hospital, or investigator.
In addition, for the CIS users, the search results display was developed to enable information specialists to read a preliminary result set, so they could identify the most appropriate trials for their callers and prepare an “information packet.”
Better Visibility for Search Forms
The 2004 redesign of the NCI website saw further changes in clinical trials searching. Based on user input, the ability to narrow a search to subtype or stage of cancer was added to the basic search form. User feedback also indicated that physicians preferred trials to be listed by phase rather than by title, so the default display of search results was changed to a listing by phase, with phase IV and phase III trials appearing before phase II and phase I trials. The most substantial change, as a result of the Clinical Trials portal review, was adding the basic search form to the top of the Clinical Trials portal home page to give more ready access to the form [15]. With continued feedback from users, the search forms will be improved further to allow more precise clinical trial searching—for example, an interactive format may be developed to help identify trials with eligibility criteria that match patient characteristics.
Patient-Oriented Clinical Trial Abstracts
In the summer of 1996, NCI collaborated with the National Alliance of Breast Cancer Organizations (NABCO) to develop patient-oriented abstracts (summaries) of clinical trial protocols for breast cancer trials. By October 1996, these clinical trial abstracts were available on the NABCO and CancerNet websites in a one-paragraph format. After seeking input from many advocacy organizations, the patient-oriented clinical trial abstract format was redesigned, writing guidelines were developed, and the project was expanded to include all cancer types. By September 1998, patient-oriented abstracts for all active clinical trials were available on CancerNet. Since that time, clinical trial abstracts have been written according to the original guidelines.
In November 2001, selected patient-oriented and corresponding health professional clinical trial abstracts were evaluated. As a result of this evaluation, several problems were identified in the guidelines for writing the patient-oriented abstracts, including a lack of specificity in some respects and inconsistent application and interpretation of the guidelines. These findings led to the recommendation that the guidelines be redefined and expanded. Consequently, a qualitative and quantitative evaluation of the needs and preferences of users of the patient-oriented abstracts was undertaken. This evaluation included the following two elements: (1) a written survey of advocacy organizations, members of NCI's Consumer Advocates in Research and Related Activities (CARRA) Program, members of the NCI Director's Consumer Liaison Group (DCLG), comprehensive cancer center directors and administrators, cancer cooperative group chairs/administrators, and oncology nurses; and (2) in-depth interviews with CIS information specialists.
Written Survey
A 10-question survey was mailed to nearly 400 organizations and individuals, with a 43% return rate. A key question focused on whether or not users could understand and act on the information provided in the clinical trial abstracts for patients. Results showed that 82% of users could explain the rationale or purpose of the clinical trial, 93% could determine if basic eligibility requirements were met, and 73% could understand the treatment plan.
The organization and layout of the clinical trial abstracts were rated “excellent” or “good” by 72% of the respondents. Three samples of text written at 5th-, 8th-, and 12th-grade reading levels were included with the survey. The different reading levels were preferred by 37%, 42%, and 20% of the respondents, respectively.
The results of the survey were better understood when viewed in the context of comments from individual respondents. Taken as a whole, the respondents' comments were varied and, at times, contradictory. Several themes, however, emerged related to language and readability, access to other resources, and pursuing participation in a clinical trial. Although 27% of the respondents indicated they could not understand the treatment plan, few specific suggestions were offered for improvement.
Interviews with CIS Information Specialists
Structured interviews were conducted with staff in six CIS offices in different geographic areas of the United States in order to obtain their perceptions of users' needs, preferences, and comprehension of the standard elements (title, rationale, purpose, eligibility criteria, treatment, and study contacts) of the patient-oriented clinical trial abstracts. The CIS information specialists interact directly with users of the abstracts by answering their questions and by guiding their use of the abstracts online during a phone call or through LiveHelp. The information specialists emphasized the need to use consumer-oriented language and the fact that users “skip” disclaimer-type information.
Based on these findings and on published principles [18], improvements to the patient-oriented abstracts were implemented as part of the 2004 redesign of the NCI website (Table 3). An example of the current abstract format [19] can be viewed online.
Table 3
Selected improvements to the patient-oriented clinical trial abstracts
Criteria
Improvement
Use of Language
Provide both simplified and health professional versions of the title. Avoid technical terms if a more common term is available (eg, “removed in surgery” instead of “resected”). Aim for an 8th-grade reading level or lower, except for drug names and medical or scientific terms defined in the website's dictionary (terms are linked to dictionary definitions).
Readability
Write sentences that are as short as the content will allow. Divide lengthy treatment descriptions into smaller paragraphs. Use bullets to separate information about different treatments.
Content Display
Emphasize how users who are interested in participating in a clinical trial can seek further information. Incorporate disclaimer information into the eligibility and trial contact information sections. Provide a boxed sidebar containing links to complementary information about clinical trials and drug information in the National Library of Medicine's MedlinePlus. Keywords in the title should not be linked to dictionary definitions. They should be linked from the purpose or treatment sections rather than the title.
Web-Friendly Cancer Information Summaries for Patients
The PDQ cancer information summaries are descriptions of the latest cancer information on treatment, supportive care, screening, prevention, genetics, and complementary and alternative medicine that are reviewed and updated monthly by cancer experts. Most of the summaries are available in two versions: one written for health professionals and a corresponding patient version written in lay language. (A small number of the summaries are available only in the health professional version.) In 2000, in response to the Chantilly meeting, work was initiated to reformat the patient-oriented information summaries. The goal was to present the information in a format and style of language that was easier to read and understand, to provide more detailed information, and to take advantage of features afforded by new Web technology.
Based on design concepts that enhance readability, as well as on strategies used in information mapping, the process of reformatting and reorganizing the patient-oriented summaries was begun. “Key Points” boxes that highlighted critical concepts and linked to explanatory information in the body text were added. Links to pop-up definitions from the website's dictionary and to clinical trials information were included. For users who wished to print documents, a printer-friendly version was added that included dictionary terms and their definitions as an appended glossary.
Usability testing was done to assess the ease of learning, efficiency in information gathering, and recall of information from the online documents. Based on testing results and Web design and usability guidelines [18], the template for the patient-oriented summaries was further refined, and the redesign has been well received by users. An example of the current summary format [20] can be viewed online.
Conclusion
NCI's website is a leading resource for cancer information on the Web, consistently appearing high on the list of retrievals using search engines such as Google, Yahoo, MSN, and AltaVista. It has been awarded the Freddie Award in the website category of the 2004 International Health and Medical Media Awards, and it placed first or as an honorable mention in seven out of eight categories in the 2005 Medicine on the Net Web Excellence Awards. Its success can be at least partly attributed to NCI's efforts to make the site highly responsive to the needs of its users.
The large volume of traffic that the site receives offers tremendous opportunities to study user patterns, gather feedback, and test new ideas and designs. Online surveys are an efficient way to solicit opinions from users, and analysis of website logs provides insight into user needs. NCI's relationships with members of the cancer research and advocacy communities also facilitate the gathering of advice, suggestions, and other feedback related to NCI information products. The growing body of Web-design literature and advice from usability experts are important to the development of new Web features, but input from the site's wide range of users promises to have the greatest impact on shaping online information from the National Cancer Institute.

online information
internet
cancer information
website
usability