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Originally published In Press as doi:10.1074/jbc.M401920200 on April 6, 2004

J. Biol. Chem., Vol. 279, Issue 25, 26698-26706, June 18, 2004
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Retinoic Acid Synthesis Controlled by Raldh2 Is Required Early for Limb Bud Initiation and Then Later as a Proximodistal Signal during Apical Ectodermal Ridge Formation*

Felix A. Mic, I. Ovidiu Sirbu, and Gregg Duester{ddagger}

From the Oncodevelopmental Biology Program, Burnham Institute, La Jolla, California 92037

Received for publication, February 22, 2004 , and in revised form, March 31, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We present evidence for the existence of two phases of retinoic acid (RA) signaling required for vertebrate limb development. Limb RA synthesis is under the control of retinaldehyde dehydrogenase-2 (Raldh2) expressed in the lateral plate mesoderm, which generates a proximodistal RA signal during limb outgrowth. We report that Raldh2-/- embryos lack trunk mesodermal RA activity and fail to initiate forelimb development. This is associated with deficient expression of important limb determinants Tbx5, Meis2, and dHand needed to establish forelimb bud initiation, proximal identity, and the zone of polarizing activity (ZPA), respectively. Limb expression of these genes can be rescued by maternal RA treatment limited to embryonic day 8 (E8) during limb field establishment, but the mutant forelimbs obtained at E10 display a significant growth defect associated with a smaller apical ectodermal ridge (AER), referred to here as an apical ectodermal mound (AEM). In these RA-deficient forelimbs, a ZPA expressing Shh forms, but it is located distally adjacent to the Fgf8 expression domain in the AEM rather than posteriorly as is normal. AER formation in Raldh2-/- forelimbs is rescued by continuous RA treatment through E10, which restores RA to distal ectoderm fated to become the AER. Our findings indicate the existence of an early phase of RA signaling acting upstream of Tbx5, Meis2, and dHand, followed by a late phase of RA signaling needed to expand AER structure fully along the distal ectoderm. During ZPA formation, RA acts early to activate expression of dHand, but it is not required later for Shh activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Retinoic acid (RA)1 functions as a ligand controlling the action of nuclear RA receptors essential for vertebrate embryonic development (1, 2). Studies of RA-deficient embryos firmly support the conclusion that RA is required for limb bud development, but the mechanism remains unclear (3-7). RA was initially proposed to play a role in limb bud development after it was observed that exogenous RA generated proximodistal duplications when administered to the blastema of regenerating axolotl limbs (8) and anteroposterior duplications when administered to the anterior region of chick limb buds (9). In the chick limb model, exogenous RA was found to induce sonic hedgehog (Shh), a known regulator of limb anteroposterior patterning expressed posteriorly in the zone of polarizing activity (ZPA) (10). However, further studies on chick limb buds have demonstrated that exogenous RA first induces the transcription factor dHand and then later Shh (11), and studies in mouse have shown that dHand is required for Shh induction in limb buds (12). This suggests that Shh activation by RA may be indirect. Endogenous RA was originally reported to be higher posteriorly in limb buds, supporting a role for RA in imparting anteroposterior information (13), but measurements by others have disputed this conclusion (14, 15). Also, the ZPA has been demonstrated to be devoid of local RA synthesis, indicating that the posterior region of limb buds does not generate RA (16, 17). Indeed, the nearest site of RA synthesis during limb bud development occurs in the proximal flanking mesoderm along the entire anteroposterior axis of the limb (18, 19). Thus, a recent conclusion that RA signaling functions differentially along the anteroposterior axis of the limb bud (7) is not consistent with the source of RA synthesis. Instead, differential RA signaling along the proximodistal axis seems more likely as supported by the early studies on axolotl limb regeneration (8) and more recent studies. By using inhibitors of RA synthesis or RA receptor antagonists, RA was reported to be required for Meis homeobox gene expression, which is normally limited to the proximal region of limb buds where it helps control proximodistal axis formation (6). Also, excess RA induces proximodistal limb reductions, and this is associated with an increase in Meis expression (20). It has been hypothesized that proximodistal limb expression of Meis genes is regulated by opposing signals, i.e. RA generated proximally acts positively and fibroblast growth factor-8 (FGF8) generated distally acts negatively (6).

RA synthesis from retinol (vitamin A) is a two-step enzymatic pathway with retinaldehyde as the intermediate. Genetic studies in mice have demonstrated that oxidation of retinol to retinaldehyde is ubiquitous during development as it is controlled by ubiquitously expressed alcohol dehydrogenase Adh3 and several additional overlapping tissue-specific enzymes (21). As for the second step, mouse genetic studies have shown that oxidation of retinaldehyde to RA is controlled by tissue-specific retinaldehyde dehydrogenase genes including Raldh1 (Aldh1a1) (22), Raldh2 (Aldh1a2) (19, 23), and Raldh3 (Aldh1a3) (24). Raldh2 is expressed in paraxial, intermediate, and lateral plate mesoderm where it generates RA for several morphogenetic processes including limb bud development (18, 19). Raldh2-/- mouse embryos display a growth defect from E8.5 to E10.5 followed by lethality at E11.5 (19, 23). RALDH2 generates most of the RA observed in midgestation mouse embryos, and although all-trans-RA and its isomer 9-cis-RA were both originally hypothesized to be important for development, retinoid rescue studies on Raldh2-/- mouse embryos have shown that only all-trans-RA is needed physiologically to correct the overall growth defect caused by a lack of RALDH2 (25). Raldh2-/- embryos rescued by continuous maternal RA treatment exhibit relatively normal limb development up to E14.5, but limited maternal RA treatment (only up to E8) results in defects in forelimb skeletal development (7) and muscle development (26).

We now provide further insight into the spatiotemporal mechanism of RA action during early forelimb bud development by examining unrescued and RA-rescued Raldh2-/- embryos. We find that RA signaling is needed at two distinct time points during early forelimb bud development. In the early phase, RA is needed for lateral plate mesoderm to initiate forelimb budding, with loss of RA being associated with loss of expression of three transcription factors important for limb development: Tbx5 needed specifically for forelimb bud initiation (27), Meis2 required for proximal limb identity (6, 20), and dHand, which provides competence to form a ZPA (11, 12). In the late phase, RA activity is localized differentially along the proximodistal axis of the developing forelimb bud (but uniformly along the anteroposterior axis), and RA is needed to allow the apical ectodermal ridge (AER) and its associated Fgf8 expression to expand fully along the distal ectoderm of the limb. This is important as Fgf8 expression in the AER is essential for maintenance of limb outgrowth (28).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Rescue of Raldh2-/- Embryos by Limited RA Treatment—Rescue of Raldh2-/- embryos by limited RA treatment was performed as described (19). Briefly, all-trans-RA was dissolved in corn oil and administered orally to timed-pregnant mice (2.5 mg/kg) at 12-h intervals on embryonic days E7.25, E7.75, and E8.25. Embryos were examined at stages E9.5 to E10.25. RA administered in this fashion was previously shown to be cleared by E9.25 (24 h after the last RA dose) through examination of the decay in {beta}-galactosidase activity induced by RA administration to mice carrying the RARE-lacZ RA-reporter gene (19). Thus, RARE-lacZ expression detected at E9.25 and later is indicative of endogenously synthesized RA, and does not represent the administered RA.

Rescue of Raldh2-/- Embryos by Continuous RA Treatment—Rescue of Raldh2-/- embryos by continuous RA treatment was performed similar to experiments described previously (25). Briefly, all-trans-RA was dissolved in corn oil and mixed with powdered food at 0.1 mg/g for treatment on E7.5 and at 0.25 mg/g for treatment on E8.5 to E10.5. Such food was prepared fresh each day. Previous studies have shown that this dietary RA treatment provides close to a physiological amount of RA to embryos as assessed by high pressure liquid chromatography analysis of treated E10.5 embryos (25).

Detection of Retinoic Acid Activity—Detection of RA activity was performed in embryos carrying the RARE-lacZ RA-reporter transgene which places lacZ (encoding {beta}-galactosidase) under the transcriptional control of a retinoic acid response element (RARE) (29). {beta}-galactosidase staining was performed for 18 h with 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside (X-gal) as substrate.

Detection of mRNA—Embryonic mRNA was detected by whole-mount in situ hybridization as described (30) except that proteinase K was omitted for examination of genes expressed in limb ectoderm (Fgf8 and Fgf4). Stained embryos were embedded in 3% agarose and sectioned at 50 µm with a vibratome. At least three embryos were examined for each expression pattern reported.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
RA Is Required for Initiation of Forelimb Bud Development and ZPA Competence—We have previously reported that Raldh2-/- embryos lack RA activity in the trunk mesoderm by examination of wild-type and Raldh2-/- embryos carrying a RARE-lacZ RA-reporter transgene (19). For comparison purposes, we show here the RARE-lacZ expression patterns of wild-type and Raldh2-/- embryos at E9.0 when the forelimb bud fields are being established (Fig. 1, A and B). The mutant lacks RA activity in the trunk mesoderm (including the lateral plate mesoderm where limb buds arise), although some RA activity is retained in the eye, heart, and neural tube possibly because of other RA-generating enzymes as suggested previously (19). At E10.25, RA activity should normally exist at the proximal base of the limbs, but the forelimb bud field of Raldh2-/- embryos still lacks RA activity and there is no sign of limb development (Fig. 1, C and D). Thus, Raldh2-/- embryos develop without any RA available to cells that establish the limb bud fields.



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  		FIG. 1.
RA is required in trunk mesoderm for establishment of the forelimb bud field. A and B, RA activity in E9.0 wild-type (WT) and Raldh2-/- embryos monitored by RARE-lacZ expression ({beta}-galactosidase activity). RA activity is lost in trunk mesoderm of Raldh2-/- embryos including the forelimb field lateral plate mesoderm but still present in eye, heart, and neural tube. C and D, RA activity is still missing in the lateral plate mesoderm of E10.25 Raldh2-/- embryos, but some activity is now present in the posterior mesonephros. E and F, Meis2 mRNA in Raldh2-/- E9.0 embryos is not detected in the somites and lateral plate mesoderm adjacent to the forelimb field, but hind-brain expression is still strong. G and H, dHand expression is not detected in the lateral plate mesoderm of an E9.5 Raldh2-/- embryo, but expression is still apparent in the heart and allantois; wild-type dHand expression extends to the posterior portion of the forelimb field. I and J, Tbx5 mRNA is absent in the forelimb field of an E9.5 Raldh2-/- embryo but still present in the eye and heart. K and L, whereas Fgf8 mRNA is detected in the distal ectoderm of an E9.5 wild-type forelimb bud, there is no detection in the forelimb field of an E9.5 Raldh2-/- embryo, although Fgf8 mRNA is present in the primitive streak. a, allantois; can, caudal neuropore; e, eye; f, forelimb bud; h, hindlimb bud; hb, hindbrain; he, heart; lpm, lateral plate mesoderm; m, mesonephros; n, neural tube; s, somite.

 
During limb bud initiation, several genes are activated in the lateral plate mesoderm that play essential roles. Meis2 encodes a homeobox transcription factor expressed in the lateral plate mesoderm and subsequently in the proximal limb mesoderm that is required as a determinant of proximal limb identity (31). In E9.0 Raldh2-/- embryos we found that Meis2 expression was not detected in the lateral plate mesoderm and somites as normal but was still present in the hindbrain (Fig. 1, E and F). dHand (Hand2) encodes a basic helix-loop-helix transcription factor expressed early in the lateral plate mesoderm that is required later for activation of Shh expression in the posterior limb bud during ZPA formation (11, 12). At E9.0, wild-type embryos expressed dHand in the lateral plate mesoderm along the posterior portion of the forelimb bud field where the ZPA will later form, but Raldh2-/- embryos exhibited no detection of dHand expression in that region although expression was still observed in the heart and allantois (Fig. 1, G and H). Tbx5 encodes a T-box transcription factor essential for forelimb bud development expressed specifically in that region of the lateral plate mesoderm that gives rise to the forelimb buds and then later throughout the forelimb bud mesoderm (27). In E9.5 Raldh2-/- embryos, Tbx5 expression in the forelimb bud field was absent although expression was still observed in the eye and heart (Fig. 1, I and J). Fgf8 is normally expressed at E9.5 in the distal ectoderm of wild-type forelimb buds, but Raldh2-/- embryos lacked Fgf8 expression in the forelimb field (Fig. 1, K and L). These findings suggest that RA signaling in the lateral plate mesoderm is required to make this tissue competent to form the limb bud fields and that RA acts upstream of genes needed for forelimb bud initiation (Tbx5), proximal identity (Meis2), and competence to form a ZPA (dHand).

RA Treatment on E7 to E8 Rescues Forelimb Bud Initiation but Not Outgrowth—Raldh2-/- embryos examined at E10.25 following limited maternal RA treatment from E7.25 to E8.25 were found to overcome the block in limb bud development. In such embryos hindlimb buds were relatively normal in appearance, but forelimb buds exhibited growth retardation (Fig. 2, A and B). Examination of RARE-lacZ expression in E10.25-rescued Raldh2-/- embryos revealed an absence of RA activity in the forelimb bud field, but some RA activity was observed in the hindlimb bud field in the vicinity of the mesonephros (Fig. 2, A and B). We have previously reported that rescued Raldh2-/- embryos retain RA activity in the mesonephros associated with expression of a related RA-generating gene, Raldh3 (19). Thus, Raldh3 or some other gene may be responsible for allowing apparently normal hindlimb bud outgrowth in rescued Raldh2-/- embryos, but evidently there is no other RA-generating enzyme that can supply RA to forelimb buds after RA treatment has ended. This suggests that there are two phases of RA action during forelimb budding and that our limited RA treatment has rescued only an early phase needed for limb bud initiation but not a late phase needed for outgrowth.



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  		FIG. 2.
RA treatment on E7 to E8 rescues forelimb bud initiation but not outgrowth. A and B, RA activity monitored by RARE-lacZ expression in E10.25 wild-type (WT) and Raldh2-/- embryos following limited maternal RA treatment from E7.25 to E8.25; in rescued Raldh2-/- embryos RA activity is absent in the lateral plate mesoderm near the forelimb bud that shows growth retardation, but RA is still present in the mesonephros adjacent to the hindlimb which shows normal growth. C and D, limited RA treatment restores Meis2 mRNA in an E10.25 rescued Raldh2-/- embryo to the lateral plate mesoderm adjacent to both forelimbs and hindlimbs. E and F, limited RA treatment restores Tbx5 mRNA to the growth-retarded forelimb bud of an E10.25 rescued Raldh2-/- embryo. G and H, Fgf10 expression is present in the growth-retarded forelimb bud of an E9.75-rescued Raldh2-/- embryo. f, forelimb bud; h, hindlimb bud; lpm, lateral plate mesoderm; m, mesonephros.

 
Examination of E10.25-rescued Raldh2-/- embryos revealed that Meis2 expression was restored to the lateral plate mesoderm adjacent to the forelimb and hindlimb bud fields (Fig. 2, C and D). Also, E10.25-rescued Raldh2-/- embryos exhibited Tbx5 expression throughout the small growth-retarded forelimb bud (Fig. 2, E and F). We also examined expression of Fgf10, which generates a mesodermal FGF signal required for limb bud initiation (32). Fgf10 is normally expressed throughout the early forelimb bud mesoderm and functions downstream of Tbx5 but upstream of AER formation (33). Rescued Raldh2-/- embryos exhibited Fgf10 expression at E9.75 throughout the small growth-retarded forelimb bud (Fig. 2, G and H). These results indicate that limited maternal RA treatment of Raldh2-/- embryos can simulate an early phase of embryonic Raldh2 function needed for initiation of forelimb and hindlimb bud development but that a late phase of Raldh2 function is also needed for outgrowth of forelimb buds. It can be concluded that this late phase of RA action during forelimb bud development is downstream of Meis2, Tbx5, and Fgf10 as all of these genes are expressed in growth-retarded mutant forelimb buds. The late phase of RA action is also downstream of dHand as previous studies have shown that a similar limited RA treatment of Raldh2-/- embryos results in expression of dHand in growth-retarded forelimb buds (7).

Raldh2 Is Required for AER Formation in Forelimb Buds—The apical ectodermal ridge is characterized as a distal thickening of the ectoderm necessary for limb proximodistal outgrowth (34). The AER is derived from distal-ventral limb ectoderm with Fgf8 expression being a morphological marker of this tissue. Fgf8 expression also marks the mature AER and is necessary for AER function during proximodistal limb outgrowth (28). The growth-retarded Raldh2-/- forelimb buds obtained at E10.25 following limited RA treatment were found to lack Fgf8 mRNA along the AER and instead displayed Fgf8 expression in a small mound located distally and centrally (Fig. 3, A and B). Transverse sections through this small distocentral mound of Fgf8 expression in a rescued Raldh2-/- forelimb bud indicated the existence of a distal ectodermal thickening containing Fgf8 mRNA, although it is somewhat smaller than the distal thickening observed in a central transverse section of a wild-type forelimb bud (Fig. 3, C and D). A transverse section posterior to the mound of Fgf8 expression in a rescued Raldh2-/- forelimb bud revealed a lack of Fgf8 mRNA and no evidence of a distal ectodermal thickening in contrast to a wild-type forelimb bud (Fig. 3, E and F). AER structure was also missing in anterior transverse sections of a rescued Raldh2-/- forelimb bud but apparent in wild type (data not shown). The small central ectodermal thickening found in rescued Raldh2-/- forelimb buds is referred to as an apical ectodermal mound rather than a ridge as it lacks any significant length along the anteroposterior axis. These findings demonstrate that continued action of Raldh2 after forelimb bud initiation is necessary to produce a complete AER.



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  		FIG. 3.
Raldh2 is required for AER formation in forelimb buds. Fgf8 mRNA was examined in E10.25 wild-type and Raldh2-/- (-/-) forelimb buds rescued by limited maternal RA treatment from E7.25 to E8.25. A and B, rescued Raldh2-/- forelimb buds display greatly reduced outgrowth and an abnormally small domain of Fgf8 mRNA located distally and centrally. AER structure was examined in transverse sections through the center (C and D) and posterior (E and F) region of the wild-type (WT) and rescued Raldh2-/- forelimb buds shown in A and B. A distal ectodermal thickening exhibiting Fgf8 mRNA is observed in wild-type forelimb buds both centrally and posteriorly representing the AER (C and E). However, in rescued Raldh2-/- forelimb buds such a distal thickening is observed centrally (D) but not posteriorly (F), demonstrating the lack of a true AER. We refer to the central thickening as an AEM instead of a ridge. A, anterior; D, dorsal; di, distal; P, posterior; pr, proximal; V, ventral.

 
Several FGF genes are expressed in the AER, but it has been conclusively demonstrated through analysis of double conditional null mutants that elimination of just Fgf8 and Fgf4 prevents limb bud proximodistal outgrowth (28). Fgf8 is normally expressed at early stages (E9.0 to 9.5) in ventral surface ectoderm and then at later stages as a band all along the anteroposterior axis of the distal ectoderm, which by E9.5 to 9.75 becomes morphologically distinct as the AER (35, 36). Fgf8 mRNA was not observed in E9.5 Raldh2-/- forelimbs rescued by limited RA treatment, whereas Fgf8 was apparent in the ventral ectoderm of wild-type forelimb buds (Fig. 4, A and B). At E9.75, Fgf8 mRNA was observed in rescued Raldh2-/- forelimbs but was limited to a small distocentral domain (Fig. 4, C and D), similar to that seen at E10.25 (Fig. 3, A and B). Thus, a loss of Raldh2 leads to a delay in the onset of forelimb bud Fgf8 expression followed by expression limited to a centrally located apical ectodermal mound.



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  		FIG. 4.
Raldh2 deficiency disrupts Fgf8, Fgf4, and Shh expression during outgrowth of forelimb buds. Wild-type (WT) and Raldh2-/- embryos rescued by limited maternal RA treatment from E7.25 to E8.25 were examined by whole-mount in situ hybridization. A and B, at E9.5 no expression of Fgf8 mRNA is detected in the rescued mutant. C and D, at E9.75 Fgf8 mRNA in the rescued mutant is limited to a small distal-central domain. E and F, at E10.25 Fgf4 mRNA is not detected in the rescued mutant. G and H, at E9.75 Shh mRNA is not detected in the rescued mutant. I and J, at E10.25 Shh mRNA in the rescued mutant is observed in a distal-central domain rather than posteriorly. A, anterior; D, dorsal; di, distal; P, posterior; pr, proximal; V, ventral.

 
Fgf4 expression is normally limited to the posterior two-thirds of the AER where it overlaps with Fgf8 expression and partially compensates for a loss of Fgf8 function (35, 36). However, loss of Fgf4 function does not affect limb bud outgrowth (37, 38), indicating the dominance of Fgf8. Raldh2-/- forelimbs rescued by limited RA treatment exhibited a complete loss of Fgf4 mRNA at E10.25 (Fig. 4, E and F) and at E9.75 (data not shown).

Shh Is Expressed in Raldh2-/- Forelimb Buds Rescued by Limited RA Treatment—Shh expression in posterior limb bud mesoderm regulates anteroposterior patterning required to specify digit number and identity (10, 39, 40). Genetic studies have shown that Fgf8 (28, 35, 36) and dHand (12) are required for proper initiation and maintenance of Shh expression in limb buds. Studies using inhibitors of RA synthesis have demonstrated a reduction in Shh expression in the ZPA suggesting that endogenous RA may play a role (3, 41), but it is unclear if RA action on Shh is direct. When Raldh2-/- embryos were rescued by limited RA treatment and examined at E9.75, the stage when forelimb bud Shh expression is normally first observed, we found that Shh was not expressed (Fig. 4, G and H). However, at E10.25 Shh expression was observed in rescued Raldh2-/- forelimb buds but in a distocentral position rather than posteriorly (Fig. 4, I and J). Shh expression in E10.25 rescued Raldh2-/- forelimb buds (Fig. 4J) was located in distocentral mesoderm adjacent to the distocentral apical ectodermal mound where Fgf8 expression was still observed (Fig. 3B). As RA activity is not present in the forelimb bud of rescued Raldh2-/- embryos (Fig. 2B), RA is not required to induce Shh expression.

Raldh2 Generates a Proximodistal RA Signal in Forelimb Buds—As the RA receptors RAR{alpha} and RAR{gamma} are both expressed uniformly throughout the limb bud during AER and ZPA formation (42), differential control of RA signaling in various regions of the limb bud must depend upon differential availability of the ligand RA. This was examined using embryos carrying the RARE-lacZ transgene to monitor RA activity. In wild-type forelimb buds, RARE-lacZ expression was observed completely across the proximodistal axis at E9.25 but at E9.75 RARE-lacZ was down-regulated in the distal region of the forelimb bud where the AER develops revealing the existence of a proximal-high to distal-low RA signal (Fig. 5, A and B). At E10.25, the distal zone lacking RARE-lacZ expression was expanded, and no RA signal was apparent in the distal third of the forelimb bud (Fig. 5C). By comparison, E10.25 Raldh2-/- forelimb buds obtained by limited RA treatment completely lacked RARE-lacZ expression (Fig. 5D). These findings provide evidence that RA signaling controlled by Raldh2 is normally occurring in distal tissue at E9.25 prior to AER formation when Fgf8 activation occurs but that by E9.75 RA signaling is not observed distally in the AER that has formed. Also, from E9.75 to E10.25 RA signaling is being down-regulated in posterodistal tissue where Shh is being activated to form the ZPA, suggesting that RA signaling is not directly needed for Shh induction.



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  		FIG. 5.
Raldh2 expressed proximally generates a RA signal along the proximodistal axis of the forelimb bud. A-D, RARE-lacZ expression observed from a dorsal whole-mount view indicates RA activity in wild-type (WT) forelimb buds at E9.25 (A), E9.75 (B), and E10.25 (C) but not in E10.25 Raldh2-/- forelimb buds rescued by limited maternal RA treatment from E7.25 to E8.25 (D). In wild-type forelimb buds, RA activity is all along the proximodistal axis at E9.25 but has begun to clear distally by E9.75. E-G, transverse sections through E9.25 wild-type forelimb buds demonstrate that Raldh2 mRNA is limited to proximal mesoderm (E), whereas RARE-lacZ expression exists all along the proximodistal axis with a proximal-dorsal high point and a distal-ventral low point (F); Fgf8 mRNA is limited to distal-ventral ectoderm (G). H and I, wild-type whole-mount forelimbs examined at E9.5 show that RA synthesis and activity normally exist across the forelimb anteroposterior axis; this is demonstrated by Raldh2 mRNA (H) and RARE-lacZ (I) expression. J, RARE-lacZ expression is absent in E9.5 Raldh2-/- forelimb buds rescued by limited maternal RA treatment from E7.25 to E8.25. A, anterior; f, forelimb bud; lbm, limb bud mesoderm; n, neural tube; P, posterior; s, somite; sp, somatopleure component of the lateral plate mesoderm.

 
RA Signal Initially Reaches the Distal Forelimb Ectoderm Where Fgf8 Is Expressed—Transverse sections of E9.25 wild-type forelimb buds demonstrated that even at this early stage RA signaling is differentially localized along the proximodistal axis. Raldh2 mRNA was limited to proximal limb mesoderm including the somatopleure component of the lateral plate mesoderm, which gives rise to much of the limb mesoderm (Fig. 5E). At E9.25, RARE-lacZ expression was observed all across the proximodistal axis of the forelimb bud but with highest expression proximally and lowest expression in the distal ectoderm where Fgf8 is expressed (Fig. 5, F and G). These findings suggest that RA synthesized by RALDH2 in proximal mesoderm is released and travels distally to the ectoderm inducing RARE-lacZ expression all along its path.

Raldh2 Generates Uniform RA Signal across the Anteroposterior Axis—Raldh2 mRNA in E9.5 wild-type embryos was detected uniformly along the anteroposterior axis of the somatopleure component of the lateral plate mesoderm adjacent to proximal forelimb mesoderm (Fig. 5H). At E9.5, RARE-lacZ expression in wild-type forelimb buds was observed uniformly across the anteroposterior axis in wild-type embryos but was absent in Raldh2-/- forelimb buds rescued by limited RA treatment (Fig. 5, I and J). It can also be seen that RA signaling exists uniformly across the anteroposterior axis of the mouse forelimb bud from E9.25 to E10.25, encompassing the stages when the AER and ZPA develop (Fig. 5, A-C).

Continuous RA Treatment Rescues Raldh2-/- AER by Providing a Distal RA Signal—Previous studies have shown that continuous maternal RA treatment from E7 to E10 substantially rescues forelimb bud outgrowth (7). We examined where RA signaling occurs during continuous RA treatment to address the mechanism. A comparison was made of RARE-lacZ expression in Raldh2-/- forelimb buds subjected to limited RA treatment from E7.25 to E8.25 or to continuous RA treatment from E7 until the point of analysis on E9.5 or E10.25. Limited RA treatment did not restore RARE-lacZ expression to Raldh2-/- forelimb buds at E9.5 (Fig. 6, A and C), but continuous RA treatment did restore substantial RARE-lacZ expression to Raldh2-/- forelimb buds (Fig. 6, B and D). The source of limb bud RA during continuous RA treatment of mutants may have been the somatopleure component of the lateral plate mesoderm (a primitive vasculature system) that exhibited very high RA activity (Fig. 6B). Importantly, continuous RA treatment resulted in Raldh2-/- forelimb buds receiving a RA signal that reached the distal-ventral ectoderm that is fated to become the AER (Fig. 6D). The limb RA signal following continuous RA treatment of Raldh2-/- embryos was higher ventrally and did not reach the dorsal limb (Fig. 6D). As the limb RA signal in wild-type embryos was higher dorsally but still reached the ventral limb (Fig. 5F), these findings together suggest that RA may act ventrally in the distal limb for AER formation.



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  		FIG. 6.
Continuous RA treatment generates a proximodistal RA signal in Raldh2-/- forelimb buds and rescues outgrowth. Comparison of forelimb bud development in Raldh2-/- embryos following limited maternal RA treatment from E7.25 to E8.25 (A, C, E, and G) or continuous maternal RA treatment from E7 up to the point of analysis on E9.5 or E10.25 (B, D, F, and H). A-D, transverse sections showing RARE-lacZ expression at E9.5 indicate that continuous RA treatment results in much higher RA activity in the somatopleure mesoderm than limited RA treatment; continuous RA treatment results in RA activity in the forelimb bud itself (arrowhead) (B) with RA activity reaching the distal-ventral region fated to become the AER (D). E-H, examination of Fgf8 mRNA shows that continuous (but not limited) RA treatment rescues Fgf8 expression at E9.5 (E and F), and increases growth and allows the AER to extend more fully along the anteroposterior axis at E10.25 (G and H). A, anterior; D, dorsal; di, distal; n, neural tube; P, posterior; pr, proximal; sp, somatopleure; V, ventral.

 
Fgf8 expression was restored to Raldh2-/- forelimb buds at E9.5 with continuous RA treatment (Fig. 6F) but not with limited RA treatment (Fig. 6E). The pattern of Fgf8 expression observed at E9.5 following continuous RA treatment was not quite restored to wild type (Fig. 4A), but it did exhibit a band across the anteroposterior axis (Fig. 6F). At E10.25, limited RA treatment resulted in a growth-restricted Raldh2-/- forelimb bud with a small mound of Fgf8 expression as described above (Fig. 6G), whereas continuous RA treatment resulted in a larger Raldh2-/- forelimb bud with an anteroposterior band of Fgf8 expression (Fig. 6H). Significant restoration of the anteroposterior band of Fgf8 expression with continuous RA treatment suggests that RA increases AER function and enhances forelimb bud outgrowth.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The studies reported here describe two distinct phases of RA signaling required for limb bud development. In the early phase, RA is synthesized by RALDH2 throughout the lateral plate mesoderm including the regions where the forelimb and hindlimb fields arise. We have shown that RA signaling in the lateral plate mesoderm is required at this stage to initiate forelimb budding and activate expression of Tbx5, Meis2, and dHand. During subsequent forelimb bud outgrowth, a late phase of RA signaling occurs that is dependent upon RA synthesized proximally by RALDH2, which forms a proximodistal signal needed for AER formation.

Previous studies have shown that zebrafish raldh2 mutants fail to initiate pectoral fin bud development and lack Tbx5 expression (43, 44), thus indicating a conserved role for RALDH2 in synthesizing RA for limb bud initiation. However, the zebrafish studies did not address whether RALDH2 is also involved in downstream events such as AER formation as we now report. An earlier report suggested that RA may affect chick AER formation as it was demonstrated that excess RA resulted in a reorganization of the AER affecting its width and height (45). We have now found that a lack of RA during mouse forelimb bud outgrowth results in a small AER that we term an apical ectodermal mound. As Fgf8 is still expressed without RA, we suggest that RA signaling regulates development of an AER structure that then allows Fgf8 expression to fully expand along the distal ectoderm. A model for RA action in limb bud development is presented (Fig. 7).



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  		FIG. 7.
Model for RA action during limb bud development. A, two phases of RA action during limb bud development are shown in relationship to several other factors known to play important roles in limb development (6, 11, 12, 27, 28, 47). In the early phase, RA acts upstream of dHand to initiate ZPA formation, and RA acts upstream of Meis2 and Tbx5 to initiate limb budding. In the late phase, RA is needed to form an AER structure that extends fully along the distal region of the limb; at this stage RA may function in parallel with Fgf8, which is needed to establish AER function. B, forelimb bud outgrowth is retarded when only the early phase of RA action occurs (in partially rescued Raldh2-/- embryos after limited RA treatment). A proximodistal RA signal normally generated by Raldh2 expressed proximally in the lateral plate mesoderm is required in the late phase of RA signaling to develop an AER fully along the anteroposterior axis of the distal ectoderm. A lack of RA signaling during the late phase does not prevent formation of the ZPA as Shh is still expressed, and a small apical ectodermal mound (AEM) expressing Fgf8 also forms. As studies by others have shown that limited RA rescue of Raldh2-/- embryos also restores dHand expression (7), dHand in mesenchyme in conjunction with Fgf8 in the AEM may be responsible for induction of Shh.

 
Early Phase of Limb RA Signaling Activates Genes in the Lateral Plate Mesoderm—We have demonstrated through loss-of-function studies that the early phase of RA synthesis by RALDH2 is necessary for lateral plate mesodermal expression of three transcription factors essential for limb development (Tbx5, Meis2, and dHand). Our findings are consistent with previous studies demonstrating that Meis2 expression in the proximal limb mesoderm is reduced when RA receptor antagonists or RA synthesis inhibitors are introduced into chick limb buds (6) and that Meis2 expression is increased following a teratogenic dose of RA (6, 20). We further demonstrate that expression of Tbx5 and Meis2 is observed in Raldh2-/- forelimb buds rescued by maternal RA treatment limited to E8 during limb field establishment. A previous study demonstrated that dHand expression is restored to Raldh2-/- forelimb buds by a similar RA treatment (7). As Tbx5, Meis2, and dHand are all expressed early in lateral plate mesoderm along with Raldh2 prior to forelimb bud initiation, induction of these genes (directly or indirectly) by RA synthesized from RALDH2 represents an early cell-autonomous event in limb bud initiation. Because RA activity later appears in the distal limb bud where Raldh2 is not expressed, this suggests that RA also acts in a cell-nonautonomous manner.

Late Phase of Forelimb Bud RA Signaling Is Required for AER Formation—In the late phase of RA signaling subsequent to limb bud initiation, we have shown that RA synthesized by RALDH2 in the proximal limb mesoderm travels to the distal ectoderm leading to generation of a proximodistal RA signal needed for AER formation in forelimb buds. We have found that continuous maternal RA treatment restores RA activity in the distal-ventral region of Raldh2-/- forelimbs (the region fated to become the AER) and that Fgf8 expression and AER function are both restored. Thus, our results provide evidence that endogenously synthesized RA functions in a cell-nonautonomous manner as a signal needed for AER formation. If RA treatment is withdrawn from Raldh2-/- embryos just prior to forelimb bud outgrowth (E8), we found that forelimb buds display a growth restriction and develop a small apical ectodermal mound expressing Fgf8 centrally instead of an AER expressing Fgf8 along its entire anteroposterior length (also Fgf4 expression is completely lost). Because of the essential role of the AER in limb outgrowth (34), the lack of a complete AER in these partially rescued Raldh2-/- forelimbs is consistent with the observed reduction in forelimb bud outgrowth. Other studies have shown that mutation of both Fgf8 and Fgf4 in mouse limb buds completely blocks AER function and arrests limb development at the early bud stage, but Fgf8-/-: Fgf4-/- limb buds still develop an AER structure, i.e. a distal ectodermal thickening (28). As a small distal ectodermal thickening containing Fgf8 expression is still observed in partially rescued Raldh2-/- forelimbs, this suggests that RA signaling regulates one or more genes distinct from Fgf8 that are involved in allowing an AER structure to fully spread out over the distal ectoderm. As RA signaling is uniform across the anteroposterior axis of the forelimb bud, this further supports a role in formation of the AER that develops distally but across the entire anteroposterior axis.

Vitamin A-deficient quail (4) and rat (5) embryos also have reduced forelimb bud outgrowth and reduced Fgf4 expression, but the effect on Fgf8 expression was less severe than that observed in rescued Raldh2-/- forelimb buds obtained with limited RA treatment. In the quail model there was evidence that Fgf8 expression (hence the AER) was often shorter along the anteroposterior axis (4), but it was not as short as we observed in Raldh2-/- forelimb buds.

RA Signaling Is Required to Activate dHand but Not Shh during ZPA Formation—ZPA formation requires at least three key genes, dHand, Fgf8, and Shh. dHAND functions as a transcriptional activator for Shh posteriorly in the ZPA (12), and FGF8 signaling provides a distal signal needed for Shh expression (28). The SHH produced then functions as a secreted ligand that generates anteroposterior patterning (10). The involvement of RA in ZPA formation is unclear. An exogenous source of RA added to the anterior limb bud stimulates formation of an ectopic ZPA resulting in limb duplications in chick embryos (9). Such RA treatment leads to induction of Shh in limb tissue lying between the source of RA and the AER where Fgf8 is expressed (10). The observation that Shh is not induced all around the RA source has been resolved by further studies demonstrating that dHand is first induced all around the RA source and then later Shh is induced, thus suggesting that Shh induction by RA is indirect (11). Our genetic findings support this conclusion as we have found that dHand expression in the limb field requires RA synthesized by RALDH2 early in the lateral plate mesoderm. Thus, our studies indicate that the previous observation of dHand induction following application of exogenous RA to anterior chick limb buds (11) reflects a normal role for RA in dHand gene activation to make limbs competent to express Shh at a later stage. Also, as Shh expression initiates in partially rescued Raldh2-/- forelimb buds during a time when RA activity is no longer detected, RA signaling is not directly needed for Shh activation.

It has been previously reported that dHand expression in partially rescued Raldh2-/- forelimb buds is expanded anteriorly rather than being limited posteriorly (7) (see Fig. 7). From these data the conclusion was made that differential RA signaling along the anteroposterior axis may participate in ZPA formation by limiting expression of dHand to the posterior limb bud (7). However, as our results indicate that RA signaling occurs uniformly along the anteroposterior axis, we suggest that some other factor is required to limit dHand expression posteriorly. In fact, other studies have shown that Gli3 expressed in the anterior limb antagonizes dHand expression (46). Also, the apparent anterior expansion of dHand expression observed in partially rescued Raldh2-/- forelimb buds may instead be the result of preferential growth of the posterior portion of these growth-retarded limbs. As for Shh expression in partially rescued Raldh2-/- forelimb buds, its appearance anteriorly and distally may be the result of the anterior expansion of dHand expression coupled with the persistent expression of Fgf8 distally in the apical ectodermal mound. We suggest that dHand and Fgf8 may normally provide sufficient posterior and distal information to limit Shh expression to the ZPA and that the involvement of RA in this process is limited to activation of dHand expression prior to limb bud initiation.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM62848 (to G. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Oncodevelopmental Biology Program, Burnham Inst., 10901 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-646-3138; Fax: 858-646-3195; E-mail: duester{at}burnham.org.

1 The abbreviations used are: RA, retinoic acid; AER, apical ectodermal ridge; AEM, apical ectodermal mound; Fgf8, fibroblast growth factor-8 gene; RALDH2, retinaldehyde dehydrogenase-2; Raldh2, RALDH2 gene; Shh, sonic hedgehog gene; ZPA, zone of polarizing activity; E8, embryonic day 8. Back


   ACKNOWLEDGMENTS
 
We thank the following for mouse cDNA probes: P. Gruss (Meis2), E. Olson (dHand), G. Martin (Fgf8 and Fgf4), A. McMahon (Shh), V. Papaioannou (Tbx5), and N. Itoh (Fgf10). We also thank J. Rossant for RARE-lacZ mice.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Zile, M. H. (2001) J. Nutr. 131, 705-708[Abstract/Free Full Text]
  2. Clagett-Dame, M., and DeLuca, H. F. (2002) Annu. Rev. Nutr. 22, 347-381[CrossRef][Medline] [Order article via Infotrieve]
  3. Stratford, T., Horton, C., and Maden, M. (1996) Curr. Biol. 6, 1124-1133[CrossRef][Medline] [Order article via Infotrieve]
  4. Stratford, T., Logan, C., Zile, M., and Maden, M. (1999) Mech. Dev. 81, 115-125[CrossRef][Medline] [Order article via Infotrieve]
  5. Power, S. C., Lancman, J., and Smith, S. M. (1999) Dev. Dyn. 216, 469-480[CrossRef][Medline] [Order article via Infotrieve]
  6. Mercader, N., Leonardo, E., Piedra, M. E., Martínez-A, C., Ros, M. A., and Torres, M. (2000) Development 127, 3961-3970[Abstract]
  7. Niederreither, K., Vermot, J., Schuhbaur, B., Chambon, P., and Dollé, P. (2002) Development 129, 3563-3574[Abstract/Free Full Text]
  8. Maden, M. (1982) Nature 295, 672-675[CrossRef][Medline] [Order article via Infotrieve]
  9. Tickle, C., Alberts, B. M., Wolpert, L., and Lee, J. (1982) Nature 296, 564-565[CrossRef][Medline] [Order article via Infotrieve]
  10. Riddle, R. D., Johnson, R. L., Laufer, E., and Tabin, C. (1993) Cell 75, 1401-1416[CrossRef][Medline] [Order article via Infotrieve]
  11. Fernandez-Teran, M., Piedra, M. E., Kathiriya, I. S., Srivatava, D., Rodriguez-Rey, J. C., and Ros, M. A. (2000) Development 127, 2133-2142[Abstract]
  12. Charite, J., McFadden, D. G., and Olson, E. N. (2000) Development 127, 2461-2470[Abstract]
  13. Thaller, C., and Eichele, G. (1987) Nature 327, 625-628[CrossRef][Medline] [Order article via Infotrieve]
  14. Tamura, K., Ohsugi, K., and Ide, H. (1990) Dev. Biol. 140, 20-26[CrossRef][Medline] [Order article via Infotrieve]
  15. Scott, W. J., Jr., Walter, R., Tzimas, G., Sass, J. O., Nau, H., and Collins, M. D. (1994) Dev. Biol. 165, 397-409[CrossRef][Medline] [Order article via Infotrieve]
  16. Wanek, N., Gardiner, D. M., Muneoka, K., and Bryant, S. V. (1991) Nature 350, 81-83[CrossRef][Medline] [Order article via Infotrieve]
  17. Noji, S., Nohno, T., Koyama, E., Muto, K., Ohyama, K., Aoki, Y., Tamura, K., Ohsugi, K., Ide, H., Taniguchi, S., and Saito, T. (1991) Nature 350, 83-86[CrossRef][Medline] [Order article via Infotrieve]
  18. Swindell, E. C., Thaller, C., Sockanathan, S., Petkovich, M., Jessell, T. M., and Eichele, G. (1999) Dev. Biol. 216, 282-296[CrossRef][Medline] [Order article via Infotrieve]
  19. Mic, F. A., Haselbeck, R. J., Cuenca, A. E., and Duester, G. (2002) Development 129, 2271-2282[Abstract/Free Full Text]
  20. Qin, P., Cimildoro, R., Kochhar, D. M., Soprano, K. J., and Soprano, D. R. (2002) Teratology 66, 224-234[CrossRef][Medline] [Order article via Infotrieve]
  21. Molotkov, A., Fan, X., Deltour, L., Foglio, M. H., Martras, S., Farrés, J., Parés, X., and Duester, G. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5337-5342[Abstract/Free Full Text]
  22. Fan, X., Molotkov, A., Manabe, S.-I., Donmoyer, C. M., Deltour, L., Foglio, M. H., Cuenca, A. E., Blaner, W. S., Lipton, S. A., and Duester, G. (2003) Mol. Cell. Biol. 23, 4637-4648[Abstract/Free Full Text]
  23. Niederreither, K., Subbarayan, V., Dollé, P., and Chambon, P. (1999) Nat. Genet. 21, 444-448[CrossRef][Medline] [Order article via Infotrieve]
  24. Dupé, V., Matt, N., Garnier, J.-M., Chambon, P., Mark, M., and Ghyselinck, N. B. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 14036-14041[Abstract/Free Full Text]
  25. Mic, F. A., Molotkov, A., Benbrook, D. M., and Duester, G. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 7135-7140[Abstract/Free Full Text]
  26. Mic, F. A., and Duester, G. (2003) Dev. Biol. 264, 191-201[CrossRef][Medline] [Order article via Infotrieve]
  27. Ahn, D.-G., Kourakis, M. J., Rohde, L. A., Silver, L. M., and Ho, R. K. (2002) Nature 417, 754-758[CrossRef][Medline] [Order article via Infotrieve]
  28. Sun, X., Mariani, F. V., and Martin, G. R. (2002) Nature 418, 501-508[CrossRef][Medline] [Order article via Infotrieve]
  29. Rossant, J., Zirngibl, R., Cado, D., Shago, M., and Giguère, V. (1991) Genes Dev. 5, 1333-1344[Abstract/Free Full Text]
  30. Wilkinson, D. G. (1992) in In Situ Hybridization: A Practical Approach (Wilkinson, D. G., ed) pp. 75-83, IRL Press, Oxford
  31. Mercader, N., Leonardo, E., Azpiazu, N., Serrano, A., Morata, G., Martinez, C., and Torres, M. (1999) Nature 402, 425-429[CrossRef][Medline] [Order article via Infotrieve]
  32. Sekine, K., Ohuchi, H., Fujiwara, M., Yamasaki, M., Yoshizawa, T., Sato, T., Yagishita, N., Matsui, D., Koga, Y., Itoh, N., and Kato, S. (1999) Nat. Genet. 21, 138-141[CrossRef][Medline] [Order article via Infotrieve]
  33. Ng, J. K., Kawakami, Y., Buscher, D., Raya, A., Itoh, T., Koth, C. M., Esteban, C. R., Rodriquez-Leon, J., Garrity, D. M., Fishman, M. C., and Belmonte, J. C. I. (2002) Development 129, 5161-5170[Abstract/Free Full Text]
  34. Saunders, J. W., Jr. (1948) J. Exp. Zool. 108, 363-403[CrossRef][Medline] [Order article via Infotrieve]
  35. Lewandoski, M., Sun, X., and Martin, G. R. (2000) Nat. Genet. 26, 460-463[CrossRef][Medline] [Order article via Infotrieve]
  36. Moon, A. M., and Capecchi, M. R. (2000) Nat. Genet. 26, 455-459[CrossRef][Medline] [Order article via Infotrieve]
  37. Sun, X., Lewandoski, M., Meyers, E. N., Liu, Y. H., Maxson, R. E., Jr., and Martin, G. R. (2000) Nat. Genet. 25, 83-86[CrossRef][Medline] [Order article via Infotrieve]
  38. Moon, A. M., Boulet, A. M., and Capecchi, M. R. (2000) Development 127, 989-996[Abstract]
  39. Litingtung, Y., Dahn, R. D., Li, Y., Fallon, J. F., and Chiang, C. (2002) Nature 418, 979-983[CrossRef][Medline] [Order article via Infotrieve]
  40. te Welscher, P., Zuniga, A., Kuijper, S., Drenth, T., Goedemans, H. J., Meijlink, F., and Zeller, R. (2002) Science 298, 827-830[Abstract/Free Full Text]
  41. Helms, J. A., Kim, C. H., Eichele, G., and Thaller, C. (1996) Development 122, 1385-1394[Abstract]
  42. Dollé, P., Ruberte, E., Kastner, P., Petkovich, M., Stoner, C. M., Gudas, L. J., and Chambon, P. (1989) Nature 343, 702-705
  43. Begemann, G., Schilling, T. F., Rauch, G. J., Geisler, R., and Ingham, P. W. (2001) Development 128, 3081-3094[Abstract/Free Full Text]
  44. Grandel, H., Lun, K., Rauch, G. J., Rhinn, M., Piotrowski, T., Houart, C., Sordino, P., Küchler, A. M., Schulte-Merker, S., Geisler, R., Holder, N., Wilson, S. W., and Brand, M. (2002) Development 129, 2851-2865[Abstract/Free Full Text]
  45. Tickle, C., Crawley, A., and Farrar, J. (1989) Development 106, 691-705[Abstract/Free Full Text]
  46. te Welscher, P., Fernandez-Teran, M., Ros, M. A., and Zeller, R. (2002) Genes Dev. 16, 421-426[Abstract/Free Full Text]
  47. Barrow, J. R., Thomas, K. R., Boussadia-Zahui, O., Moore, R., Kemler, R., Capecchi, M. R., and McMahon, A. P. (2003) Genes Dev. 17, 394-409[Abstract/Free Full Text]

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