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DEVELOPMENTAL DYNAMICS 213:39–49 (1998) Graded Retinoid Responses in the Developing Hindbrain S.F. GODSAVE, C.H. KOSTER, A. GETAHUN, M. MATHU, M. HOOIVELD, J. VAN DER WEES, J. HENDRIKS, AND A.J. DURSTON* Netherlands Institute for Developmental Biology, Utrecht, The Netherlands ABSTRACT The purpose of this study was to make an explicit test of the idea that a retinoid could act as a morphogen, differentially activating genes and specifying anteroposterior (a-p) level in the developing vertebrate central nervous system (CNS). Our approach was to characterize the concentration-dependent effects of retinoic acid (RA) on the neural expression of a set of a-p patterning genes, both in vivo and in an in vitro system for neural patterning. Our results indicate that a retinoid is unlikely to specify a-p level along the entire CNS. Instead, our data support the idea that the developing hindbrain may be patterned by a retinoid gradient. Sequentially more posterior hindbrain patterning genes were induced effectively by sequentially higher RA concentration windows. The most posterior CNS level induced under our RA treatment conditions corresponded to the most posterior part of the hindbrain. Dev. Dyn. 1998;213:39–49. r 1998 Wiley-Liss, Inc. Key words: retinoids; Hoxb genes; Xenopus; anteroposterior patterning INTRODUCTION There is evidence that anteroposterior (a-p) axis formation in the vertebrate central nervous system (CNS) occurs in two steps (for review, see Durston et al., 1998). A neural induction or activation signal in the gastrula first induces competent ectoderm to develop into anterior neural tissue. A transformation signal then converts the posterior part of the newly induced anterior neuroectoderm into presumptive hindbrain and spinal cord. Neural transformation activity is present as a gradient in the gastrula and in early neurula-stage amphibian embryo. High levels of transforming activity induce spinal cord, and lower levels induce hindbrain (Nieuwkoop et al., 1952). Retinoids have been proposed as candidates for the neural transformation signal in early embryos (for review, see Durston et al., 1998). Retinoic acid (RA) and several other acidic retinoids are potent teratogens that can suppress development of the forebrain and other anterior structures and can cause enlargement of the posterior CNS (Durston et al., 1989; Sive et al., 1990; Papalopulu et al., 1991; Ruiz i Altaba and Jessell, 1991; Pijnappel et al., 1993; Simeone et al., 1995; Blumberg et al., 1996). Active retinoids have been identified in Xenopus gastrulae while neural induction and a-p r 1998 WILEY-LISS, INC. patterning of the CNS are in progress (Durston et al., 1989; Pijnappel et al., 1993; Chen et al., 1994; Creech Kraft et al., 1994; Blumberg et al., 1996). They are reported to be concentrated in the late gastrula organizer (source of transformation signals) and to be present as a posterior-to-anterior concentration gradient in the neurula (Chen et al., 1994). A molecular basis for retinoid posteriorization is indicated by the finding that some posterior neural patterning genes, including some of the Hox genes, are direct retinoid targets through enhancers that contain RA response elements (Langston and Gudas, 1992; Pöpperl and Featherstone, 1993; Marshall et al., 1994; Studer et al., 1994; Frasch et al., 1995; Ogura and Evans, 1995a,b; Doerksen et. al., 1996; Morrison et al., 1996; Dupé et al., 1997; Langston et al., 1997). The purpose of the present study was to test whether a retinoid gradient could mediate neural transformation, such that sequentially higher retinoid concentrations specify sequentially more posterior zones in the developing neural plate through differential regulation of a-p patterning genes. Surprisingly, this idea has never been tested explicitly before, despite a large number of publications about retinoid effects on development. We therefore made a systematic investigation of the effects of the active retinoid, RA, on early neural patterning. Our results indicated that RA does not modulate the whole of neural transformation, but that an RA-like retinoid may be involved in two separable aspects of the transformation process. RA can transform presumptive forebrain and midbrain into hindbrain and can also modulate hindbrain patterning. We detected an anterior-to-posterior gradient of RA sensitivity within the developing hindbrain, suggesting that a retinoid gradient may pattern this part of the CNS in vivo. RESULTS AND DISCUSSION The idea that a retinoid gradient could mediate CNS patterning was tested by characterizing the effects of a range of RA concentrations on the neural expression of a set of a-p patterning genes (for details, see Experimental Procedures). If a retinoid gradient does convey Grant sponsor: Netherlands Science Organization Life Sciences; Grant number: 805–33–021; Grant number: 417442; Grant sponsor: EU Biotech Programme; Grant number: ERBBIO4CT960378. *Correspondence to: A.J. Durston, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. Received 24 February 1998; Accepted 20 May 1998 40 GODSAVE ET AL. Fig. 1. Effect of retinoic acid (RA) treatments on expression of Otx-2, En-2, and Krox-20 in whole tailbud stage embryos as shown by in situ hybridization. A: Expression of otx-2 in the forebrain and midbrain of control embryos. There is also some expression in other anterior structures and associated with the otic vesicles (o). B,C: Expression of Otx-2 in stage 29–31 tailbud embryos following RA treatment beginning at the early gastrula stage: 1027 M RA (B) and 1026 M RA (C). Note the smaller expression domain in B compared with the control (A). However, very anterior structures, including nasal pits, still developed in these embryos (data not shown). D: Expression of en-2 at the midbrain-hindbrain border in untreated embryos. E,F: Expression of en-2 in stage 29–31 embryos following addition of 1027 M RA (E) and 5 3 1027 M RA (F) to the culture medium at stage 10. In E, note the extended domain of en-2 expression along the anteroposterior (a-p) axis and the lack of transcripts dorsally in the brain. There is no localized expression of otx-2 (C) or en-2 (F) following treatment with 2.5 3 1027 M (not shown) or higher RA concentrations. G–I: Krox-20 expression in tailbud stage embryos. Control embryo is shown in G. Expression is visible in rhombomeres 3 and 5 (r3 and r5) and in r5-associated neural crest. H shows an embryo that was treated with 1028 M RA: The two Krox-20 stripes are broader and closer together. In I, an embryo that was treated with 1027 M RA shows only one clear stripe of Krox-20 expression. This is presumably the r5 stripe, because it is associated with Krox-20 positive neural crest cells. Krox-20 expression is undetectable in embryos treated with 2.5 3 1027 M RA or higher (not shown). positional information, then we would expect increasing concentration windows of RA to activate the expression of increasingly more posterior markers. These experiments employed whole embryos as well as an in vitro system for CNS patterning, as described below. Anterior development was inhibited, and the expression of the forebrain and midbrain markers, Otx-2 and En-2, as well as hindbrain markers back to Hoxb-3 (rhombomeres 5 and 6; r5 and r6) could be downregulated by RA (for details, see Figs 1, 2, Table 1). The posteriorization caused by RA was mediated at least partly by posterior transformation of the developing CNS rather than just anterior deletion. We note, for example, that repression of anterior markers by increasing concentrations of RA (up to 2.5 3 1027 M) was accompanied by sequential extension of the expression zones of Hoxb-3 and Hoxb-4 until these reached the front end of the embryo. This indicates that the presumptive anterior CNS is transformed entirely to a state resembling posterior hindbrain by high concentrations of RA. In contrast to its action on presumptive forebrain, midbrain, and hindbrain domains, continuous RA treat- Neural Transformation Is Not Simply Mediated by a Retinoid Gradient RA treatment of whole embryos posteriorized the developing CNS, as reported previously (for review, see Durston et al., 1998). This RA-induced posteriorization shows some resemblances to the natural neural transformation process, but it also shows striking differences. In situ hybridization to markers for different levels along the developing CNS (shown and described in detail in Figs. 1 and 2) revealed that markers for the forebrain, midbrain, and hindbrain are RA sensitive. GRADED RETINOID RESPONSES IN THE HINDBRAIN Figure 2. 41 42 GODSAVE ET AL. ment had relatively little effect on the expression domains of 58 Hoxb genes in the spinal cord. High RA concentrations (2.5 3 1027 M to 1026 M) caused posterior truncations and a corresponding shortening of the 58 Hoxb gene expression domains, but lower concentrations had no detectable effect, and the effects of RA were generally much less than on hindbrain development (Fig. 2). These data match previous findings in Xenopus and mouse embryos that RA has little effect on neural Hoxb-9 expression (Conlon and Rossant, 1992; Dekker et al., 1992b). They are also consistent with findings from loss-of-function experiments using vitamin A deficiency (Maden et al., 1996) or dominant-negative retinoid receptors (Kolm et al., 1997; van der Wees et al., 1998; but see also Blumberg et al., 1997) in emphasizing that retinoids are important for development of hindbrain rather than spinal cord. This characteristic contrasts with the nature of the natural neural transformation signal, which induces spinal cord at the highest intensities (Nieuwkoop et al., 1952; Sala, 1955). We conclude that neural transformation is likely to be mediated by a complex of different signals and that retinoids could mediate formation and patterning of the hindbrain part of the developing CNS. It seems likely, however, that the retinoid responses of neural markers are somewhat condition dependent, because RA responses have been reported in other studies for the expression of relatively 58 posterior Hox genes (IzpisuaBelmonte et al., 1991; Sharpe, 1991; Conlon and Rossant, 1992; Dekker et al., 1992b; López and Carrasco, 1992). We observed sharp RA thresholds for inhibition of the development of particular a-p zones. For example, the forebrain and midbrain marker, Otx-2, was expressed in an almost normal pattern following treatment with 1027 M RA or less, but was totally undetectable following treatment with 2.5 3 1027 M RA or more. Similarly sharp thresholds were observed for repression of other markers (Figs. 1, 2, Table 1). The pattern of the thresholds that was observed, as described below, sug- Fig. 2. (Previous page.) A–U: Effect of RA treatments on Hoxb gene expression in whole embryos, as shown by wholemount in situ hybridization. Expression patterns are shown of Hoxb-1 (A), Hoxb-3 (E), Hoxb-4 (I), Hoxb-5 (M), Hoxb-7 (P), and Hoxb-9 (S) in untreated, tailbud stage 29–31 embryos. B–D, F–H, J–L, N, O, Q, R, T, and U show embryos that were treated with RA from stage 10 and fixed at stage 29–31. B–D: Hoxb-1 expression in embryos treated with 1027 M RA (B), 5 3 1027 M RA (C), and 1026 M RA (D). Note that the expression of Hoxb-1 occurs adjacent to the otic vesicle (o) in both control (A) and 1027 M RA-treated embryos (B). Higher levels of RA (2.5 3 1027 M or more) caused a loss of localized Hoxb-1 expression. This was also the case when embryos were given 30-min pulse treatments with high concentrations of RA (not shown). F–H: Hoxb-3 expression following treatment with 1027 M RA (F), 5 3 1027 M RA (G), and 1026 M RA (H). J–L: Hoxb-4 transcripts in embryos treated with 1027 M RA (J), 5 3 1027 M RA (K), and 1026 M RA (L). RA causes Hoxb-3 and Hoxb- 4 to be expressed in extended domains along the a-p axis. This is already evident in embryos treated with 1028 M RA (not shown) and occurs to increasingly greater extents with increasing RA concentrations. This effect is correlated with a loss of rhombomeric segmentation in the hindbrain. In F, the strongest Hoxb-3 expression is anteriorized by 1027 M gested that retinoid effects on the hindbrain and on the forebrain and midbrain occur through separable mechanisms. A Gradient of RA Sensitivity in the Developing Hindbrain We detected anterior-to-posterior sequences of repression and activation in the developing hindbrain. The most striking feature in vivo was an anterior-toposterior sequence of RA repression thresholds. Embryos treated with less than 1027 M RA expressed all hindbrain markers. At 1027 M RA, the r3 stripe of Krox-20 was absent, but all other hindbrain markers remained. At 2.5 or 5 3 1027 M RA, Hoxb-1 (r4) and the posterior stripe of Krox-20 (r5) were also repressed. At 1026 M RA, Hoxb-3 (r5 and r6) was also repressed. No RA inhibition was ever seen for Hoxb-4 (r7 and r8) or for more posteriorly expressed genes. It is notable that this sequence of hindbrain inhibition thresholds begins at an RA concentration (1027 M) that is lower than the repression threshold for forebrain and midbrain development, suggesting that these aspects are separable (Figs. 1, 2, Table 1). Along with repressing gene expression, RA induced expression of hindbrain markers (but not of forebrain or midbrain markers) in vivo. This was especially obvious for Krox-20, Hoxb-3, and Hoxb-4 in whole embryos (Figs. 1, 2; see also Conlon and Rossant, 1992). Low RA concentrations have also been shown to induce anterior expansion of Hoxb-1 expression in murine and chicken embryos (Morriss-Kay et al., 1991; Conlon and Rossant, 1992; Marshall et al., 1992; Sundin and Eichele, 1992; Wood et al., 1994; Simeone et al., 1995). We did not see this in our experiments, possibly because induced Hoxb-1 expression was too diffuse to be detected by in situ hybridization, although this was detectable by RNAse protection (see Fig. 3). RA-mediated induction was also obvious for a range of markers in explants in vitro (see below). RA, so that it overlaps with Hoxb-1 expression adjacent to the otic vesicle. There are also dispersed spots of expressing cells visible anterior to the otic vesicle and weak staining in the post-otic hindbrain. In embryos that were treated with 2.5 3 1027 M RA (not shown) or with 5 3 1027 RA (G), Hoxb-3 expression extends up to the anterior end of the brain. No localized Hoxb-3 expression is visible in H. In J, strong Hoxb-4 expression is anteriorized by 1027 M RA so that it extends up to the otic vesicle, and weaker expression continues more anteriorly. In K and L and in embryos that were treated with 2.5 3 1027 M RA (not shown), expression extends up to the anterior end of the central nervous system (CNS). Note that somites continued to express Hoxb-4 in RA-treated embryos. N,O: Effect of 1027 M RA (N) and 1026 M RA (O) on Hoxb-5 expression. Q,R: Effect of 1027 M RA (Q) and 1026 M RA (R) on Hoxb-7 expression in embryos. T,U: Effect of 1027 M RA (T) and 1026 M RA (U) on Hoxb-9 expression. High concentrations of RA caused tail truncations and a corresponding shortening of the 58 Hoxb gene expression domains. The spinal cord also appears somewhat thickened (O,R,U). There was no clear anteriorization of the 58 Hoxb genes, Hoxb-5 through Hoxb-9, within the hindbrain in these embryos. GRADED RETINOID RESPONSES IN THE HINDBRAIN 43 TABLE 1. Concentration-Dependent Effects of Retinoic Acid on Anteroposterior Marker Expression in Whole Embryos: Summary of In Situ Hybridization Dataa aThe left hand column is a schematic representation of the central nervous system (CNS). The zones where each marker gene is expressed most strongly in untreated embryos are shown in the second column headed ‘‘Control.’’ For simplicity, Hoxb-3 (strongest expression in rhombomeres 5 and 6; r5 and r6) is shown adjacent to r6, and Hoxb-5 and Hoxb-7 (anterior expression border in the posterior hindbrain) are shown adjacent to the spinal cord. The effects of different concentrations of retinoic acid (RA) on marker gene expression in whole embryos (see Figs. 1 and 2) are shown in the right hand set of columns as follows: 1, expression detectable in the CNS; 11 and 111, expression in an extended zone along the CNS; 2, no expression detectable in the CNS. fb, Forebrain; mb, midbrain; hb, hindbrain; r1–r8, rhombomeres 1–8; SC, spinal cord. RA induction obviously occurred at concentrations below those required for repression of the same genes, indicating RA concentration windows for inducing gene expression. We note further that extended gene expression zones, as observed here, are a prediction of gradient models in which anterior and posterior gene expression boundaries are set by activation and inhibition threshold concentrations of a morphogen and in which ectopic addition of a signaling molecule causes flattening of the gradient. The phenotypes described above were observed at tailbud stage 29–31, when morphological markers facilitated identification of a-p level. Because RA has teratogenic effects at much earlier stages, we examined the timing of RA effects on gene expression. RA-induced changes in gene expression occurred early and were stable; they were very similar in embryos that were harvested at the early neurula stage (Fig. 4, stage 14) and in larvae harvested at stage 29–31 (described above). Fig. 3. RNase protection analysis of Otx-2 and 38 Hoxb gene expression in fragments of control and RA-treated embryos. Our in situ hybridization data showed that high concentrations of RA abolished hindbrain-localized expression of Hoxb-1 and Hoxb-3. Similar treatments were previously shown by RNase protection analysis to cause increased expression of these genes (Dekker et al., 1992a,b; Leroy and De Robertis, 1992). Here, we investigated the localization of RA-induced transcripts. RNA from untreated (CON) tailbud (stage 28) embryos and from embryos that were treated with 1026 M RA from stage 10 onward (RA) was analyzed for expression of Otx-2 (otx), Hoxb-1 (b1), and Hoxb-3 (b3). Xom 62/9 (xhom) was used as an internal control for the loading. The RNA samples analyzed were from whole embryos (WE) and from embryos that were cut into three fragments: an anterior piece (Ant) that included all tissues up to the level of the hindbrain/spinal cord border, a posterior dorsal piece (PD) that was cut just ventral to the somites, and the remaining posterior ventral structures (PV). Otx-2 expression was restricted to the anterior fragment in control embryos and was absent in RA-treated embryos. In control embryos, Hoxb-1 and Hoxb-3 were expressed most strongly in the fragments that included dorsal tissue both in the fragment containing hindbrain (Ant) and in the more posterior dorsal fragment (PD). There was also very weak expression in the posterior ventral fragment, in which bands were clearly visible only after longer exposure of the autoradiograph. In RA-treated embryos, there was a clear up-regulation of both Hoxb-1 and Hoxb-3 in all fragments, suggesting that there is widespread, RA-inducible expression that is not detectable by using in situ hybridization. The effects of RA on whole embryos are complex, and RA induction is difficult to detect against the background of endogenous gene expression. We therefore used an in vitro system to examine RA posteriorization of developing anterior neural tissue directly: animal caps from embryos injected with mRNA for the anterior neural inducer noggin (Lamb et al., 1993). The results were assayed by using RNAse protection assays (Fig. 5) and with wholemount in situ hybridization (Fig. 6). We found that noggin injection induced expression of the general neural marker N-CAM. These neutralized explants also expressed the forebrain marker Otx-2 but not six (hindbrain and/or spinal cord expressed) Hox genes that were examined. This supports previous conclusions that noggin-induced neurectoderm has anterior specification (Lamb et al., 1993). RA treatment of these explants down-regulated Otx-2 but not N-CAM, 44 GODSAVE ET AL. Fig. 4. Effect of RA treatment at the early gastrula stage on Hoxb-3 expression in early neurula stage embryos, as shown by wholemount in situ hybridization. A: Hoxb-3 transcripts in a control stage 14 embryo. B,C: Hoxb-3 expression in stage 14 early neurulae following treatment at the early gastrula stage (stage 10) with 1027 M RA (B) and with 1026 M RA (C). Anterior is to the left in all cases. The expression patterns are very similar to those seen at stage 29–31 (Fig. 2). RA-induced effects on the Hoxb-1, Hoxb-4, and Hoxb-9 expression patterns also showed very little change between the early neurula stage and the tailbud stage (not shown). and induced expression of some Hox genes, confirming previous findings that RA posteriorizes noggin explants (Papalopulu and Kintner, 1996). All Hox genes expressed predominantly in the hindbrain (Hoxb-1, Hoxb-3, Hoxb-4) were strongly inducible in noggin explants by continuous RA treatment. Hoxb-5, which is expressed in the posterior hindbrain as well as the spinal cord, was also inducible. Two other 58 posterior Hox genes were far less inducible. Hoxb-7 was not induced at all, and Hoxb-9 was induced at a low level. Intriguingly, the apparent cut-off point in inducibility found here, between Hoxb-5 and Hoxb-7, coincides with the division between the Antennapedia and Bithorax Fig. 5. RA-induced activation of Hoxb gene expression in noggininjected animal caps. RNase protection assays were used to analyze the expression of otx-2 (Otx), neural cell adhesion molecule (NCAM), and six of the Hoxb genes: Hoxb-1 (b-1), Hoxb-3 (b-3), Hoxb-4 (b-4), Hoxb-5 (b-5), Hoxb-7 (b-7), and Hoxb-9 (b-9) in noggin-injected (Noggin AC) and control (NIC AC) animal caps in the absence of RA (C) or following addition of 1026 M RA (6), 1027 M RA (7), or 1028 M RA (8) at the early gastrula stage (stage 10). Animal caps were dissected out at stage 10, and the caps and control whole embryos (WE) were harvested at stage 23–25 for analysis. The data presented were obtained from a number of experiments. Expression of each gene was tested in at least two experiments, and the results obtained were consistent. Otx-2 was expressed in noggin-injected animal caps and was also detectable in control caps (see also Lamb and Harland, 1995; Itoh and Sokol, 1997), and, in both cases, this expression was down-regulated by RA. No Hoxb gene expression was detectable in non-RA-treated animal caps. However, expression of Hoxb-1, Hoxb-3, Hoxb-4, and Hoxb-5, but not of Hoxb-7 or Hoxb-9, was detectable following exposure of noggin-induced caps to 1028 M RA. The expression levels of the induced genes increased with exposure to increasing concentrations of RA. Hoxb-4 expression was induced to levels several-fold higher than expression of the other RA-inducible genes. Low levels of Hoxb-9 expression were detectable in noggin-induced animal caps that were treated with high concentrations of RA. Activation of Hox gene expression also occurred in control animal caps in response to RA (see also Kolm and Sive, 1995). Hoxb-1 through Hoxb-5 were expressed in animal caps that were treated with 1026 M RA. Only noggin-injected animal caps expressed the neural marker N-CAM, and expression levels were increased by the addition of RA. complexes in Drosophila (Akam, 1989; Duboule and Dollé, 1989; Kappen and Ruddle, 1993). The five RA-inducible hindbrain Hox genes showed a colinear, anterior-to-posterior sequence of concentra- Fig. 6. A–Q: RA-mediated transformation in vitro. Wholemount in situ hybridization analysis of animal cap explants. Control, whole, stage 26 embryos were hybridized with antisense probes for Otx-2 (A), En-2 (B), and Hoxb-4 (C). D–O: Animal cap explants taken at stage 10 from noggin-injected, fertilized eggs and cultured to stage 26. D–F: Expression of Otx-2 (D), en-2 (E), and Hoxb-4 (F) in explants cultured in the absence of RA. Only Otx-2 transcripts were detected in the noggin-induced explants. The level of Otx-2 expression was very variable, but a high percentage of explants (22/30; 73%) contained detectable Otx-2 transcripts. In D, only the explant marked with an asterisk was unstained. G–I: Expression of Otx-2 (G), en-2 (H), and Hoxb-4 (I) in noggin-expressing explants that were treated with 1027 M RA from stage 10 onward. Expression of Otx-2 was down-regulated in many explants (only 9/24 showed detectable Otx-2 expression; 37%), but no induction of en-2 or Hoxb-4 was evident. We also assayed for Hoxb-3 and Hoxb-9 expression in RA-treated explants. However, no expression was detected at any of the RA concentrations tested. The in situ hybridization technique appears to be less sensitive than RNAse protection assays, in which 38 Hoxb gene expression was already detectable in explants treated with low concentrations of RA. J–L: Expression of Otx-2 (J), en-2 (K), and Hoxb-4 (L) following treatment with 5 3 1027 M RA. There was a further decrease in the number of Otx-2-expressing explants at this RA concentration to 3/23 (13%), and there was an induction of Hoxb-4 expression in a low percentage of cases (4/22; 18%). In L, Hoxb-4 expression is visible (arrow) in one of the explants. M–O: Expression of Otx-2 (M), en-2 (N), and Hoxb-4 (O) following treatment with 1026 M RA. Only Hoxb-4 is expressed detectably. A very high percentage of explants were Hoxb-4 positive (21/23; 91%). The expression in many of the explants appears to be at a much higher level than in the CNS of control embryos. This expression was apparently unpatterned in contrast to the effects of fibroblast growth factor (FGF) on noggin-treated, anterior neural tissue (Cox and Hemmati-Brivanlou, 1995; Lamb and Harland, 1995). P: Animal caps dissected from uninjected embryos and hybridized to an antisense Otx-2 probe. No transcripts were detectable. Q: Animal caps dissected from uninjected embryos and treated with 1026 M RA did not hybridize to a Hoxb-4 probe at detectable levels. 46 GODSAVE ET AL. Fig. 7. Hoxb-1 through Hoxb-5 are induced colinearly by RA in noggin-injected animal caps: Hoxb-1 is induced to 50% of its maximal expression level by a lower concentration of RA than are successively more 58 Hoxb genes. Hoxb gene transcript levels were determined relative to those of the internal control, Xom 62/9, by phosphorimager analysis of RNase protection assay gels (see Fig. 5). Expression levels induced by 1026 M RA were taken to represent 100% expression. Expression levels of Hoxb-7 and Hoxb-9 were undetectable and very low, respectively, and are not included here. Fig. 8. RA concentration-dependent induction of Hoxb gene expression in noggin-injected animal caps. Phosphorimager analysis of RNase protection gels (see Fig. 5) was used to determine the expression levels of six Hoxb genes relative to the expression of the internal standard, Xom 62/9. The total expression units are arbitrary. Hoxb-7 and Hoxb-9 show very little response to the RA treatment. However, there is a partial colinearity in the degree of induction by 1026 M RA of the more 38 Hoxb genes. Hoxb-1 is induced the least, and progressively more 58 genes are induced to progressively higher levels, with the exception that Hoxb-4 is induced to much higher levels than all of the other Hoxb genes tested. tion dependences for induction. Hoxb-1 (r4) was induced to 50% maximal expression by 2.6 3 1028 M RA, Hoxb-3 (r5 and r6) was induced by 2.0 3 1027 M, Hoxb-4 (r7 and r8) was induced by 2.1 3 1027 M, and Hoxb-5 (r8; spinal cord) was induced by 3.5 3 1027 M (Fig. 7). Besides colinearity in the RA-concentration-dependent half maximal induction of Hox genes, we observed that the maximal levels of induction were successively greater for successively more 58 RA-inducible Hoxb genes. Hoxb-4 expression could be induced to levels several fold higher than expression of the other induced Hoxb genes (Fig. 8). It is not yet known how different combinations and expression levels of Hox genes affect a-p-specification. Our findings suggest, however, that an integral, RAsensitive mechanism patterns the hindbrain. Toward a Model for Retinoid Patterning of the Hindbrain The data described above show that a retinoid may be involved in an early subdivision of the CNS, transforming presumptive anterior CNS into neural tissue with hindbrain specification. Subsequently, a gradient of a retinoid morphogen may be involved in providing an informational code for a-p level in the developing hindbrain, because different hindbrain patterning genes are induced and repressed by different RA concentrations that correlate with their axial expression levels. GRADED RETINOID RESPONSES IN THE HINDBRAIN 47 The data suggest the existence of sequentially higher RA concentration windows for induction of sequentially more posterior hindbrain genes, resembling the induction of successively more dorsoanterior markers by successively higher concentration windows of activin (Green et al., 1992, 1994). Retinoid patterning activity in the hindbrain is likely to be mediated at least in part through direct interactions of retinoid receptors with Hox genes. RA response elements have been found in the vicinity of several paralogue group 1 and group 4 Hox genes (Langston and Gudas, 1992; Pöpperl and Featherstone, 1993; Marshall et al., 1994; Studer et al., 1994; Frasch et al., 1995; Ogura and Evans, 1995a,b; Doerksen et al., 1996; Morrison et al., 1996; Dupé et al., 1997; Langston et al., 1997). The question arises whether a gradient of endogenous active retinoid that could pattern the developing hindbrain is available in vivo during embryogenesis. At the gastrula and early neurula stages, when neural Hox gene expression patterns are established, Xenopus embryos contain active retinoids at physiologically relevant concentrations (Chen et al., 1992, 1994; Pijnappel et al., 1993; Creech Kraft et al., 1994; Blumberg et al., 1996). It has also been reported that the late (posterior) organizer regions of vertebrate embryos (chicken, mouse, and Xenopus) are sources of active retinoids (Chen et al., 1992, 1994; Hogan et al., 1992) and that Xenopus embryos contain a tenfold posterior-to-anterior concentration gradient of an active retinoid at the early neurula stage (Chen et al., 1994). The RA concentrations at which different hindbrain patterning genes were induced or repressed in Xenopus embryos or explants in this study (1028 to 1026 M) are high, suggesting that RA itself is not the morphogen in vivo. However, there are selectively active retinoids that are available in early Xenopus embryos and that posteriorize the early embryo with very high efficiency (Pijnappel et al., 1993; unpublished observations). ously (Godsave et al., 1994). RA (Acros) was prepared as a 1022 M stock in dimethyl sulfoxide (DMSO) and stored at 280°C in small aliquots. Twenty to thirty embryos were exposed to RA continuously from stage 10 onward in the dark in 7 ml tap water containing 25 (g/ml gentamycin and a range of concentrations of RA (from 1028 M to 1026 M). Concentrations higher than 1026 M were toxic. Continuous exposure to RA was used to ensure that the treatment lasted for the entire period of a-p patterning in the CNS. In most cases, the effects on gene expression were analyzed at tailbud stage 29–31, when morphological landmarks are available to facilitate identification of gene expression zones. Embryos were demembranated manually and fixed for wholemount in situ hybridization in MEMFA (Harland, 1991) for 2 hours. They were washed once in 100% methanol and stored at 220°C in fresh methanol. Embryos used for RNAse protections were frozen in liquid nitrogen and stored at 280°C. General Conclusions In Situ Hybridization In conclusion, our data show that RA can differentially regulate genes of the Hoxb cluster in neural tissue. RA concentration-dependent induction of Hoxb and other a-p patterning genes suggest a mechanism whereby retinoids can contribute to setting up a series of expression zones of genes involved in providing a-p positional information. It is clear that patterning of the main body axis occurs in several steps and that retinoids may be involved at several stages. It is also clear that other signals are required to generate the complete range of axial pattern. The Xenopus embryo is an excellent model system for further investigations in this area. The in situ hybridization probes used were Otx-2, a gene that is expressed in the forebrain and midbrain (Fig. 1A; Blitz and Cho, 1995; Pannese et al., 1995); en-2, a marker of the midbrain-hindbrain border (Fig. 1D; Hemmati-Brivanlou et al., 1991; Eizema et al., 1994); Krox-20, which is expressed in r3 and r5 of the hindbrain (Fig. 1G; Bradley et al., 1992); and six of the Hoxb genes, Hoxb-1, Hoxb-3, Hoxb-4, Hoxb-5, Hoxb-7, and Hoxb-9 (Fig. 2A,E,I,M,P,S; Godsave et al., 1994; Godsave and Durston, 1997). Hoxb-1 through Hoxb-4 are expressed in sequential zones along the hindbrain. The rhombomeres in which expression of these Hoxb genes is strongest are r4 for Hoxb-1, r5 and r6 for Hoxb-3, and r7 and r8 for Hoxb-4. The anterior expression boundaries of Hoxb-5 and Hoxb-7 are near the posterior end of the hindbrain, and expression extends along the spinal cord, whereas Hoxb-9 expression is restricted to the spinal cord. EXPERIMENTAL PROCEDURES Embryos and RA treatments Xenopus embryos were obtained by in vitro fertilization, dejellied, cultured, and staged as described previ- Explants Fertilized eggs were dejellied, transferred into 3% ficoll in 25% MMR solution (Newport and Kirschner, 1982), and microinjected in the animal hemisphere with 150 pg noggin D58 mRNA (Smith and Harland, 1992). At the early blastula stage, embryos were transferred into 20% MMR and cultured overnight at 14°C to stage 10. The resulting early gastrulae were placed in Flickinger medium (Flickinger, 1949) containing 25 µg/ml gentamycin, the vitelline membranes were removed by using sharpened forceps, and animal caps were dissected out by using electrolytically sharpened tungsten needles. Explants were cultured individually in 1% agarose-coated wells of 96-well plates, in 100 µl Flickinger medium containing a range of concentrations of retinoic acid, at 20°C until control embryos reached stage 23–27. They were then fixed for in situ hybridization as for whole embryos or frozen in liquid nitrogen for RNAse protection analysis. 48 GODSAVE ET AL. Digoxigenin-labeled probes were generated by in vitro transcription of linearized templates, incorporating digoxigenin-11-UTP according to the manufacturer’s instructions (Boehringer-Mannheim, Indianapolis, IN). Antisense probes for Otx-2, En-2, Hoxb-3, Hoxb-4, and Hoxb-9 were as described in Godsave and Durston (1997). Antisense Krox-20 probe was as described by Bradley et al. (1992). Antisense Hoxb-1 probe was a 900-nucleotide (nt) sequence recognizing a part of the homeobox and more 58 coding sequence. The antisense Hoxb-5 probe was a 1,200-nt sequence complimentary to the complete coding region of the Hoxb-5 gene. The Hoxb-7 probe recognized a 300-nt coding sequence upstream from the homeobox. A subclone of XlHbox2 cDNA (Wright et al., 1987) was used as the template. A sense Hoxb-3 probe was used as control for nonspecific in situ hybridization staining (results not shown). The in situ hybridization procedure was as described by Harland (1991), with slight modifications as described by Godsave and Durston (1997). Pigmented embryos and explants were bleached following staining and fixation in MEMFA (see Godsave and Durston, 1997). RNase Protection Analysis RNA was isolated from embryonic material by using proteinase K, as described by Ruiz i Altaba (1993). RNAse protection assays were performed as described by Dekker et al. (1992a) by using a32P (UTP) to label the antisense RNA probes. The Otx-2 probe was that described by Pannese et al. (1995), the neural cell adhesion molecule (N-CAM) probe was that described by Mayor et al. (1995), and the Hoxb probes were those described by Dekker et al. (1992a). ACKNOWLEDGMENTS We are grateful to Richard Morgan and Pim Pijnappel for critical reading of the paper. We thank Richard Harland for the noggin-expression clone; Ken Cho and Maria Pannese for otx-2 in situ and RNAse protection clones, respectively; David Wilkinson for the Krox-20 clone; and Richard Morgan for the N-CAM clone. S.F.G. and J.v.d.W. were funded by Netherlands Science Organization (NWO) Life Sciences research grants 805–33– 021 and 417442, respectively. A.J.D. thanks the EU Biotech Programme (contract ERBBIO4CT960378) for support. REFERENCES Akam M. Hox and HOM: Homologous gene clusters in insects and vertebrates. Cell 1989;57:347–349. Blitz I, Cho KWY. Anterior neurectoderm is progressively induced during gastrulation: The role of the Xenopus homeobox gene orthodenticle. Development 1995;121:993–1004. Blumberg B, Bolado J, Derguini F, Craig AG, Moreno TA, Chakravarti D, Heyman RA, Buck J, Evans RM. Novel retinoic acid receptor ligands in Xenopus embryos. Proc. Natl. Acad. Sci. U.S.A. 1996;93: 4873–4878. Blumberg B, Bolado J, Moreno TA, Kintner C, Evans RM, Papalopulu N. An essential role for retinoid signaling in antero-posterior neural patterning. Development 1997;124:373–379. Bradley LC, Snape A, Bhatt S, Wilkinson DG. The structure and expression of the Xenopus Krox-20 gene: Conserved and divergent patterns of expression in rhombomeres and neural crest. Mech. Dev. 1992;40:73–84. Chen YP, Huang L, Russo AF, Solursh M. Retinoic acid is enriched in Hensen’s node and is developmentally regulated in the early chicken embryo. Proc. Natl. Acad. Sci. U.S.A. 1992;89:10056–10059. Chen Y, Huang L, Solursh M. A gradient of retinoids in the early Xenopus laevis embryo. Dev. Biol. 1994;161:70–76. Conlon RA, Rossant J. Exogenous retinoic acid rapidly induces anterior ectopic expression of murine Hox-2 genes in vivo. Development 1992;116:357–368. Cox WG, Hemmati-Brivanlou A. Caudalization of neural fate by tissue recombination and bFGF. Development 1995;121:4349–4358. Creech Kraft J, Schuh T, Juchau MR, Kimelman D. Temporal distribution, localisation and metabolism of all-trans-retinol, didehydroretinol, and all-trans retinal during Xenopus development. Biochem. J. 1994;301:111–119. Dekker E-J, Pannese M, Houtzager E, Boncinelli E, Durston A. Colinearity in the Xenopus Hox-2 complex. Mech. Dev. 1992a;40: 3–12. Dekker E-J, Pannese M, Houtzager E, Timmermans A, Boncinelli E, Durston A. Xenopus Hox-2 genes are expressed sequentially after the onset of gastrulation and are differentially induced by retinoic acid. Development 1992b;(Suppl.):195–202. Doerksen LF, Bhattacharya A, Kannan P, Pratt D, Tainsky MA. Functional interaction between a RARE and an AP-2 binding site in the regulation of the human HOX A4 gene promoter. Nucleic Acids Res. 1996;24:2849–2856. Duboule D, Dollé P. The structural and functional organization of the murine HOX gene family resembles that of Drosophila homeotic genes. EMBO J. 1989;8:1497–1505. Dupé V, Davenne M, Brocard J, Dollé P, Mark M, Dierich A, Chambon P, Rijli FM. In vivo functional analysis of the Hoxa-1 38 retinoic acid response element (38 RARE). Development 1997;124:399–410. Durston AJ, Timmermans JPM, Hage WJ, Hendriks HFJ, de Vries NJ, Heideveld M, Nieuwkoop PD. Retinoic acid causes an anteroposterior transformation in the developing central nervous system. Nature 1989;340:140–144. Durston AJ, van der Wees J, Pijnappel WWM, Godsave SF. Retinoids and related signals in early development of the vertebrate central nervous system. Curr. Top. Dev. Biol. 1998: in press. Eizema K, Koster JG, Stegeman BI, Baarends WM, Lanser PH, Destree OHJ. Comparative analysis of Engrailed-1 and Wnt-1 expression in the developing central nervous system of Xenopus laevis. Int. J. Dev. Biol. 1994;38:623–632. Flickinger RA. A study of the metabolism of amphibian neural crest cells during their migration and pigmentation in vitro. J. Exp. Zool. 1949;112:165–185. Frasch M, Chen X, Lufkin T. Evolutionary-conserved enhancers direct region-specific expression of the murine Hoxa-1 and Hoxa-2 loci in both mice and Drosophila. Development 1995;121:957–974. Godsave SF, Durston AJ. Neural induction and patterning in embryos deficient in FGF signalling. Int. J. Dev. Biol. 1997;41:57–65. Godsave S, Dekker E-J, Holling T, Pannese M, Boncinelli E, Durston A. Expression patterns of Hoxb genes in the Xenopus embryo suggest roles in anteroposterior specification of the hindbrain and in dorsoventral patterning of the mesoderm. Dev. Biol. 1994;166:465– 476. Green JBA, New HV, Smith JC. Responses of embryonic Xenopus cells to activin and FGF are separated by multiple dose thresholds and correspond to distinct axes of the mesoderm. Cell 1992;71:731–739. Green JBA, Smith JC, Gerhart JC. Slow emergence of a multithreshold response to activin requires cell-contact-dependent sharpening but not prepattern. Development 1994;120:2271–2278. Harland R. In situ hybridization: An improved whole mount method for Xenopus embryos. Methods Cell Biol. 1991;36:685–695. Hemmati-Brivanlou A, de la Torre JR, Holt C, Harland R M. Cephalic expression and molecular characterization of Xenopus En-2. Development 1991;111:715–724. Hogan BLM, Thaller C, Eichele G. Evidence that Hensen’s node is a site of retinoic acid synthesis. Nature 1992;359:237–241. Itoh K, Sokol SY. Graded amounts of Xenopus disheveled specify GRADED RETINOID RESPONSES IN THE HINDBRAIN discrete anteroposterior cell fates in prospective ectoderm. Mech. Dev. 1997;61:113–125. Izpisúa-Belmonte J-C, Tickle C, Dollé P, Wolpert L, Duboule D. Expression of the homeobox Hox-4 genes and the specification of position in chick wing development. Nature 1991;350:585–589. Kappen C, Ruddle FH. Evolution of a regulatory gene family: HOM/ HOX genes. Curr. Opin. Gen. Dev. 1993;3:931–938. Kolm PJ, Sive HL. Regulation of the Xenopus labial homeodomain genes, HoxA1 and HoxD1: Activation by retinoids and peptide growth factors. Dev. Biol. 1995;167:34–49. Kolm PJ, Apekin V, Sive H. Xenopus hindbrain patterning requires retinoid signalling. Dev. Biol. 1997;192:1–16. Lamb TM, Harland RM. Fibroblast growth factor is a direct neural inducer, which combined with noggin generates anterior-posterior neural pattern. Development 1995;121:3627–3636. Lamb TM, Knecht AK, Smith WC, Stachel SE, Economides AN, Stahl N, Yancopoulos GD, Harland RM. Neural induction by the secreted polypeptide noggin. Science 1993;262:713–718. Langston AW, Gudas LJ. Identification of a retinoic acid responsive enhancer 38 of the murine homeobox gene Hox-1.6. Mech. Dev. 1992;38:217–228. Langston AW, Thompson JR, Gudas LJ. Retinoic acid-responsive enhancers located 38 of the Hox A and Hox B homeobox gene clusters. J. Biol. Chem. 1997;272:2167–2175. Leroy P, De Robertis EM. Effects of lithium chloride and retinoic acid on the expression of genes from the Xenopus Hox-2 complex. Dev. Dyn. 1992;194:21–32. López SL, Carraso AE. Retinoic acid induces changes in the localization of homeobox proteins in the antero-posterior axis of Xenopus laevis embryos. Mech. Dev. 1992;36:153–164. Maden M, Gale E, Kostetskii I, Zile M. Vitamin A-deficient quail embryos have half a hindbrain and other neural defects. Curr. Biol. 1996;6:417–426. Marshall H, Nonchev S, Sham MH, Muchamore I, Lumsden A, Krumlauf R. Retinoic acid alters hindbrain Hox code and induces transformation of rhombomeres 2/3 into a 4/5 identity. Nature 1992;360:737–741. Marshall H, Studer M, Popperl H, Aparicio S, Kuroiwa A, Brenner S, Krumlauf R. A conserved retinoic acid response element required for early expression of the homeobox gene Hoxb-1. Nature 1994;370:567– 571. Mayor R, Morgan R, Sargent MG. Induction of the prospective neural crest of Xenopus. Development 1995;121:767–777. Morrison A, Moroni MC, Ariza-McNaughton L, Krumlauf R, Mavilio F. In vitro and transgenic analysis of a human HOXD-4 retinoidresponsive enhancer. Development 1996;122:1895–1907. Morriss-Kay GM, Murphy P, Hill RE, Davidson DR. Effects of retinoic acid excess on expression of Hox-2.9 and Krox-20 and on morphological segmentation in the hindbrain of mouse embryos. EMBO J. 1991;10:2985–2995. Newport J, Kirschner M. A major developmental transition in early Xenopus embryos: 1. Characterization and timing of cellular changes at the midblastula stage. Cell 1982;30:675–686. Nieuwkoop PD, Boterenbrood EC, Kremer A, Bloemsma FFSN, Hoessels ELMJ, Meyer G, Verheyen FJ. Activation and organization of the central nervous system in amphibians. J. Exp. Zool. 1952;120: 1–31. Ogura T, Evans RM. A retinoic acid-triggered cascade of HOXB1 gene activation. Proc. Natl. Acad. Sci. U.S.A. 1995a;92:387–391. Ogura T, Evans RM. Evidence of two distinct retinoic acid response pathways for HOXB1 gene regulation. Proc. Natl. Acad. Sci. U.S.A. 1995b;92:392–396. 49 Pannese M, Polo C, Andreazzoli M, Vignali R, Kablar B, Barsacchi G, Boncinelli E. The Xenopus homologue of Otx2 is a maternal homeobox gene that demarcates and specifies anterior body regions. Development 1995;121:707–720. Papalopulu N, Kintner C. A posteriorising factor, retinoic acid, reveals that anteroposterior patterning controls the timing of neuronal differentiation in Xenopus neuroectoderm. Development 1996;122: 3409–3418. Papalopulu N, Clarke JDW, Bradley L, Wilkinson D, Krumlauf R, Holder N. Retinoic acid causes abnormal development and segmental patterning of the anterior hindbrain in Xenopus embryos. Development 1991;113:1145–1158. Pijnappel WWM, Hendriks HFJ, Folkers GE, van den Brink CE, Dekker EJ, Edelenbosch C, van der Saag PT, Durston AJ. The retinoid ligand 4-oxo-retinoic acid is a highly active modulator of positional specification. Nature 1993;366:340–344. Pöpperl H, Featherstone MS. Identification of a retinoic acid response element upstream of the murine Hox-4.2 gene. Mol. Cell. Biol. 1993;13:257–265. Ruiz i Altaba A. RNAase protection assays. In: Stern CD, Holland PWH, eds. Essential Developmental Biology; A Practical Approach. Oxford: IRL Press, 1993:213–222. Ruiz i Altaba A, Jessell TM. Retinoic acid modifies the pattern of cell differentiation in the central nervous system of neurula stage Xenopus embryos. Development 1991;112:945–958. Sala M. Distribution of activating and transforming influences in the archenteron roof during the induction of the nervous system in amphibians. 1. Distribution in cranial-caudal direction. Kon. Ned. Akad. Wet. Proc. Series C 1995;58:635–647. Sharpe CR. Retinoic acid can mimic endogenous signals involved in transformation of the Xenopus nervous system. Neuron 1992;7:239– 247. Simeone A, Avantaggiato V, Moroni MC, Malvilio F, Arra C, Cotelli F, Nigro N, Acampora D. Retinoic acid induces stage-specific anteroposterior transformation of rostral central nervous system. Mech. Dev. 1995;51:83–98. Sive HL, Draper BW, Harland RM, Weintraub H. Identification of a retinoic acid-sensitive period during primary axis formation in Xenopus laevis. Genes Dev. 1990;4:932–942. Smith WC, Harland RM. Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos. Cell 1992;70:829–840. Studer M, Popperl H, Marshall H, Kuroiwa A, Krumlauf R. Role of a conserved retinoic acid response element in rhombomere restriction of Hoxb-1. Science 1994;265:1728–1732. Sundin O, Eichele G. An early marker of axial pattern in the chick embryo and its respecification by retinoic acid. Development 1992; 114:841–852. van der Wees J, Schilthuis JG, Koster CH, Diesveld-Schipper H, Folkers GE, van der Saag PT, Dawson MI, Shudo K, van der Burg B, Durston AJ. Inhibition of retinoic acid receptor-mediated signalling alters positional identity in the developing hindbrain. Development 1998; 125: 545–556. Wood H, Pall G, Morriss-Kay G. Exposure to retinoic acid before or after the onset of somitogenesis reveals separate effects on rhombomeric segmentation and 38 HoxB gene expression domains. Development 1994;120:2279–2285. Wright CVE, Cho KWY, Fritz A, Burglin TR, De Robertis EM. A Xenopus laevis gene encodes both homeobox-containing and homeobox-less transcripts. EMBO J. 1987;6:4083–4094.