Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 10142, 67404 Illkirch Cedex, CU de Strasbourg, France
* Present address: Departments of Medicine and Molecular and Cellular Biology, Center for Cardiovascular Development, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
These authors contributed equally to this work
Author for correspondence (e-mail: dolle{at}igbmc.u-strasbg.fr)
Accepted 30 April 2002
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SUMMARY |
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Key words: Aldh1a2, dHand, Hox genes, Limb, Mouse development, Raldh2, Retinaldehyde dehydrogenase, Retinoids, Sonic hedgehog
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INTRODUCTION |
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Retinoic acid (RA), the active derivative of vitamin A (retinol), is believed to be required for limb development. RA-impregnated beads placed under the anterior limb margin induce digit duplications similar to ZPA grafts (Tickle et al., 1982). Furthermore, local administration of synthetic molecules acting as RA receptor (RAR) antagonists, or inhibitors of RA synthesis, severely inhibit limb outgrowth in chick (Helms et al., 1996
; Stratford et al., 1996
). Nutritional vitamin A deficiency (VAD) also has marked effects on limb growth and patterning in quail embryos (Stratford et al., 1999
). Evaluating the role of retinoids in mammalian limb development is complicated by the fact that severe VAD results in female infertility. Whereas classical studies of VAD rodents did not report limb defects (e.g. Wilson et al., 1953
), a recent study in which severely deficient rat embryos were obtained following RA supplementation during early gestation reported relatively mild limb hypoplasia (Power et al., 1999
). In the mouse, due to partial functional redundancy, limb defects only occur if two of the RARs are disrupted in combination. RAR
/RAR
mutants have skeletal limb defects with no major reductions in limb size (Lohnes et al., 1994
), whereas RARß/RAR
mutants show impaired interdigital cell death (Dupé et al., 1999
). Finally, administration of teratological doses of RA to pregnant dams during embryonic limb bud differentiation generates skeletal defects (Kwasigroch and Kochhar, 1980
), whereas early administration prior to gastrulation can induce the development of supernumerary hindlimbs (Rutledge et al., 1994
; Niederreither et al., 1996
). Thus, while the overall importance of RA to mammalian limb outgrowth is still unclear, one common outcome of both avian and rodent studies is that RA deficiency prevents the proper establishment of the Fgf4-Shh AER-ZPA signaling loop (Power et al., 1999
; Stratford et al., 1999
).
We have used Raldh2 (Aldh1a2 Mouse Genome Informatics) knockout mouse mutants to investigate the contribution of RA synthesized by the embryo from maternal retinol to limb morphogenesis. The Raldh2 gene codes for a retinaldehyde dehydrogenase that catalyzes the second oxidative step in the biosynthesis of RA from retinol (Zhao et al., 1996). Raldh2 is responsible for most of the RA-synthesizing activity during early mouse embryogenesis (E7.5-9.5), as seen from the failure of Raldh2/ embryos to activate RA-responsive transgenes, except in the developing retina (Niederreither et al., 1999
). These mutant embryos, which die at E9.5-10.5 from severe cardiac defects, exhibit axial truncation due to impaired somite growth, as well as hindbrain defects (Niederreither et al., 1999
; Niederreither et al., 2000
; Niederreither et al., 2001
). Furthermore, they display no sign of limb bud outgrowth; however, no conclusion concerning the importance of local RA synthesis in this process can be drawn from this observation, as development of these embryos may be arrested shortly before or at the onset of limb induction. Using several modes of RA supplementation, we now demonstrate a critical role of endogenous RA synthesis in forelimb morphogenesis, and describe the morphological and molecular consequences of this endogenous RA deficiency.
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MATERIALS AND METHODS |
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RESULTS |
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When RA was provided ad libitum at a concentration of 100 µg/g maternal food from E7.5 to E8.5, about 5% of the Raldh2/ mutants survived until E18.5. These mutants exhibited alterations in craniofacial morphology, lack of cephalic flexure and cardiovascular defects (Fig. 1D, and data not shown). Fetal forelimb outgrowth was strikingly reduced, whereas the hindlimbs appeared normal (Fig. 1D). Closer examination of left mutant minilimbs revealed what appeared to be a single digit with a nail-like structure (Fig. 1D, inset). Two digits could be formed on the right side under these conditions (data not shown).
We next determined the extent to which we could rescue mutant forelimb development under maximal subteratogenic RA supplementation. RA was administered at 100 µg/g food from E7.5 to E8.5, and then at 250 µg/g food from E8.5 to E14.5. A marked increase in mutant forelimb size was observed, and in some cases the right forelimb appeared close to its wild-type size (Fig. 1E and data not shown). However, the number and pattern of digits formed was variable. In some cases 4 or 5 digits were seen. Some mutants exhibited mirror-image digit duplications. Fig. 1E shows an example of complete digit duplication on the left side, whereas the right side limb has a lobster claw defect (arrowhead). Other mutants exhibited less severe alterations, such as polydactyly with 6 symmetrical digits (Fig. 1F), or five digits with an abnormally long first digit (Fig. 1G, arrowhead). No abnormalities of the forelimbs were observed in wild-type or heterozygote littermates. Increasing RA concentration to 400 µg/g food from E8.5-E14.5 did not improve forelimb growth or patterning, but did result in teratogenic effects in both wild-type and mutant embryos, including exencephaly and lumbosacral truncations, and embryonic lethality in some mothers (data not shown). No further experiment was therefore conducted at concentrations higher than 250 µg/g. RA supplementation was also started at E6.5 (100 µg/g) to test whether abnormal forelimb development in mutants could be due to lack of RA prior to E7.5. These early treatments did not improve the mutant forelimb phenotype (data not shown).
In order to determine the critical period for RA-dependent forelimb outgrowth, a range of RA treatment levels and times where tested (Table 1). In all cases RA was administered from E7.5 to E8.5 at 100 µg/g food, as these levels are necessary to rescue heart development in the mutants (lower levels at this stage severely reduced mutant survival, whereas higher levels resulted in teratogenic effects). Embryos were collected at E14.5 and skeletal patterns were analyzed after whole-mount Alcian Blue cartilage staining. The RA rescue of mutant forelimb development appeared to be both stage- and dose-dependent (see Table 1 and Fig. 2A for examples of forelimb patterns obtained under various treatment conditions). Short-term treatments (E7.5-E8.5: experimental group 1, Table 1) resulted in highly truncated mutant forelimbs. These contained a minute scapular blastema, no recognizable humerus, a single, indistinct stylopodal/zeugopodal element, and 1 or 2 rudimentary digits (Fig. 2A). Extending the duration of RA treatment to E9.5 (100 µg/g food: group 2, Table 1) had little effect on overall forelimb growth, while development along the AP axis was improved, as seen by the occasional presence of a humeral and/or of two separate radial and ulnar cartilages (Table 1). Most mutants, though, exhibited no (or a highly hypoplastic) humerus and a single radial/ulnar zeugopodal cartilage (Fig. 2A,C,D), and their dactyly was slightly improved (2-3 digits; Table 1, Fig. 2A,D). Interestingly, increasing RA to the maximal non-teratogenic tolerated dose (250 µg/g food from E8.5 to E9.5: group 3, Table 1) provided a better rescue of skeletal patterning along both the PD and AP axes. Most forelimbs had well formed scapula and humerus; however, the resulting limbs remained truncated and many cases of single, ulna-like zeugopodal cartilages were seen, whereas dactyly was usually rescued to 4-6 digits (Table 1, Fig. 2A,D).
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RA treatments were also extended to E10.5 at low (100 µg/g food) and high (250 µg/g food) doses (groups 4 and 5, Table 1). The forelimb phenotypes of mutants treated at the lower dose did not markedly differ from those of group 2, which received the same dosage until E9.5 (see Table 1, and data not shown). Further improvement of the overall forelimb growth was obtained under high RA concentration (Fig. 2A, group 5). Most mutant limbs in this group had a well formed scapula and humerus, as well as distinct radius and ulna cartilages (Table 1, Fig. 2A,F,G). The digit number ranged from 3 (on the left side) to 6 digits (Table 1). Mutant forelimbs with 5 digits showed a characteristic pattern alteration: the first digit was as long as the other digits (Fig. 2F), while it is normally shorter (two phalanges) in wild type (Fig. 2B). Such a symmetrical arrangement of all digits was also seen in polydactylous mutant limbs (Fig. 2G).
In both chick and mouse embryos, Raldh2 expression is strongly induced in both lateral plate and somitic trunk mesoderm shortly before limb buds are induced (Niederreither et al., 1997; Swindell et al., 1999
). This may possibly generate a surge of RA critical for forelimb bud outgrowth, consistent with HPLC data indicating elevated RA levels in the chick wing bud (Thaller and Eichele, 1987
). To reproduce such a RA surge, an additional series of experiments was performed (group 6, Table 1) in which a RA dose was administered by maternal oral gavage (30 µg/g body weight at E8 8:00 am) in addition to food administration under the same conditions as group 5. RA administered by maternal gavage has been shown to act in the embryo within 2-4 hours (e.g. Mendelsohn et al., 1994
). Thus, the RA surge is expected to occur at
E8.5 (noon), when embryos are at the 5-8 somite stage. Although this condition did not fully restore normal humerus and radius/ulna patterning, it resulted in a lower incidence of hypodactylous mutant forelimbs (Table 1). Again, some of the mutants were polydactylous or had abnormal first digits.
Molecular alterations in the RA-rescued Raldh2/ forelimbs
Numerous molecular signals act in coordination to control limb outgrowth and patterning (for a review, see Johnson and Tabin, 1997). Molecular defects underlying altered Raldh2/ forelimb development were investigated both under severe growth deficiency conditions (Table 1, groups 1,2) or under a condition (Table 1, group 3) that better rescues forelimb growth, but results in patterning defects. In all cases, RA administration was stopped at least 24 hours before molecular analysis (e.g. at E9.5 for an E10.5 analysis).
We first analyzed whether the administered RA might interfere with the expression pattern of an RA-responsive (RARE-hsp68-lacZ) (Rossant et al., 1991) reporter transgene (Fig. 3A-L) or with endogenous expression of an RA-responsive gene (RARß; Fig. 3M-R). The expression pattern of the RARE-hsp68-lacZ was not detectably altered in wild-type embryos analyzed at E9.5 after dietary RA supplementation from E7.5 to E9.5 (250 µg/g food; Fig. 3B; 100 µg/g food: data not shown), when compared to untreated embryos (Fig. 3A). During initiation of limb budding, the reporter transgene was expressed at lower levels in lateral plate mesoderm than in somitic mesoderm or neural tube, in both untreated (Fig. 3E) and RA-treated (Fig. 3F) wild-type embryos. Transgene activity became restricted to the proximal limb mesenchyme as soon as outgrowth started (Fig. 3H). At E10.5, proximally restricted transgene expression was similarly seen in untreated (Fig. 3J) and RA-treated (E7.5 to E10.5; Fig. 3K) wild-type embryos. However, the RA-responsive transgene was clearly downregulated in the RA-rescued Raldh2/ embryos. E9.5 mutants treated with 250 µg/g food exhibited weak and/or patchy expression in both the spinal cord and trunk mesoderm (Fig. 3C,G), whereas mutants treated at a lower dose (100 µg/g food) showed almost complete downregulation of the transgene (Fig. 3D,I); note the persistent transgene activity in the ventral region of the spinal cord and hindbrain neuroepithelium, which may indicate the presence of another RA-generating activity (K. N., J. V., P. C. and P. D., unpublished data). Raldh2/ embryos treated with RA until E10.5 (250 µg/g) exhibited weak transgene activity in the trunk and proximal forelimb mesoderm (data not shown). However, the reporter transgene was selectively activated in the mesonephric area, near the base of the hindlimb buds (Fig. 3L), suggesting the presence of another local RA-producing activity (see Discussion).
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Fibroblast growth factors (FGFs) are key effectors of limb growth and AER function. Fgf8 is specifically expressed in the surface ectoderm, and subsequently in the AER of the developing limb buds, and its conditional mutation in mouse severely impairs limb development (Lewandowski et al., 2000; Moon and Capecchi, 2000
). Raldh2 mutant embryos under short-term (E7.5-E8.5) RA supplementation showed a reduced forelimb domain of Fgf8 expression, which did not define a proper AER domain as in control littermates (Fig. 4A-C). Upon longer (E7.5-E9.5) RA rescue, Raldh2/ embryos exhibited a range of AER alterations: FGF8 was often expressed at higher levels in the anterior portion of the mutant AER (whereas its expression is more prominent posteriorly in the E10.5 wild-type AER; Fig. 4D), which sometimes appeared wider or abnormally bifurcated (Fig. 4E,F). In some mutants, the AER was disrupted centrally (Fig. 4G), leading in extreme cases to the development of two separate distal outgrowths (Fig. 4H).
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Sonic hedgehog (Shh) is a key molecular determinant of AP limb patterning and its expression has been shown to be retinoid-dependent in other VAD systems (Stratford et al., 1996; Stratford et al., 1999
; Power et al., 1999
). Wild-type Shh expression is specific to the posterior mesenchyme of the developing limbs, i.e. the ZPA region (Fig. 5A). Shh expression was either undetectable, or markedly reduced, in Raldh2/ forelimb buds expected to develop as minilimbs (Fig. 5B,C). When present, its expression was seen along the distal margin instead of being posteriorly restricted (Fig. 5B). Even under better rescue conditions, Shh was expressed in distal mesenchyme at lower levels than in wild type (Fig. 5D-F), and reversal of its transcript distribution, with highest expression towards the anterior limb margin, was observed in some mutants (Fig. 5E,F). We also analyzed Bmp2 expression, whose posterior expression in wild-type limbs (Fig. 5G) is dependent on Shh (Drossopoulou et al., 2000
; Chiang et al., 2001
). Bmp2 was expressed at appropriate levels in Raldh2/ minilimbs, but showed no posterior asymmetry (Fig. 5H). Mutant limbs that achieved better outgrowth showed a ring-like Bmp2 transcript pattern along the AP axis (Fig. 5I).
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The basic helix-loop-helix factor dHand has been implicated in the establishment of the limb ZPA and the induction of posterior Shh expression (Fernandez-Teran et al., 2000; Charité et al., 2000
). dHand is first expressed along the whole lateral plate mesoderm and then, during limb outgrowth (E9.5), is selectively downregulated in the anterior region of the buds (see Fig. 6D). In wild-type E10.5 forelimb buds, dHand is expressed along the posterior mesoderm in a domain that outflanks that of Shh (Fig. 6A). Instead of being posteriorly restricted, we found that dHand was expressed along the whole distal margin of the severely deficient Raldh2/ forelimbs (Fig. 6B). In mutant limbs that underwent better growth, dHand was expressed throughout the mesoderm (Fig. 6C). To see whether these abnormal patterns result from misregulation during early limb budding, mutant embryos were analyzed at E9-E9.5. Interestingly, dHand downregulation occurred normally during the initial phase of limb budding in mutants (Fig. 6E, bracket). Thus, a graded posterior to anterior expression pattern could be established in the Raldh2/ buds (Fig. 6F). However, owing to their size deficiency, mutant limb buds expressed dHand in a domain encompassing a larger portion of the buds, compared to wild type (compare Fig. 6D and F). Thus, in most severely deficient buds, only the proximal, anterior margin was devoid of dHand transcripts (Fig. 6G, arrow), which accounts for dHand expression throughout the distal mesoderm during further outgrowth (Fig. 6C).
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Although the forelimbs of the RA-rescued Raldh2/ mice do not show obvious dorsoventral (DV) patterning defects (e.g. Fig. 1E-G), we analyzed the expression of Wnt7a and Engrailed-1 (En1), two determinants of limb DV polarity that are specifically expressed in the dorsal and ventral limb bud ectoderm, respectively (Kimmel et al., 2001), and whose expression is misregulated in the wing buds of VAD quail embryos (Stratford et al., 1999
). Wnt7a was properly expressed along the dorsal ectoderm of the Raldh2/ forelimb buds; however, the presence of cells ectopically expressing Wnt7a was detected in the anteriormost ventral ectoderm (Fig. 7K), especially in the most severely growth-deficient limbs (Fig. 7L). In contrast, En1 expression was not detectable in the prospective ventral forelimb ectoderm of E9.5 Raldh2/ mutants (compare Fig. 7M and N; note also the diminished expression in dermomyotome). At E10.5, En1 was expressed throughout the ventral ectoderm of the Raldh2/ forelimb buds (Fig. 7N, inset) while, at this stage, its expression became restricted to the AER in wild-type littermate embryos (Fig. 7M, inset). En1 acts both as a repressor of Wnt7a ventral expression and as a determinant of proper AER formation (Kimmel et al., 2000
). Its delayed timing of activation may contribute to the AER defects seen in Raldh2/ forelimb buds (Fig. 4), and to the abnormal anterior expression of Wnt7a.
The AbdominalB-related Hoxd genes (Hoxd9-d13) are sequentially activated along both the PD and AP axes of the limb buds and, thus, control the morphogenesis of defined skeletal elements (Zakany and Duboule, 1999). Before limb budding, only Hoxd9 is expressed along the flank mesoderm up to the prospective forelimb level. Hoxd9 expression was detected in the flank of E9.5 Raldh2/ embryos even in the absence of any RA supplementation. However, these embryos showed no increase of Hoxd9 expression in the putative forelimb territory, as normally seen in wild-type embryos (data not shown). Hoxd9 upregulation took place in the forelimb buds of the RA-supplemented mutant embryos, even if highly hypoplastic (data not shown). Likewise, Hoxd11, d12 and d13 were expressed at normal levels in mutant forelimb buds (Fig. 8, and data not shown). However, their spatial transcript distributions were abnormal. In E9.5 mutant buds, lack of posterior restriction was observed for both Hoxd11 (compare Fig. 8A and B) and Hoxd12 (Fig. 8F and G). Over the next day, expression of these genes is specifically upregulated in the posterodistal region of the autopod in wild type (Sordino et al., 1995
), leading to an apparent double expression domain for Hoxd11 (Fig. 8C) and 2 regions with different signal intensities for Hoxd12 (Fig. 8H). This autopodal upregulation was clearly present in mutant embryos (Fig. 8D,E,I,J). It was seen, however, along the whole margin of the autopod (see the strong Hoxd12 expression at the level of the abnormal anterior outgrowth: Fig. 8J, arrowhead) or was even reversed in polarity (see Hoxd11 strong expression along the anterior margin, and weak expression along the posterior margin, of the autopod in Fig. 8D and E).
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DISCUSSION |
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While exogenously supplied RA could ensure forelimb growth in the rescued mutants (most likely because FGF signaling is operational; see below), it was unable to ensure normal AP patterning. Specific activation of Shh in the posterior limb bud mesoderm is an essential feature of the ZPA, the main determinant of AP limb patterning (Riddle et al., 1993). Although some of the severely affected Raldh2/ forelimbs lacked detectable Shh expression, most often they showed a delocalized expression towards the distal tip of the limb rudiment and, sometimes, a reversed pattern with higher levels towards the anterior limb margin. Fgf4, the maintenance of which depends on Shh in a positive feedback loop (Zuniga et al., 1999
; Chiang et al., 2001
), was either downregulated or expressed ectopically at the anterior margin. Accordingly, the Raldh2/ forelimbs exhibited marked AP patterning defects. In its most severe form, the Raldh2/ phenotype consisted of a single undefined stylopodal/zeugopodal element and a digit rudiment. This phenotype is remniscent of, although not similar to, that of Shh/ null mutants, which have a polarized stylopodal element (humerus) and a distinct, non-polarized zeugopodal element and single digit (Chiang et al., 2001
; Kraus et al., 2001
). We therefore propose that the Raldh2/ forelimb abnormalities result from a lack of Shh asymmetrical posterior expression, rather than from a lack of Shh function. This interpretation is supported by the different outcomes seen in both mutants on the expression of downstream genes: Bmp2 expression in posterior mesenchyme is substantially reduced in Shh/ mutants (Chiang et al., 2001
), whereas its expression expands symmetrically in anterior and posterior mesenchyme in Raldh2/ mutants. Likewise, Hoxd11 and Hoxd12 expression is severely reduced in Shh/ limb buds, whereas these genes are expressed along both the anterior and posterior margins sometimes at higher levels at the anterior margin of the Raldh2/ forelimb autopod.
We suggest that the Raldh2 function in the flank and/or the proximal forelimb bud mesoderm is to synthesize RA required for efficient posterior activation of Shh. Interestingly, HPLC data have indicated higher RA levels in the posterior region of the chick limb bud (Thaller and Eichele, 1987) even though Raldh2 does not exhibit a preferential posterior distribution (Niederreither et al., 1997
; Swindell et al., 1999
). Such an uneven RA distribution could possibly be ascribed to the action of RA-metabolizing enzymes (MacLean et al., 2001
). It is however likely that a posteriorly restricted factor such as dHand (Fernandez-Terran et al., 2000
; Charité et al., 2000
) is also required to specifically activate Shh. We found no misregulation of dHand in the flank mesoderm of E9 Raldh2/ mutants, consistent with the idea that it acts upstream or in parallel with RA signaling. However, dHand expression encompassed a relatively larger area of the early forelimb bud mesoderm in Raldh2/ than in wild-type embryos. This could contribute to the inability of Raldh2/ embryos to properly restrict Shh expression to the posterior margin of their forelimb buds, and thus to establish a functional ZPA.
Even though the presence of a RA response element has been noted in the Shh promoter region (Chang et al., 1997), the effects of RA could be complex and several genes may be involved. Hoxb8, which is normally expressed up to the prospective posterior forelimb mesoderm, is thought to be directly controlled by RA and can be activated ectopically up to 4 hours after implantation of a RA bead in the anterior chick wing bud mesoderm (Lu et al., 1997
; Stratford et al., 1997
). However, we found no downregulation or misexpression of Hoxb8 in the flank mesoderm of Raldh2/ embryos (our unpublished data) and Stratford et al. (Stratford et al., 1999
) reported that Hoxb8 is expressed at normal levels and is even anteriorly expanded in the lateral mesoderm of VAD quail embryos. Interestingly, Ogura et al. (Ogura et al., 1996
) have provided evidence that some of the RA effects in establishing the ZPA activity could be Shh-independent, as they have shown that a yet unidentified factor(s) induced by RA in P19 embryonal carcinoma cells can act in combination with Shh to generate a strong ZPA activity in a chick wing bud assay.
Exogenous RA can allow proper forelimb proximodistal patterning in Raldh2 mutants
The homeobox gene Meis2 has been implicated as a determinant of proximal limb structures (Capdevila et al., 1999; Mercader et al., 2000
). Mercader et al. (Mercader et al., 2000
) found that addition of RA ectopically can reprogram distal limb mesodermal cells to express more proximal gene combinations, including that of Meis2. Administration of a RAR antagonist leads to a rapid downregulation of Meis2 expression, followed by selective lack of proximal elements. It was further postulated that proximal Meis2 expression is antagonized and thus prevented in distal mesoderm through the FGF signaling pathway (Mercader et al., 2000
). Although Meis2 expression may be subtly downregulated in the trunk mesoderm of Raldh2/ embryos that were rescued at a low RA concentration (100 µg/g food: Table 1, group 2), its transcripts were clearly detected in the proximal mesoderm of the mutant minilimbs. Under better rescue conditions, Meis2 proximodistal expression boundary was located, as in wild-type embryos, at the level of the stylopod-zeugopod boundary. This leads us to conclude that, if RA synthesized by Raldh2 is involved in Meis2 regulation, its lack of synthesis can be readily compensated for in mutant embryos by maternal RA supplementation. As this supplementation is unlikely to reproduce the tissue distribution of enzyme-mediated RA synthesis, it appears that the positioning of the Meis2 expression boundary may be more critically dependent on FGF-mediated distal repression than on RA-mediated proximal activation. Several lines of evidence indicate that the FGF signaling pathway is operational in the RA-rescued Raldh2/ forelimbs: Fgf10 and Fgf8 are expressed at relatively normal levels in the mesoderm and AER, respectively, of the RA-rescued limbs, and they can elicit the expression of target genes such as Sprouty4 in distal limb mesoderm. We also note that in the mutant limbs, the gene for the BMP antagonist Gremlin is expressed distally as in wild-type embryos. Thus, in contrast to the case of the AP axis, we conclude that the control of gene expression along the limb PD axis is not critically dependent on the precise tissue distribution of Raldh2-synthesized RA, as this control can be essentially achieved in mutant embryos through exogenous RA supplementation.
Retinoic acid and hindlimb development
Hindlimb development was not detectably altered in the Raldh2 mutants, whatever the RA rescue conditions used. This could be because (1) maternally administered RA fully rescues hindlimb development; (2) another RA-synthesizing enzyme may be critically involved in RA synthesis within the hindlimb field; (3) hindlimb development may be prominently controlled by other inducing/growth promoting factors.
We and others (Grün et al., 2000) have observed that Raldh3 is expressed in the mesonephros of wild-type embryos, adjacent to the hindlimb buds. This expression is present in RA-rescued Raldh2/ embryos (our unpublished data). Strong activation of the RARE-hsp68-lacZ reporter transgene is seen within the same region in mutant embryos (Fig. 3). Thus, Raldh3 could be responsible for the local RA synthesis required for proper hindlimb development. An alternative possibility may implicate Wnt signaling, which has recently been involved in the initiation of limb development (Kawakami et al., 2001
). Whereas Wnt2b, the candidate for forelimb induction, is expressed at rather low levels in the forelimb field, several Wnt genes (including Wnt8a/8c or Wnt3a) are expressed in the region where hindlimbs are induced. Owing to a preponderant Wnt influence, hindlimb development may require lower RA levels to proceed normally, and these levels may be reached in Raldh2 mutants by maternal supplementation. In any event, hindlimb development is unlikely to be RA independent, as both the forelimbs and hindlimbs are affected in VAD quail embryos (Stratford et al., 1999
).
In conclusion, we have reported that correct patterning and outgrowth of the forelimb depends on endogenous RA production by Raldh2, which can only be partially rescued in knockout mutants by sustained RA supplementation from E7.5 to (at least) E9.5. As Raldh2 null mutants do not survive past this stage without RA supplementation, only conditional mutation of Raldh2 in the lateral mesoderm will indicate if forelimb outgrowth is entirely abolished, and hindlimb development is affected, in its absence.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Büscher, D. Bosse, B., Heymer, J. and Rüther, U. (1997). Evidence for genetic control of Sonic hedgehog by Gli3 in mouse limb development. Mech. Dev. 62, 175-182.[Medline]
Capdevila, J., Tsukui, T., Esteban, C. R., Zappavigna, V. and Izpisua Belmonte, J. C. (1999). Control of vertebrate limb outgrowth by the proximal factor Meis2 and distal antagonism of BMPs by Gremlin. Mol. Cell 4, 839-849.[Medline]
Charité, J., McFadden, D. G. and Olson, E. N. (2000). The bHLH transcription factor dHAND controls Sonic hedgehog expression and establishment of the zone of polarizing activity during limb development. Development 127, 2461-2470.
Chang, B. E., Blader, P., Fischer, N., Ingham, P. W. and Strähle, U. (1997). Axial (HNF3beta) and retinoic acid receptors are regulators of the zebrafish sonic hedgehog promoter. EMBO J. 16, 3955-3964.
Chiang, C., Litingtung, Y., Harris, M. P., Simandl, B. K., Li, Y., Beachy, P. A. and Fallon, J. F. (2001). Manifestation of the limb prepattern: limb development in the absence of sonic hedgehog function. Dev. Biol. 236, 421-435.[Medline]
Décimo, D., Georges-Labouesse, E. and Dollé, P. (1995). In situ hybridization to cellular RNA. In Gene Probes 2, a Practical Approach (eds. B. D. Hames and S. J. Higgins), pp. 183-210. New York: Oxford University Press.
Dollé, P., Ruberte, E., Kastner, P., Petkovich, M., Stoner, C. M., Gudas, L. J. and Chambon, P. (1989). Differential expression of genes encoding alpha, beta and gamma retinoic acid receptors and CRABP in the developing limbs of the mouse. Nature 342, 702-705.[Medline]
Donovan, M., Olofsson, B., Gustafson, A. L., Dencker, L. and Eriksson, U. (1995). The cellular retinoic acid binding proteins. J. Steroid Biochem. Mol. Biol. 53, 459-465.[Medline]
Drossopoulou, G., Lewis, K. E., Sanz-Ezquerro, J. J., Nikbakht, N., McMahon, A. P., Hofmann, C. and Tickle, C. (2000). A model for anteroposterior patterning of the vertebrate limb based on sequential long- and short-range Shh signalling and Bmp signalling. Development 127, 1337-1348.
Dupé, V., Ghyselinck, N. B., Thomazy, V., Nagy, L., Davies, P. J. A., Chambon, P. and Mark, M. (1999). Essential roles of retinoic acid signaling in interdigital apoptosis and control of BMP-7 expression in mouse autopods. Dev. Biol. 208, 30-43.[Medline]
Fernandez-Teran, M., Piedra, M. E., Kathiriya, I. S., Srivastava, D., Rodriguez-Rey, J. C. and Ros, M. A. (2000). Role of dHAND in the anterior-posterior polarization of the limb bud: implications for the Sonic hedgehog pathway. Development 127, 2133-2142.
Fromental-Ramain, C., Warot, X., Lakkaraju, S., Favier, B., Haack, H., Birling, C., Dierich, A., Dollé, P. and Chambon, P. (1996). Specific and redundant functions of the paralogous Hoxa-9 and Hoxd-9 genes in forelimb and axial skeleton patterning. Development 122, 461-472.
Gorry, P., Lufkin, T., Dierich, A., Rochette-Egly, C., Décimo, D., Dollé, P., Mark, M., Durand, B. and Chambon, P. (1994). The cellular retinoic acid binding protein I is dispensable. Proc. Natl. Acad. Sci. USA 91, 9032-9036.[Abstract]
Grün, F., Hirose, Y., Kawauchi, S., Ogura, T. and Umesono, K. (2000). Aldehyde dehydrogenase 6, a cytosolic retinaldehyde dehydrogenase prominently expressed in sensory neuroepithelia during development. J. Biol. Chem. 275, 41210-41218.
Helms, J. A., Kim C. H., Eichele, G. and Thaller, C. (1996). Retinoic acid signaling is required during early chick limb development. Development 122, 1385-1394.
Jegalian, B. G. and De Robertis E. M. (1992). Homeotic transformations in the mouse induced by overexpression of a human Hox3.3 transgene. Cell 71, 901-910.[Medline]
Johnson, R. L. and Tabin, C. J. (1997). Molecular models for vertebrate limb development. Cell 90, 979-990.[Medline]
Kawakami, Y., Capdevila, J., Buscher, D., Itoh, T., Esteban, C. R. and Izpisua Belmonte, J. (2001). WNT sigals control FGF-dependent limb initiation and AER induction in the chick embryo. Cell 104, 891-400.[Medline]
Kimmel, R. A., Turnbull, D. H., Blanquet, V., Wurst, W., Loomis, C. A. and Joyner, A. L. (2001). Two lineage boundaries coordinate vertebrate apical ectodermal ridge formation. Genes Dev. 14, 1377-1389.
Kraus, P., Fraidenraich, D. and Loomis, C. A. (2001). Some distal limb structures develop in mice lacking Sonic hedgehog signaling. Mech. Dev. 100, 45-58.[Medline]
Kwasigroch, T. E. and Kochhar, D. M. (1980). Production of congenital limb defects with retinoic acid: phenomenological evidence of progressive differentiation during limb morphogenesis. Anat. Embryol. (Berl). 161, 105-113.[Medline]
Lampron, C., Rochette-Egly, C., Gorry, P., Dollé, P., Mark, M., Lufkin, T., LeMeur, M. and Chambon, P. (1995). Mice deficient in cellular retinoic acid binding protein II (CRABPII) or in both CRABPI and CRABPII are essentially normal. Development 121, 539-548.
Laufer, E., Nelson, C. E., Johnson, R. L., Morgan, B. A. and Tabin, C. (1994). Sonic hedgehog and Fgf-4 act through a signaling cascade and feedback loop to integrate growth and patterning of the developing limb bud. Cell 79, 993-1003.[Medline]
Lewandoski, M., Sun, X. and Martin, G. R. (2000). Fgf8 signalling from the AER is essential for normal limb development. Nat. Genet. 26, 460-463.[Medline]
Lohnes, D., Mark, M., Mendelsohn, C., Dollé, P., Dierich, A., Gorry, P., Gansmuller, A. and Chambon, P. (1994). Function of the retinoic acid receptors (RARs) during development. I. Craniofacial and skeletal abnormalities in RAR double mutants. Development 120, 2723-2748.
Lu, H. C., Revelli, J. P., Goering, L., Thaller, C. and Eichele, G. (1997). Retinoid signaling is required for the establishment of a ZPA and for the expression of Hoxb-8, a mediator of ZPA formation. Development 124, 1643-1651.
MacLean, G., Abu-Abed, S., Dollé, P., Tahayato, P., Chambon, P. and Petkovich, M. (2001) Cloning of a novel retinoic-acid metabolizing cytochrome P450, Cyp26B1, and comparative expression analysis with Cyp26A1 during early murine development. Mech. Dev. 107, 195-201.[Medline]
Maden, M., Ong, D. E., Summerbell, D. and Chytil, F. (1989). Spatial distribution of cellular protein binding to retinoic acid in the chick limb bud. Nature 335, 733-735.
Masuya, H., Sagai, T., Moriwaki, K. and Shiroishi, T. (1997). Multigenic control of the localization of the zone of polarizing activity in limb morphogenesis in the mouse. Dev. Biol. 182, 42-51.[Medline]
Mendelsohn, C., Larkin, S., Mark, M., LeMeur, M., Clifford, J., Zelent, A. and Chambon, P. (1994). RAR beta isoforms: distinct transcriptional control by retinoic acid and specific spatial patterns of promoter activity during mouse embryonic development. Mech. Dev. 45, 227-241.[Medline]
Mercader, N., Leonardo, E., Piedra, M. E., Martinez-A., C., Ros, M. A. and Torres, M. (2000). Opposing RA and FGF signals control proximodistal vertebrate limb development through regulation of Meis genes. Development 127, 3961-3970.
Minowada, G., Jarvis, L. A., Chi, C. L., Neubuser, A., Sun, X., Hacohen, N., Krasnow, M. A. and Martin, G. R. (2000) Vertebrate Sprouty genes are induced by FGF signaling and can cause chondrodysplasia when overexpressed. Development 126, 4465-4475.
Moon, A. M. and Capecchi, M. R. (2000). Fgf8 is required for outgrowth and patterning of the limbs. Nature Genet. 26, 455-459.[Medline]
Moon, A. M., Boulet, A. M. and Capechi, M. R. (2000). Normal limb development in conditional mutants of Fgf4. Development 127, 989-996.
Niederreither, K., Ward, S. J., Dollé, P. and Chambon, P. (1996). Morphological and molecular characterization of retinoic acid-induced limb duplications in mice. Dev. Biol. 176, 185-198.[Medline]
Niederreither, K., McCaffery, P., Dräger, U. C., Chambon, P. and Dollé, P. (1997). Restricted expression and retinoic acid-induced downregulation of the retinaldehyde dehydrogenase type 2 (RALDH-2) gene during mouse development. Mech. Dev. 62, 67-78.[Medline]
Niederreither, K., Subbarayan, V., Dollé, P. and Chambon, P. (1999). Embryonic retinoic acid synthesis is essential for early mouse post-implantation development. Nature Genet. 21, 444-448.[Medline]
Niederreither, K., Vermot, J., Schuhbaur, B., Chambon, P. and Dollé, P. (2000). Retinoic acid synthesis and hindbrain patterning in the mouse embryo. Development 127, 75-85.
Niederreither, K., Vermot, J., Messaddeq, N., Schuhbaur, B., Chambon, P. and Dollé, P. (2001). Embryonic retinoic acid synthesis is essential for heart morphogenesis in the mouse. Development 128, 1019-1031.
Niswander, L., Tickle, C., Vogel, A., Booth, I. and Martin, G. R. (1993) FGF-4 replaces the apical ectodermal ridge and directs outgrowth and patterning of the limb. Cell 75, 579-587.[Medline]
Niswander, L., Jeffrey, S., Martin, G. R. and Tickle, C. (1994). A positive feedback loop coordinates growth and patterning in the vertebrate limb. Nature 371, 609-612.[Medline]
Ogura, T., Alvarez, I. S., Vogel, A., Rodriguez, C., Evans, R. M. and Izpisua Belmonte, J. C. (1996). Evidence that Shh cooperates with a retinoic acid inducible co-factor to establish ZPA-like activity. Development 122, 537-542.
Power, S. C., Lancman, J. and Smith, S. M. (1999). Retinoic acid is essential for Shh/Hoxd signaling during rat limb outgrowth but not for limb initiation. Dev. Dyn. 216, 469-480.[Medline]
Riddle, R. D., Johnson, R. L., Laufer, E. and Tabin, C. (1993). Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75, 1401-1416.[Medline]
Rossant, J., Zirngibl, R., Cado, D., Shago, M. and Giguère, V. (1991). Expression of a retinoic acid response element-hsplacZ transgene defines specific domains of transcriptional activity during mouse embryogenesis. Genes Dev. 5, 1333-1344.[Abstract]
Rutledge, J. C., Shourbaji A. G., Hughes, L. A., Polifka, J. E., Cruz, Y. P., Bishop, J. B. and Generoso, W. M. (1994). Limb and lower-body duplications induced by retinoic acid in mice. Proc. Natl. Acad. Sci. USA 91, 5436-5440.[Abstract]
Sekine, K., Ohuchi, H., Fujiwara, M., Yamasaki, M., Yoshizawa, T., Sato, T., Yagishita, N., Matsui, D., Koga, Y., Ithoh, N. and Kato, S. (1999). Fgf10 is essential for limb and lung formation. Nature Genet. 21, 138-141.[Medline]
Sordino, P., van der Hoeven, F. and Duboule, D. (1995). Hox gene expression in teleost fins and the origin of vertebrate digits. Nature 375, 678-681.[Medline]
Stratford, T., Horton, C. and Maden, M. (1996). Retinoic acid is required for the initiation of outgrowth in the limb bud. Curr. Biol. 6, 1124-1133.[Medline]
Stratford, T. H., Kostakopoulou, K. and Maden, M. (1997). Hoxb-8 has a role in establishing early anterior-posterior polarity in chick forelimb but not hindlimb. Development 124, 4225-4234.
Stratford, T., Logan, C., Zile, M. and Maden, M. (1999). Abnormal anteroposterior and dorsoventral patterning of the limb bud in the absence of retinoids. Mech. Dev. 81, 115-125.[Medline]
Sun, X., Lewandoski, M., Meyers, E. N., Liu, Y. H., Maxson, R. E. and Martin, G. R. (2000). Conditional inactivation of Fgf4 reveals complexity of signalling during limb bud development. Nature Genet. 25, 83-86.[Medline]
Swindell, E. C., Thaller, C., Sockanathan, S., Petkovich, M., Jessell, T. M. and Eichele, G. (1999). Complementary domains of retinoic acid production and degradation in the early chick embryo. Dev. Biol. 216, 282-296.[Medline]
Tickle, C., Alberts, B., Wolpert, L. and Lee, J. (1982). Local application of retinoic acid to the limb bond mimics the action of the polarizing region. Nature 296, 564-566.[Medline]
Thaller, C. and Eichele, G. (1987). Identification and spatial distribution of retinoids in the developing chick limb bud. Nature 327, 625-628.[Medline]
Wilson, J. G., Roth, C. B. and Warkany, J. (1953). An analysis of the syndrome of malformations induced by maternal vitamin A deficiency. Effects of restoration of vitamin A at various times during gestation. Amer. J. Anat. 92, 189-217.
Zakany, J. and Duboule, D. (1999). Hox genes in digit development and evolution. Cell Tissue Res. 296, 19-25.[Medline]
Zhao, D., McCaffery, P., Ivins, K. J., Neve, R. L., Hogan, P., Chin, W. W. and Dräger, U. C. (1996). Molecular identification of a major retinoic acid-synthesizing enzyme, a retinaldehyde dehydrogenase. Eur. J. Biochem. 15, 15-22.
Zuniga, A., Haramis, A. P. G., McMahon, A. P. and Zeller, R. (1999). Signal relay by BMP antagonism controls the SHH/FGF4 feedback loop in vertebrate limb buds. Nature 401, 598-602.[Medline]