1 Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, PA, USA
2 Department of Molecular Genetics, University of Texas MD Anderson Cancer Center, Houston, TX, USA
3 Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
4 Procter & Gamble Pharmaceuticals, Mason, OH, USA
*Author for correspondence (e-mail: crenshab{at}mail.med.upenn.edu)
Accepted August 21, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Apical ectodermal ridge, Bone morphogenetic protein receptor, Dorsal-ventral patterning, Engrailed 1, Limb development, loxP/cre conditional knockout, Mouse
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Limbs grow and develop with proximal structures forming first, and then progressively distal structures forming with time. The AER forms as a specialized epithelial structure at the distal DV border of the developing limb bud (Tickle and Altabef, 1999). Classical transplantation experiments demonstrate a crucial role for the AER in limb outgrowth and this, in turn, leads to the proper formation of structures along the PD axis (Saunders, 1948). Removal of the AER very early in limb development drastically stunts the growth of the limb bud. If the AER is removed at progressively later stages of development, then the more distal structures are formed (Saunders, 1948). Fibroblast growth factors (FGFs) mediate the function of the AER. Several FGFs are expressed normally in the AER, including Fgf2, Fgf4, Fgf8, Fgf9 and Fgf17 (Cohn et al., 1995; Niswander and Martin, 1993; Sun et al., 2000). Beads soaked in FGFs can largely replace the function of the AER (Niswander et al., 1993), and induce ectopic limbs in ovo (Cohn et al., 1995). Previous transplantation experiments have demonstrated that limb mesoderm induces the AER (Carrington and Fallon, 1984; Saunders and Reuss, 1974). However, the molecular mechanisms that mediate this induction are not well understood.
Complex interactions between ectoderm and mesoderm regulate DV patterning during limb development (Chen and Johnson, 1999). Before limb bud formation, inductive signals from the mesoderm are required to establish DV pattern in the overlying ectoderm of the limb field (Geduspan and MacCabe, 1987; Geduspan and MacCabe, 1989; Michaud et al., 1997). Using transplantation analyses in chick/quail chimeras, Le Douarin and colleagues demonstrated that signals that ventralize limb ectoderm emanate from the lateral mesoderm, whereas signals that dorsalize limb ectoderm are derived from more medial somitic mesoderm (Michaud et al., 1997). However, the molecular mechanisms that mediate these mesoderm derived signals are unknown.
Once the presumptive limb ectoderm is induced by the mesoderm, it then plays a primary role in specifying DV pattern as the limb bud forms (Carrington and Fallon, 1984; Michaud et al., 1997; Saunders and Reuss, 1974). The molecular mechanisms that regulate DV patterning by the limb ectoderm have been characterized by molecular genetic analyses, which are summarized in Fig. 8A,B. The homeodomain gene, Engrailed 1 (En1), is expressed in the ventral ectoderm of the limb, where it specifies ventral limb identity (Davis et al., 1991; Gardner and Barald, 1992; Loomis et al., 1996). Null mutations in the En1 gene dorsalize the ventral limb ectoderm and subsequently the distal region of the limb (Cygan et al., 1997; Loomis et al., 1996; Loomis et al., 1998). These genetic analyses suggest a model in which En1 is the first step in specifying DV patterning in the ectoderm of the limb (Fig. 8B). Knockout analyses demonstrate that En1 specifies ventral limb identity, at least in part, by suppressing the expression of the growth factor, Wnt7a (Cygan et al., 1997; Loomis et al., 1998). Wnt7a expression is normally restricted to the dorsal ectoderm (Dealy et al., 1993; Parr et al., 1993), and null mutations in the Wnt7a gene give a double ventral phenotype, which includes the formation of ventral foot pads on the dorsal limb instead of claws and ventralization of dorsal muscles and tendons (Parr and McMahon, 1995). Therefore, DV patterning in the limb ectoderm is specified by domains of En1 expression ventrally, and Wnt7a expression dorsally.
|
We now describe our analyses of a conditional knockout of the most widely expressed type I BMP receptor, BMPR-IA. We specifically abrogated expression of BMPR-IA in the pre-hindlimb ectoderm to determine the role of BMP signaling during limb ectoderm development. BMP receptors are serine/threonine kinases that require both a type I and type II receptor subunit to function efficiently (Massague, 1998). Previous knockout analyses have demonstrated that BMPR-IA, encoded by the Bmpr gene (Bmpr1a Mouse Genome Informatics), plays a crucial role in BMP signaling. A null mutation of the Bmpr gene results in early embryonic lethality around the time of gastrulation (Mishina et al., 1995). Therefore, genetic analyses of BMPR-IA function during limb development requires a conditional knockout of the gene. To overcome the early embryonic lethal phenotype of the Bmpr knockout (Mishina et al., 1995), we used the loxP/Cre approach to induce tissue-specific mutations in the Bmpr gene (Nagy, 2000).
Using the loxP/Cre mutagenesis approach, we demonstrate that BMP signaling is required for the formation of the organizing centers that regulate PD and DV patterning in the limb. Abrogation of BMPR-IA signaling interferes with the formation of the AER, and abolishes the expression of FGFs that mediate AER function, as well as other AER molecular markers. Surprisingly, the Bmpr conditional knockout results in the formation of double dorsal hindlimbs. This alteration dramatically affects the overall expression of DV regulatory genes; En1 gene expression is lost in ventral limb ectoderm, and ectopic expression of Wnt7a and Lmx1b is observed. Finally, we examine the expression profile of the BMPR-IA ligands, BMP4 and BMP7, which are detected early in the lateral mesoderm at a time in which the mesoderm induces DV pattern in the overlying ectoderm. These data demonstrate that BMP signaling is required to establish DV patterning in the limb ectoderm. Furthermore, this is the first demonstration that BMP signaling is required for the formation of critical organizing centers during limb development.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Southern analysis of Cre-mediated rearrangement of Bmpr gene
Genomic DNA was isolated from the tissues indicated in Fig. 1C from an 18.5 dpc embryo, digested with NheI/SacI, and probed with the H23 probe, which encompasses the third exon (Fig. 1D; box above exon 3 of modified alleles). Each of the Bmpr alleles and the Cre-mediated rearrangements of the alleles can be distinguished by Southern analyses of genomic DNA using this strategy.
Histological techniques
Staining for lacZ expression was accomplished as described previously (Phippard et al., 1999). Histological analyses of neonatal and adult limbs were accomplished by Hematoxylin and Eosin staining of paraffin sections. When necessary, the limbs were decalcified using Cal ExII (Fisher Scientific). In situ hybridization was accomplished by a modification (T. A. Sanders and C. W. Ragsdale, unpublished) of previously described techniques (Wilkinson, 1992). The mouse Wnt7a and Shh probes were a kind gift from Andrew McMahon (Parr et al., 1993; Echelard et al., 1993); the mouse Lmx1b probe was a kind gift from Randy Johnson (Chen et al., 1998); and the mouse En1 probe was kind gift from Alexandra L. Joyner (Logan et al., 1992). The Fgf8, Fgf4, Bmp2, Bmp4 and Bmp7 probes were generated by PCR amplification of 0.5 kb regions of the cDNAs that do not cross hybridize.
Phospho-SMAD1 immunohistochemistry
Immunohistochemical analyses were carried out on paraffin sectioned material (7 µm) that had been fixed overnight in 4% paraformaldehyde/phosphate-buffered saline at 4°C prior to embedding in paraffin. Sections were processed by immunoperoxidase labeling using the Vectastain ABC Kit (Vector Labs). The immunoperoxidase signal was amplified with the TSA Indirect Tyramide Signal Amplification Kit (Perkin Elmer Life Science) according to the manufacturers instructions with the following modifications. For antigen unmasking, sections on slides were heated in a microwave oven in 10 mM sodium citrate pH 6.0 for 4 minutes. Microwave power settings were adjusted to maintain temperature just below boiling during heating. The sections were then treated to quench endogenous peroxidase activity with 2% H2O2. The specimens were blocked in 5% normal goat serum for 1 hour at room temperature and incubated overnight at 4°C with the Phospho-Smad1 antibody (Cell Signalling Technology) diluted 1:3000 in 5% normal goat serum. The specimens were then incubated in 0.05% blocking reagent supplied in the tyramide kit for 30 minutes at room temperature to further eliminate nonspecific binding. All further incubations were done in the 0.05% blocking reagent as per manufacturers instructions.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue-specific expression of the Cre recombinase gene was driven by the Brn4/Pou3f4 promoter region in a Brn4-Cre transgenic pedigree (Fig. 1A). Previous analyses demonstrated that the Brn4 proximal 5' flanking region directs expression to the neural tube (Heydemann et al., 2001). The Brn4-Cre pedigree used in this study efficiently induced Cre-mediated rearrangement of the Bmpr gene in tissues derived from the neural tube, such as spinal cord, hindbrain and forebrain (Fig. 1C-D). In addition, Southern analyses and the mutant limb phenotype demonstrated that this Brn4-Cre pedigree was ectopically expressed in the limbs (Fig. 1C-D). We have not detected expression of the endogenous Brn4 gene (Phippard et al., 1999; Phippard et al., 1998) or transgenes containing the 6 kb Brn4 flanking region (Heydemann et al., 2001) in the limb. Therefore, the ectopic expression of the Brn4-Cre transgene in the ventrolateral ectoderm encompassing the limb field is probably a consequence of the site of transgene integration. This transgene integration site does not appear to have interrupted a gene necessary for limb development, because the transgene can be homozygosed without any detectable limb phenotype. Furthermore, molecular markers of limb development are not affected in non-mutant animals containing the Brn4-Cre transgene.
Cre-mediated induction of mutation eliminates BMP signaling by 10.0 dpc
To determine the spatial and temporal expression of the Brn4-Cre pedigree in the limb, we intercrossed the Brn4-Cre pedigree with the ROSA reporter strain, which activates the expression of the lacZ gene upon Cre-mediated recombination (Soriano, 1999). As shown in Fig. 2A, Brn4-Cre-mediated expression of lacZ is initially detected at 9.75 dpc in the ventrolateral ectoderm in a region encompassing both of the embryonic limb anlage. At this stage of embryogenesis, the forelimb bud has begun to form, but the hindlimb bud, which typically forms about a half a day later than the forelimb, has not formed yet (Fig. 2A). The expression of the Brn4-Cre transgene is restricted to the ectoderm of the limb with the highest degree of lacZ reporter expression detected in the AER. Abundant lacZ reporter expression was also detected in the ventral limb ectoderm, and some expression was detected in the dorsal limb ectoderm (Fig. 2B). Significant Brn4-Cre-mediated induction of reporter transgene expression was detected at the earliest stages of hindlimb bud formation (Fig. 2C). The induction of reporter gene expression increased in the limb ectoderm, particularly in the pre-AER region, as the limb develops (Fig. 2C-E). The overall pattern of Cre-mediated lacZ expression was identical in the forelimb at this stage, but the abundance of reporter expression was reduced in comparison to the hindlimb (Fig. 2G,H). These data demonstrate that the Brn4-Cre transgene expression is activated simultaneously throughout the ventrolateral ectoderm encompassing the limb fields/bud. However, the forelimb bud begins to form before Cre-mediated expression, whereas the hindlimb forms after Cre-mediated lacZ expression is induced.
|
|
To corroborate that BMP signaling is abrogated at 10.0 dpc, we examined the expression of Msx2, whose expression is often induced by BMP signaling (Hogan, 1996). Msx2 expression is not detected in mutant limb ectoderm at 10.0 dpc, although its expression is still detected in the dorsal neural tube (Fig. 3O,P). No differences in Msx2 expression were detected at 9.75 dpc (data not shown). Attempts to detect loss of BMPR-IA expression directly by in situ hybridization or by immunohistochemistry did not yield reliable results. However, three different criteria, namely, ROSA reporter expression, phosphorylation of SMAD1 and expression of Msx2, demonstrate that the Bmpr gene is mutated resulting in loss of BMP signaling between 9.75 dpc and 10.0 dpc.
Limb phenotype in Bmpr mutants
Conditional inactivation of the Bmpr gene using the Brn4-Cre transgenic pedigree resulted in a severe limb phenotype (Fig. 4). Although the phenotype was variable, the most severely affected animals (8/42 hindlimbs) demonstrated complete agenesis of the hindlimb (Fig. 4A). The forelimbs typically demonstrated subtle malformations, which occasionally resulted in an ectopic distal phalange (Fig. 4B,D). However, the hindlimbs of the mutant animals were more severely affected, presumably because the Brn4-Cre transgene was expressed before limb bud formation in the hindlimb, but after initial forelimb bud formation. The mutant hindlimbs were grossly malformed (Fig. 4C,F,G,I). A common feature of the hindlimb malformations was polysyndactyly (Fig. 4C,F,G,I). This phenotype included fusion of digits (syndactyly), as well as partial duplication of distal segments of the digits (Fig. 4F,G,I). The typical hindlimb had fewer digits (Fig. 4C,F). However, the mutant limbs rarely demonstrated supernumerary (polydactyly) digits (Fig. 4G,I). In addition, hematoma at the distal tips of the digits was commonly observed (Fig. 4C). Transverse sections through the hindlimbs of moderately affected mutants demonstrated a loss of ventral structures (Fig. 4H,I,L,M). This was most clearly illustrated by the loss/transformation of the prominent ventral flexor digitorum profundus tendon, and an overall mirror image symmetry of the mesenchyme in the dorsal/ventral plane (Fig. 4H,I,L,M). Skeletal preparations demonstrated a loss of the sesamoid process, which is a ventral bone structure (Fig. 4J,K). Additionally, malformed hair follicles were found on both the ventral and dorsal surfaces of the foot, whereas follicles were normally restricted to the dorsal limb (data not shown). Finally, eccrine glands, which were normally found on the ventral side of the limb, were drastically reduced in number on the ventral side of the mutant hindlimb (data not shown). These data demonstrate that the morphology of the hindlimb displayed a double dorsal phenotype.
|
|
|
Malformations of the ZPA appear secondary to AER malformations
To examine the status of the anterior/posterior limb organizer, the zone of polarizing activity (ZPA) (Tickle, 1981; Wolpert, 1969), we analyzed the expression of sonic hedgehog (Shh) (Krauss et al., 1993; Riddle et al., 1993) in the Bmpr conditional mutants. We observed a variable expression of Shh, which reflected the variability observed with Fgf8 expression and AER formation (see below). Because Shh expression requires the expression of the Fgfs to maintain appropriate expression levels (Laufer et al., 1994; Sun et al., 2000), we hypothesized that the variability of Shh expression is dependent on FGF expression in these mutants. To test this directly, we double-labeled embryos with probes against Fgf8 and Shh (Fig. 5C-F). Because the distribution of Fgf8 expression in the mutants was largely stochastic, the domain of Shh expression correlated with the amount of Fgf8 expression (Fig. 5D-F). However, occasionally a modest domain of Fgf8 expression would lie directly above the posterior margin of the hindlimb resulting in a robust patch of Shh expression (data not shown). These observations are consistent with previous data suggesting that the maintenance of Shh expression is dependent on the AER.
Specification of ventral hindlimb is disrupted in Bmpr mutants
To assess dorsal/ventral patterning of the limb, we examined the expression of the transcription factor gene, Lmx1b. This gene is normally expressed in the dorsal mesenchyme of both the hindlimb bud (Fig. 6A, Fig. 8) and the forelimb bud (Fig. 6C, Fig. 8). In the Bmpr conditional mutant, Lmx1b was expressed in both the dorsal and ventral mesoderm of the hindlimb, consistent with the double dorsal phenotype observed by histological analyses (Fig. 6B). Expression of Lmx1b was restricted to dorsal mesoderm in the mutant forelimb (Fig. 6D) indicating that dorsal/ventral patterning was not disrupted in the forelimb.
|
Because previous studies demonstrated that Wnt7a expression is repressed in the ventral limb ectoderm by En1 (Cygan et al., 1997; Loomis et al., 1996; Loomis et al., 1998), we examined En1 gene expression in the Bmpr conditional mutants. As shown in Fig. 6G,H, En1 expression was virtually lost in mutant embryos. We did not detect substantial En1 expression at any stage of embryogenesis from 10.0-11.5 dpc, although En1 expression in the forelimb was normal in the same mutant embryos (data not shown).
Unlike the AER phenotype, the double dorsal phenotype, as assessed by molecular markers, was completely penetrant. All 58 embryos examined with molecular markers of dorsal/ventral patterning demonstrated a double dorsal phenotype in the hindlimb (En1, Lmx1b and Wnt7a probes on embryos 10.25-11.5 dpc). Therefore, these data indicate that BMPR-IA signaling during early limb development is required for the expression of En1 and therefore, the establishment of ventral limb patterning (Fig. 8).
Expression of Bmp4 and Bmp7 correlates with dorsal/ventral patterning and AER formation in the mouse
To assess the role of previously characterized ligands of BMPR-IA during limb development, we examined the expression pattern of Bmp2, Bmp4 and Bmp7 in the hindlimb at timepoints just before and during the period in which the molecular phenotypes of the Bmpr mutants first appeared (Fig. 7). We have observed an extremely dynamic pattern of Bmp gene expression during this period. Although Bmp2 is expressed in the AER at later stages of development (Lyons et al., 1990), we did not detect appreciable levels of expression in the hindlimb during the 9.75-10.0 dpc timeframe (data not shown). At 9.75 dpc, before hindlimb bud formation, Bmp4 is highly expressed throughout the ventral-lateral part of the embryo, including expression in the lateral mesoderm (Fig. 7A). By 10.0 dpc, the hindlimb bud has begun to form, and Bmp4 expression has been downregulated in the limb mesoderm (Fig. 7D). Expression levels remain high in the pre-AER region in a pattern that correlates well with the expression of the AER marker, Fgf8 (Fig. 7D,E). Bmp7 demonstrates a more restricted pattern of expression than Bmp4 during the 9.75-10.0 dpc time period. At 9.75 dpc, Bmp7 expression is detected in the ventral region of the embryo, but is restricted to the outermost region of the embryo within or in close apposition to the ectoderm (Fig. 7B). Interestingly, this expression domain correlates well with the expression domain of Msx2 at this stage of development (Fig. 7C). By 10.0 dpc, the expression of Bmp7 has been largely downregulated (Fig. 7F). However, Msx2 expression retains an expression pattern roughly comparable with that observed at 9.75 dpc (Fig. 7G). These data demonstrate that Bmp4 and Bmp7 are expressed in a temporal and spatial sequence that is consistent with the specification of early ventral limb ectoderm, and then rapidly downregulated after limb bud formation is initiated.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mutant phenotype is much more profound in the hindlimb than in the forelimb of the Bmpr mutants. Although we cannot rule out the possibility that En1 expression and DV patterning is differentially regulated in the fore- and hindlimbs, the difference in phenotype between the fore- and hindlimbs most likely results from the difference in the timing and extent of Bmpr gene inactivation in the limbs (see Fig. 2A). We hypothesize that DV patterning is established in the forelimb prior to extensive Bmpr inactivation, whereas gene inactivation occurs before DV patterning during hindlimb formation. These data would suggest that DV patterning is established in a narrow temporal window just prior to the initial outgrowth of the limb bud. In addition, our observations are consistent with those of Niswander and colleagues, who have shown that BMP signaling is required for DV patterning and AER formation in both the fore- and hindlimbs of chickens (Pizette et al., 2001).
Specification of ventral limb identity
Classical transplantation experiments have demonstrated that mesoderm dictates the DV patterning before limb bud formation, whereas DV patterning is regulated by the ectoderm after the limb bud forms (Chen and Johnson, 1999). In the chicken, the transition of control over DV patterning from the lateral mesoderm to the somatic ectoderm occurs between stages HH14 and HH16 (Geduspan and MacCabe, 1987; Geduspan and MacCabe, 1989). The first indication of limb bud formation occurs with the condensation of lateral mesoderm opposite somites 15-16 at stages HH16 (Hamburger and Hamilton, 1951). The transition from mesodermal to ectodermal control of DV patterning is demonstrated by the fact that a 180° rotation of prospective limb ectoderm at stage HH14 results in a limb with normal DV polarity. However, ectodermal inversion at HH16 results in formation of a limb with reversed DV polarity (Geduspan and MacCabe, 1987; Geduspan and MacCabe, 1989). The acquisition of DV polarity by the ectoderm is accompanied by the expression of genetic regulatory genes, En1 and Wnt7a, at HH15-16 (Davis et al., 1991; Dealy et al., 1993; Gardner and Barald, 1992; Riddle et al., 1995). Mutational analyses in mice have clearly demonstrated that En1 and Wnt7a play complementary roles in specifying ventral and dorsal limb ectoderm, respectively (Fig. 8) (Cygan et al., 1997; Loomis et al., 1996; Loomis et al., 1998; Parr and McMahon, 1995). Therefore, one would predict that candidate factors which regulate the initial induction of DV patterning would be expressed in the lateral mesoderm before limb bud formation and would be required for the induction of factors that specify DV patterning in the overlying ectoderm. Our data demonstrate that ligands of BMPR-IA fulfill these criterion, because Bmp4 and Bmp7 are expressed in lateral mesoderm and the overlying ectoderm before limb bud formation, and BMPR-IA signaling is required for the induction of En1.
Although classical transplantation analyses have been undertaken in chickens and amphibians, analyses of the cellular and molecular events regulating limb morphogenesis suggest that mechanisms of DV patterning and AER formation are conserved between vertebrates (Johnson and Tabin, 1997; Martin, 1998; Vogt and Duboule, 1999). Specifically, regarding the role of BMP signaling, observations from Niswander and her colleagues (Pizette et al., 2001) are similar, if not identical, to those we have observed in mice. Therefore, our model for BMP signaling will incorporate embryological and molecular observations from all vertebrate model systems.
Although the role of ectoderm in specifying DV patterning after limb bud formation has been well characterized, the precise role of mesoderm before limb bud formation has been less clear, and the molecular mechanisms are completely unknown. In the 1970s, transplantation experiments characterizing the ability of lateral mesoderm to specify early DV patterning were conflicting (Michaud et al., 1997). However, more recent transplantation and cell marking experiments suggest that paraxial mesoderm plays a role in specifying dorsal limb ectoderm, whereas lateral mesoderm specifies ventral limb ectoderm (Altabef et al., 1997; Michaud et al., 1997). The conclusions from these studies is incorporated into the model schematized in Fig. 8C,D. For example, Michaud et al. demonstrated that an 180° inversion of lateral mesoderm alone at pre-limb bud stages (HH12-13) (dark blue in Fig. 8C) does not result in an inversion of limb polarity (Michaud et al., 1997). However, inversions of both lateral and somitic mesoderm (dark blue and red/orange, respectively, in Fig. 8C) does invert DV polarity of the resulting limbs. In addition, transplantation of an ectopic somite into lateral mesoderm, such that the lateral mesoderm was flanked by the endogenous somite medially and the ectopic somite laterally, resulted in the formation of a bidorsal limb. These data and other data suggested that a dorsalizing signal was emanating from the somitic mesoderm, and a ventralizing signal emanated from the lateral mesoderm.
If the dorsalizing signal emanates from the somitic mesoderm, how then does this give rise to dorsal limb ectoderm? Both transplantation (Michaud et al., 1997) and cell labeling experiments (Altabef et al., 1997), demonstrate that ectoderm overlying the somites are fated to become dorsal limb ectoderm (depicted as purple ectoderm in Fig. 8C,D). Tickle and colleagues have undertaken cell fate mapping experiments that demonstrate a large fraction of dorsal limb ectoderm originates from the ectoderm overlying the somites (Altabef et al., 1997). These data suggest that the ectoderm overlying the somites is fated to become dorsal limb ectoderm, and then morphogenetic movements result in the translocation of this ectoderm such that it covers the dorsal limb (as schematized in Fig. 8D).
A molecular model for epithelial-mesenchymal interactions during early limb development
Our model hypothesizes that BMP signaling pathway mediates the initial induction of DV pattern in the presumptive limb ectoderm before limb bud formation. Bmp4 and Bmp7 are expressed in the lateral mesoderm and the overlying ectoderm just prior to the induction of limb bud formation, and therefore are correctly positioned in both time and space to induce ventral limb ectoderm (Fig. 7, Fig. 8). Furthermore, we have demonstrated that BMPR-IA signaling is required for the induction of En1, and subsequently the specification of ventral limb identity. Finally, the dorsalizing effect of somites, we hypothesize, is mediated by the expression of the BMP antagonist, noggin, in the myotomal compartment of the somite (Capdevila and Johnson, 1998; Hirsinger et al., 1997; Marcelle et al., 1997; McMahon et al., 1998; Reshef et al., 1998; Tonegawa and Takahashi, 1998). Noggin expression in the somites could explain the ability of somites to dorsalize the ectoderm in transplantation experiments because of their ability to inhibit BMP signals from the lateral mesoderm. Noggin expressed in the somites could neutralize the effects of BMPs in the ectoderm that is in close apposition to the somites (Fig. 8C). Alternatively, the somitic mesoderm could induce the expression of an unidentified BMP antagonist in dorsal ectoderm, which suppresses the BMP signaling within the dorsal ectoderm itself.
Finally, the mesoderm-derived inductive signal would have to be downregulated as the lateral mesoderm loses its ability to induce the overlying ectoderm and as the dorsally fated ectoderm moves over the limb mesenchyme. Our analyses demonstrate that the expression of Bmp4 and Bmp7 are rapidly downregulated in most of the lateral mesenchyme as the limb bud is formed (Fig. 7D,F), although Bmp4 expression is maintained in the distal limb where BMP signaling is required for AER formation.
Role of BMPR-IA signaling during AER formation
Our data demonstrate a crucial role for BMPR-IA signaling in the formation of the AER. It is conceivable that the AER defects are secondary to DV patterning defects. However, this seems unlikely because the penetrance of the DV patterning defect is complete, whereas the AER defect is quite variable. This difference in penetrance argues that AER formation and DV patterning are two independent processes. Our argument is further bolstered by analyses of the eudiplopodia chick mutant that suggest that AER formation is not strictly dependent on establishment of a DV border at the distal tip of the limb (Laufer et al., 1997).
Classical studies have shown that lateral mesoderm induces the AER (Carrington and Fallon, 1984; Saunders and Reuss, 1974). As Bmp4 and Bmp7 are expressed in lateral mesoderm, they are candidates for the initial inductive event required for AER formation.
There are mutants, such as limbless, in which both AER formation and DV patterning are affected. One parsimonious explanation is that the limbless gene product may be epistatic to BMP signaling and therefore affects both independent processes because they are mediated by one signaling pathway. An additional mutant that affects both DV patterning and AER formation is the En1 knockout. In this case, there are important differences between the Bmpr mutant and the En1 mutant. Most importantly, the En1 knockout does not abrogate AER formation, but does affect the positioning of the AER. En1 mutants demonstrate a ventral extension of the AER (Loomis et al., 1996; Loomis et al., 1998). The AER, where it is formed in the Bmpr mutant, does not demonstrate the ventral extension seen in En1 mutants (Fig. 5; data not shown). Michaud et al. also did not observe a ventral extension of the AER in double dorsal phenotypes generated by transplantation experiments in chick (Michaud et al., 1997). These data, in conjunction with our data, suggest that the ventral AER extension is not a consistent feature of the double dorsal phenotype.
In summary, the conditional knockout of the gene for the most widely expressed type I BMP receptor, BMPR-IA, results in limb malformations that are due to the disruption of AER formation and loss of DV patterning. This is the first demonstration that BMPR-IA signaling is essential for these early events in limb morphogenesis.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altabef, M., Clarke, J. D. and Tickle, C. (1997). Dorso-ventral ectodermal compartments and origin of apical ectodermal ridge in developing chick limb. Development 124, 4547-4556.
Arango, N. A., Lovell-Badge, R. and Behringer, R. R. (1999). Targeted mutagenesis of the endogenous mouse Mis gene promoter: in vivo definition of genetic pathways of vertebrate sexual development. Cell 99, 409-419.[Medline]
Capdevila, J. and Johnson, R. L. (1998). Endogenous and ectopic expression of noggin suggests a conserved mechanism for regulation of BMP function during limb and somite patterning. Dev. Biol. 197, 205-217.[Medline]
Carrington, J. L. and Fallon, J. F. (1984). The stages of flank ectoderm capable of responding to ridge induction in the chick embryo. J. Embryol. Exp. Morphol. 84, 19-34.[Medline]
Chen, H. and Johnson, R. L. (1999). Dorsoventral patterning of the vertebrate limb: a process governed by multiple events. Cell Tissue Res. 296, 67-73.[Medline]
Chen, H., Lun, Y., Ovchinnikov, D., Kokubo, H., Oberg, K. C., Pepicelli, C. V., Gan, L., Lee, B. and Johnson, R. L. (1998). Limb and kidney defects in Lmx1b mutant mice suggest an involvement of LMX1B in human nail patella syndrome. Nat. Genet. 19, 51-55.[Medline]
Cho, H., Mu, J., Kim, J. K., Thorvaldsen, J. L., Chu, Q., Crenshaw, E. B., 3rd, Kaestner, K. H., Bartolomei, M. S., Shulman, G. I. and Birnbaum, M. J. (2001). Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta). Science 292, 1728-1731.
Cohn, M. J., Izpisua-Belmonte, J. C., Abud, H., Heath, J. K. and Tickle, C. (1995). Fibroblast growth factors induce additional limb development from the flank of chick embryos. Cell 80, 739-746.[Medline]
Crossley, P. H., Minowada, G., MacArthur, C. A. and Martin, G. R. (1996). Roles for FGF8 in the induction, initiation, and maintenance of chick limb development. Cell 84, 127-136.[Medline]
Cygan, J. A., Johnson, R. L. and McMahon, A. P. (1997). Novel regulatory interactions revealed by studies of murine limb pattern in Wnt-7a and En-1 mutants. Development 124, 5021-5032.
Davis, C. A., Holmyard, D. P., Millen, K. J. and Joyner, A. L. (1991). Examining pattern formation in mouse, chicken and frog embryos with an En-specific antiserum. Development 111, 287-298.[Abstract]
Dealy, C. N., Roth, A., Ferrari, D., Brown, A. M. and Kosher, R. A. (1993). Wnt-5a and Wnt-7a are expressed in the developing chick limb bud in a manner suggesting roles in pattern formation along the proximodistal and dorsoventral axes. Mech. Dev. 43, 175-186.[Medline]
Dreyer, S. D., Zhou, G., Baldini, A., Winterpacht, A., Zabel, B., Cole, W., Johnson, R. L. and Lee, B. (1998). Mutations in LMX1B cause abnormal skeletal patterning and renal dysplasia in nail patella syndrome. Nat. Genet. 19, 47-50.[Medline]
Echelard, Y., Epstein, D. J., St-Jacques, B., Shen, L., Mohler, J., McMahon, J. A. and McMahon, A. P. (1993). Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75, 1417-1430.[Medline]
Gardner, C. A. and Barald, K. F. (1992). Expression patterns of engrailed-like proteins in the chick embryo. Dev. Dyn. 193, 370-388.[Medline]
Geduspan, J. S. and MacCabe, J. A. (1987). The ectodermal control of mesodermal patterns of differentiation in the developing chick wing. Dev. Biol. 124, 398-408.[Medline]
Geduspan, J. S. and MacCabe, J. A. (1989). Transfer of dorsoventral information from mesoderm to ectoderm at the onset of limb development. Anat. Rec. 224, 79-87.[Medline]
Hamburger, V. and Hamilton, H. L. (1951). A series of normal stages in the development of chick embryos. J. Morphol. 88, 49-92.
Heydemann, A., Nguyen, L. C. and Crenshaw III, E. B. (2001). A regulatory region of the Brn4/Pou3f4 promoter directs expression to developing forebrain and neural tube. Dev. Brain Res. 128, 83-90.[Medline]
Hirsinger, E., Duprez, D., Jouve, C., Malapert, P., Cooke, J. and Pourquie, O. (1997). Noggin acts downstream of Wnt and Sonic Hedgehog to antagonize BMP4 in avian somite patterning. Development 124, 4605-4614.
Hogan, B., Beddington, R., Costantini, F. and Lacy, E. (1994). Manipulating the mouse embryo: a laboratory manual. Plainview, NY: Cold Spring Harbor Laboratory Press.
Hogan, B. L. (1996). Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes Dev. 10, 1580-1594.[Medline]
Johnson, R. L. and Tabin, C. J. (1997). Molecular models for vertebrate limb development. Cell 90, 979-990.[Medline]
Krauss, S., Concordet, J. P. and Ingham, P. W. (1993). A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos. Cell 75, 1431-1444.[Medline]
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]
Laufer, E., Dahn, R., Orozco, O. E., Yeo, C. Y., Pisenti, J., Henrique, D., Abbott, U. K., Fallon, J. F. and Tabin, C. (1997). Expression of Radical fringe in limb-bud ectoderm regulates apical ectodermal ridge formation. Nature 386, 366-373.[Medline]
Logan, C., Hanks, M. C., Noble-Topham, S., Nallainathan, D., Provart, N. J. and Joyner, A. L. (1992). Cloning and sequence comparison of the mouse, human, and chicken engrailed genes reveal potential functional domains and regulatory regions. Dev. Genet. 13, 345-358.[Medline]
Loomis, C. A., Harris, E., Michaud, J., Wurst, W., Hanks, M. and Joyner, A. L. (1996). The mouse Engrailed-1 gene and ventral limb patterning. Nature 382, 360-363.[Medline]
Loomis, C. A., Kimmel, R. A., Tong, C. X., Michaud, J. and Joyner, A. L. (1998). Analysis of the genetic pathway leading to formation of ectopic apical ectodermal ridges in mouse Engrailed-1 mutant limbs. Development 125, 1137-1148.
Lyons, K. M., Pelton, R. W. and Hogan, B. L. (1990). Organogenesis and pattern formation in the mouse: RNA distribution patterns suggest a role for bone morphogenetic protein-2A (BMP-2A). Development 109, 833-844.[Abstract]
Mahmood, R., Bresnick, J., Hornbruch, A., Mahony, C., Morton, N., Colquhoun, K., Martin, P., Lumsden, A., Dickson, C. and Mason, I. (1995). A role for FGF-8 in the initiation and maintenance of vertebrate limb bud outgrowth. Curr. Biol. 5, 797-806.[Medline]
Marcelle, C., Stark, M. R. and Bronner-Fraser, M. (1997). Coordinate actions of BMPs, Wnts, Shh and noggin mediate patterning of the dorsal somite. Development 124, 3955-3963.
Martin, G. R. (1998). The roles of FGFs in the early development of vertebrate limbs. Genes Dev. 12, 1571-1586.
Massague, J. (1998). TGF-beta signal transduction. Annu. Rev. Biochem. 67, 753-791.[Medline]
McMahon, J. A., Takada, S., Zimmerman, L. B., Fan, C. M., Harland, R. M. and McMahon, A. P. (1998). Noggin-mediated antagonism of BMP signaling is required for growth and patterning of the neural tube and somite. Genes Dev 12, 1438-1452.
Michaud, J. L., Lapointe, F. and Le Douarin, N. M. (1997). The dorsoventral polarity of the presumptive limb is determined by signals produced by the somites and by the lateral somatopleure. Development 124, 1453-1463.
Mishina, Y., Suzuki, A., Ueno, N. and Behringer, R. R. (1995). Bmpr encodes a type I bone morphogenetic protein receptor that is essential for gastrulation during mouse embryogenesis. Genes Dev. 9, 3027-3037.[Abstract]
Nagy, A. (2000). Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99-109.[Medline]
Niswander, L. and Martin, G. R. (1993). FGF-4 and BMP-2 have opposite effects on limb growth. Nature 361, 68-71.[Medline]
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]
Parr, B. A. and McMahon, A. P. (1995). Dorsalizing signal Wnt-7a required for normal polarity of D-V and A-P axes of mouse limb. Nature 374, 350-353.[Medline]
Parr, B. A., Shea, M. J., Vassileva, G. and McMahon, A. P. (1993). Mouse Wnt genes exhibit discrete domains of expression in the early embryonic CNS and limb buds. Development 119, 247-261.
Phippard, D., Lu, L., Lee, D., Saunders, J. C. and Crenshaw III, E. B. (1999). Targeted mutagenesis of the POU-domain gene, Brn4/Pou3f4, causes development defects in the inner ear. J. Neurosci. 19, 5980-5989.
Phippard, D. J., Heydemann, A., Lechner, M., Lu, L., Lee, D., Kyin, T. and Crenshaw III, E. B. (1998). Changes in the subcellular localization of the Brn4 gene product precede mesenchymal remodeling of the otic capsule. Hear. Res. 120, 77-85.[Medline]
Pizette, S., Abate-Shen, C. and Niswander, L. (2001). BMP controls proximodistal outgrowth, via induction of the apical ectodermal ridge, and dorsoventral patterning of the vertebrate limb. Development 128, 4463-4474.
Reshef, R., Maroto, M. and Lassar, A. B. (1998). Regulation of dorsal somitic cell fates: BMPs and Noggin control the timing and pattern of myogenic regulator expression. Genes Dev. 12, 290-303.
Riddle, R. D., Ensini, M., Nelson, C., Tsuchida, T., Jessell, T. M. and Tabin, C. (1995). Induction of the LIM homeobox gene Lmx1 by WNT7a establishes dorsoventral pattern in the vertebrate limb. Cell 83, 631-640.[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]
Saunders, J. W., Jr (1948). The proximo-distal sequences of origin of the parts of the chick wing and the role of ectoderm. J. Exp. Zool. 108, 363-404.
Saunders, J. W. J. and Reuss, C. (1974). Inductive and axial properties of prospective wing-bud mesoderm in the chick embryo. Dev. Biol. 38, 41-50.[Medline]
Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70-71.[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. Nat. Genet. 25, 83-86.[Medline]
Tickle, C. (1981). The number of polarizing region cells required to specify additional digits in the developing chick wing. Nature 289, 295-298.[Medline]
Tickle, C. and Altabef, M. (1999). Epithelial cell movements and interactions in limb, neural crest and vasculature. Curr. Opin. Genet. Dev. 9, 455-460.[Medline]
Tonegawa, A. and Takahashi, Y. (1998). Somitogenesis controlled by Noggin. Dev. Biol. 202, 172-182.[Medline]
Vogel, A., Rodriguez, C., Warnken, W. and Izpisua Belmonte, J. C. (1995). Dorsal cell fate specified by chick Lmx1 during vertebrate limb development. Nature 378, 716-720.[Medline]
Vogel, A., Rodriguez, C. and Izpisua-Belmonte, J. C. (1996). Involvement of FGF-8 in initiation, outgrowth and patterning of the vertebrate limb. Development 122, 1737-1750.
Vogt, T. F. and Duboule, D. (1999). Antagonists go out on a limb. Cell 99, 563-566.[Medline]
Wilkinson, D. G. (1992). In situ hybridization: a practical approach. In A Practical Approach Series (ed. D. Rickwood and B. D. Hames), pp. 75-83. New York: Oxford University Press.
Wolpert, L. (1969). Positional information and the spatial pattern of cellular differentiation. J. Theor. Biol. 25, 1-47.[Medline]