Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS/INSERM/ULP/Collège de France, B.P. 163, 67404 ILLKIRCH Cedex, C.U. de STRASBOURG, France
*Author for correspondence (e-mail: marek{at}igbmc.u-strasbg.fr)
Accepted March 13, 2001
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SUMMARY |
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Key words: Nuclear receptors, Rhombomeres, Hox genes, kreisler, Embryo culture, Mouse, Vitamin A
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INTRODUCTION |
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An extensive functional redundancy between RARs accounts for the observation that RAR (, ß or
)-null mutants exhibit only few developmental defects, whereas altogether the phenotypes of mutants that lack both RAR
and RARß (A
/Aß mutants), RAR
and RAR
(A
/A
mutants) and RARß and RAR
(Aß/A
mutants) recapitulate all the abnormalities characteristic of the fetal vitamin A-deficiency (VAD) syndrome (Wilson et al., 1953; Kastner et al., 1995). Of the three types of RAR double-null mutants, those that lack RAR
and RAR
are overall the most severely affected. Many A
/A
mutants die in utero in contrast to A
/Aß and Aß/A
mutants, which survive until birth. Moreover, near-term (embryonic day (E)18.5) A
/A
fetuses are markedly growth deficient and exhibit evident external malformations, whereas E18.5 A
/Aß and Aß/A
fetuses are externally undistinguishable from their wild-type littermates (Lohnes et al., 1994; Mendelsohn et al., 1994; Ghyselinck et al., 1997; Luo et al., 1996).
In the first part of this work, we have established the timing of the appearance of defects previously observed in A/A
mutants at fetal stages of development (essentially E18.5; Lohnes et al., 1994) by determining the phenotype of early embryos. Comparison of this phenotype with that of A
/Aß embryos (Dupé et al., 1999) has revealed major differences in the patterning of the hindbrain. In order to gain further insights into the developmental mechanisms that underlie these differences, we have studied the fate of the hindbrain when the RA-signaling pathway was blocked with a synthetic RA-antagonist, at different developmental stages.
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MATERIALS AND METHODS |
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Embryo culture and retinoid treatments
Embryos collected at E7.0 (primitive streak stage of gastrulation) or E8.0 (2-4 somite stages) (Kaufman, 1992; Downs and Davies, 1993), were cultured for 3 to 48 hours as described by Copp and Cockroft (1990). All-trans-RA (Sigma) or the pan-RAR synthetic retinoid antagonist BMS493 (Bristol-Myers-Squibb, Princeton, NJ; Wendling et al., 2000; Chazaud et al., 1999; Mollard et al., 2000), diluted in ethanol, were added to the culture medium at final concentrations of 0.1 µM for RA and 1 or 5 µM for BMS493. In control cultures, the retinoid vehicle (i.e. ethanol) was added at the same final concentration (0.1%).
External morphology, histology and in situ hybridization
Following fixation in Bouins fluid, E9.5 embryos were rapidly rinsed in 70% ethanol, then in PBS. They were stained for 3 minutes in Acridine Orange (10 µg/ml in PBS, Sigma) (Zucker et al., 1995). Excess of stain was removed with PBS and the embryos were visualized under a fluorescence microscope (FITC filter). The embryos were postfixed in Bouins fluid and processed for histology. Whole-mount in situ hybridization (ISH) was performed as previously described (Décimo et al., 1995) using digoxigenin-labeled riboprobes for kreisler (Mafb Mouse Genome Informatics; Cordes and Barsh, 1994), Krox20 (Egr2 Mouse Genome Informatics; Wilkinson et al., 1989), Hoxd4 (Featherstone et al., 1988) and EphA2 (Epha2 Mouse Genome Informatics; Ruiz and Robertson, 1994).
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RESULTS |
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Treatment with the pan-RAR antagonist BMS493 at E8.0 generates a posterior expansion of rhombomere 5 and rhombomere 6 identities
The external morphology of the vast majority of E8.0+24 hours (n=70) control embryos was identical to that of E8.75 embryos in vivo (Wendling et al., 2000, and data not shown). However, in these controls, only the five rostral rhombomeres could be identified (Fig. 4A,C,E,G; data not shown). The majority of E8.0+24 hours BMS493-treated embryos (55 out of 70) showed a specific morphological enlargement of R5 (Fig. 4B,D,F,H).
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Treatments at E8.0 with 5 µM of BMS493 did not alter Krox20 and Hoxb1 expression in R3 and R4, respectively (Fig. 4B,D). Krox20 and kreisler were expressed throughout the enlarged R5 (Fig. 4B,F), and kreisler expression extended more posteriorly than in controls into the R6 region (n=6/6) (compare Fig. 4E with 4F). Moreover, expression of Hoxd4 was abolished in the hindbrain, while it remained strongly expressed in the prospective spinal cord (n=3/3; Fig. 4H). Altogether, these data indicate that a state of functional RA deficiency started at the two- to four-somite stages (i.e. about 12 hours prior to the formation of rhombomere boundaries) causes a posterior expansion of R5 and R6 characters, and the loss of an R7 character. The phenotype induced in the R3-R7 region upon treatment with BMS493 at E8.0 is clearly distinct from that of A/A
embryos, but closely related to that of A
/Aß embryos (Dupé et al., 1999 and see below).
To determine more precisely the time at which RA signaling is required for the determination of kreisler expression domain, E8.0 embryos were cultured for a short period (3 hours) in the presence of either 1 µM or 5 µM BMS493, then processed for ISH. Exposure to BMS493 resulted in a dose-dependent expansion of kreisler expression in the neuroectoderm caudal to the otic sulcus (arrows in Fig. 5A-C), suggesting that kreisler expression at E8.0 is normally repressed by RA.
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Interestingly, in E7.0+48 hours embryos treated with a lower concentration of BMS493 (1 µM), the pR5 stripe of Krox20 expression was reduced, whereas the pR3 stripes slightly expanded caudally (compare pR3 and pR5, Fig. 6A,B). This observation indicates that the single broad expression domain of Krox20 observed upon treatment with 5 µM BMS493 indeed corresponds to a pR3. It also supports the view that the caudal enlargement of R3 and R4 characters in the treated embryos occurs at the expense of R5 and R6.
The loss of kreisler expression that occurred at E7.0 on treatment with the pan-RAR antagonist at 5 µM could be relieved upon simultaneous addition of 0.1 µM RA to the culture medium, thus demonstrating that this loss actually arose as a consequence of a block in RA signaling (Fig. 6H).
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DISCUSSION |
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It is noteworthy that the aforementioned defects of E18.5 A/A
mutants had not been reported in VAD animals at fetal stages of development (Wilson et al., 1953). Therefore, they might have been caused by a relief of the RA-independent transcriptional repression exerted by co-repressor-associated unliganded RAR/RXR heterodimers (reviewed in Chambon, 1996; Glass and Rosenfeld, 2000). However, the present analysis of A
/A
embryos indicates that these defects actually reflect a state of RA-deficiency, as they are similar to those exhibited by RALDH2-null embryos. Indeed, RALDH2-null mutants, which lack the first RA-generating enzyme expressed during ontogenesis, are most probably devoid of RA (Niederreither et al., 1999). With one exception (see below), the spectrum of abnormalities observed in E9.5 A
/A
embryos is strikingly similar to that of RALDH2-null embryos, even though some defects are either more penetrant, or more severe in these latter mutants. Indeed, axial rotation is defective in all RALDH2-null embryos, but in only a minority of A
/A
embryos; the 2nd pharyngeal arch and forelimb buds are absent in RALDH2-null embryos, but severely reduced in A
/A
embryos; and the entire neural tube fails to close in some RALDH2-null embryos, whereas this defect is restricted to the hindbrain in A
/A
embryos.
The status of the heart represents the only notable difference between the phenotypes of A/A
and RALDH2-null mutants. In A
/A
and A
/Aß+/-/A
embryos, the heart tube shows normal (rightward) looping and displays well-defined inflow tract (including the primitive atrium; A in Fig. 1), primitive ventricle (V, Fig. 1) and outflow tract (OT, Fig. 1; O. W., N. B. G., P. C. and M. M., unpublished histological data). Likewise, A
/Aß embryos also display normal heart looping (Ghyselinck et al., 1997). In contrast, the heart of RALDH2-null embryos forms a medial dilated structure with poorly defined chambers, and a markedly hypoplastic inflow tract (Niederreither et al., 1999; Niederreither et al., 2001). These data suggest that the process of cardiac looping requires only low levels of signaling through RAR/RXR heterodimers, rather than a unique role of a given RAR isotype in this process. It is noteworthy that a role of RXR homodimers in RA-mediated cardiac looping is very unlikely, as the shape of the heart is normal in mutant fetuses that lack RXR ligand-dependent transactivation functions (Mascrez et al., 1998). A block in RA-signaling transduction (BRST) generated at E7.0 through treatment with the pan-RAR antagonist, results in an absence of externally visible cardiac chambers. In contrast, the same block started at E7.5, does not alter cardiac chamber formation (Chazaud et al., 1999; Niederreither et al., 2001; Zile et al., 2000; O. W., N. B. G., P. C. and M. M., unpublished). These data suggest that, during normal embryogenesis, the cardiogenic mesoderm requires RA as early as E7.5 (i.e. prior to the appearance of the primitive medial heart tube) to form a loopable primordium.
With the exception of hypoplasia of the 3rd pharyngeal arch, the defects observed in E9.5 A/A
embryos are absent in A
/Aß embryos. In fact, E9.5 A
/Aß embryos display only discrete defects, which are restricted to the caudal hindbrain (R5, R6 and R7) and pharyngeal arches 3, 4 and 6 (compare Fig. 7A with 7B; Dupé et al., 1999). Therefore, the morphogenetic effects of RA during early development (i.e. E7.5 to E9.5) are transduced by RAR
and/or RAR
.
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The dramatic effects of RAR and
inactivations on hindbrain segmentation can be understood in terms of mis-specification of pro-rhombomeric identities (Maden, 1999; Gavalas and Krumlauf, 2000; Barrow et al., 2000; and see below). The A
/A
caudal hindbrain has apparently acquired an anterior character, as it expresses a combination of R3 and R4 molecular markers (i.e. Krox20 and Hoxb1) instead of expressing kreisler (the earliest marker of the normal R5/R6 territory). It is noteworthy that a very similar anterior transformation of caudal hindbrain identities has been extensively documented in RALDH2-null mice (Fig. 7D-F; Niederreither et al., 1999; Niederreither et al., 2000). These observations indicate that RAR
and/or RAR
mediate the RA signal required to determine the identity of R5 and R6. Interestingly, since the nucleus of the abducens nerve differentiates from the neuroectoderm of R5 and R6, the agenesis of this structure in E18.5 A
/A
mutants (Lohnes et al., 1994) is likely to be a direct consequence of the absence of R5 and R6 at E8.5.
Distinct hindbrain phenotypes in RAR/RARß and RAR
/RAR
double null mutants are related to different time windows of RA action
The timecourse analysis of the alterations induced by the pan-RAR antagonist indicates that RA signaling is involved at distinct stages of the specification of the R5/R6 territory: first to permit its formation, and subsequently to define the position of its caudal boundary. A BRST generated on treatment at E7.0 yields a posterior expansion of neuroectodermal territories that carry R3 and R4 identities, and concomitant disappearance of territories that correspond to R5 and R6, leading to a phenocopy of the hindbrain patterns observed in A/A
mutants (Fig. 7D,F,G). It is noteworthy that such an early BRST is unlikely to be effective before E7.5, which corresponds to the onset of embryonic RA synthesis (Rossant et al., 1991; Ang et al., 1996; Niederreither et al., 1997). In contrast, a BRST at E8.0 does not affect the patterning of the first 4 rhombomeres, but induces a caudal expansion of territories carrying R5 and R6 identities and loss of Hoxd4 expression in R7. Thus, a BRST at E8.0 leads to a phenocopy of hindbrain patterning defects previously described in A
/Aß embryos, which include apparently normal R3 and R4, an increase in the size of R5, anteriorization of R6 identity, and loss of an R7 character (i.e. Hoxd4 expression; Dupé et al., 1999; compare Fig. 7B with 7C). RAR
, RARß and RAR
are expressed uniformly throughout the prospective hindbrain at E7.5, whereas 24 hours later, RARß expression becomes restricted to the posterior part of this structure (Ang and Duester, 1997). Altogether these results suggest (1) that RAR
and/or RAR
transduce the RA-signal that, at E7.5, is required to specify the prospective R5/R6 territory; and (2) that a caudal increase in RA-signaling at E8.0, probably mediated by RARß, sets up the caudal boundary of this territory.
The hindbrain patterning defects observed in cultured embryos depend on the severity of the BRST. At E7.0, intermediate levels of BRST, achieved by either a low concentration of the pan-RAR antagonist or the presence of both a high concentration of this antagonist and RA, allow the formation of small domains of either kreisler or Krox20 expressions. A more robust BRST abolishes the formation of these expression domains. Along the same lines, gradual enlargement of the kreisler expression domain parallels the level of inhibition of RA-signaling. Therefore, precise thresholds of RA signaling are apparently required to commit enough cells towards R5 and R6 fates at E7.0, and subsequently to restrict the size of the prospective R5/R6 territory at E8.0. These data suggest that the enzymatic activities, which in vivo determine RA availability (RALDHs and CYP26; Maden, 1999; Abu-Abed et al., 2001; and references therein), must be tightly controlled during the development of the embryonic hindbrain in order to generate such thresholds.
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ACKNOWLEDGMENTS |
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