Bmp2, Bmp4 and Bmp7 Are Co-Required in the Mouse AER for Normal Digit Patterning but Not Limb Outgrowth

Outgrowth and patterning of the vertebrate limb requires a functional apical ectodermal ridge (AER). The AER is a thickening of ectodermal tissue located at the distal end of the limb bud. Loss of this structure, either through genetic or physical manipulations results in truncation of the limb. A number of genes, including Bmps, are expressed in the AER. Previously, it was shown that removal of the BMP receptor Bmpr1a specifically from the AER resulted in complete loss of hindlimbs suggesting that Bmp signaling in the AER is required for limb outgrowth. In this report, we genetically removed the three known AER-expressed Bmp ligands, Bmp2, Bmp4 and Bmp7 from the AER of the limb bud using floxed conditional alleles and the Msx2-cre allele. Surprisingly, only defects in digit patterning and not limb outgrowth were observed. In triple mutants, the anterior and posterior AER was present but loss of the central region of the AER was observed. These data suggest that Bmp ligands expressed in the AER are not required for limb outgrowth but instead play an essential role in maintaining the AER and patterning vertebrate digits.


Introduction
The vertebrate limb begins as a bud of lateral plate mesenchyme surrounded by surface ectoderm. In mice, over the course of ,4 days this undifferentiated bud of tissue forms all structures found in a normal limb [1]. The molecular pathways responsible for the formation of the limb have been an active area of investigation for decades and a number of factors have been identified that are required for limb development.
Outgrowth of the limb is controlled by the Apical Ectodermal Ridge (AER). This structure resides at the distal end of the limb bud and is composed of a stratified columnar of ectodermal cells [2,3]. Removal of this structure results in truncation of the forming limb [4,5,6]. A class of AER-specific factors required for limb bud outgrowth is Fgfs (Fgf4, Fgf8, Fgf9 and Fgf17). Fgf8 appears to be the major factor responsible for limb bud outgrowth since removal of this gene from the AER, but none of the other AER-expressed Fgfs, results in defects in limb patterning [7,8]. Removal of both Fgf4 and Fgf8 results in an absence of limb bud outgrowth, indicating that Fgf genes can partially compensate for one another [9]. Fgf8 is the most broadly and highly expressed Fgf gene in the AER, which may explain why removal of this gene, but none of the other AER-expressed Fgfs, produces a visible phenotype [10].
In addition to Fgfs a number of additional genes are expressed in the AER including members of the Bmp family. During limb formation, Bmp genes are expressed in the limb bud mesenchyme and AER. In the mesenchyme, they have been shown to be required for initiating chondrogenesis and limb patterning [11].
The early removal of all Bmp signaling in the AER, through conditional deletion of the Tgfb receptor Bmpr1a, resulted in limb truncations [12]. These data indicate that this signaling pathway is required for limb outgrowth but the source of the BMP ligand could not be identified in these experiments. Previously, we reported that removal of Bmp2 and Bmp4 in the AER, surprisingly, did not cause limb truncations but instead resulted in polydactyly, an increase in cell proliferation, and a decrease in cell death in the limb suggesting that Bmp expression in the underlying mesenchyme or Bmp7 expression in the AER may compensate for loss of Bmp2/4 expression in the AER [13]. In these experiments Bmp7 was still expressed in the AER and could potentially compensate for loss of Bmp2 and Bmp4 in this structure.
In the current study, triple mutant mice in which Bmp2, Bmp4 and Bmp7 were removed from the AER were created and analyzed. In all triple mutants, limb outgrowth occurred and a stylopod, zeugopod and autopod were present however; defects in autopod patterning were observed. These data suggest that Bmps expressed in the underlying mesenchyme can at least partially activate the Bmp signaling pathway in the AER and that AER-expressed Bmps are only required for digit patterning and not limb outgrowth.

Statement of Ethical Approval
This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida (Protocol number: 201005047).
Generation of Embryos that Lacked Bmp2, Bmp4 and Bmp7 in the AER Animals were handled in accordance with the University of Florida Institutional Animal Care and Use Committee. Mice containing conditional floxed alleles of Bmp2, Bmp4, Bmp7 or Msx2-cre have been described previously [11,14,15,16,17,18]. Bmp7 f/+ ; Bmp2 f/f , Bmp4 f/f , Msx2-cre and Bmp7 f/f ; Bmp2 f/f , Bmp4 f/f , Msx2-cre embryos were generated by mating Bmp7 f/+ ; Bmp2 f/f , Bmp4 f/f , Msx2-cre males with Bmp7 f/f ; Bmp2 f/f , Bmp4 f/f females. Control embryos lacked the Msx2-cre allele and contained different combinations of floxed Bmp alleles. Bmp2, Bmp4 and the Msx2-cre alleles are all located on the same chromosome. Recombination of the Bmp floxed alleles and Msx2-cre onto the same chromosome has been described previously [13]. Triple homozygous mice for all three floxed Bmp alleles (Bmp7 f/f ; Bmp2 f/f , Bmp4 f/f ) did not contain any visible defects. All mouse strains were on a mixed genetic background.

RNA in situ Hybridization, b-galactosidase Staining, and Skeleton Preparations
Whole-mount RNA in situ hybridization and b-galactosidase staining were performed as described previously [19,20,21]. Skeleton preparations were performed as previously described [22].

Immunohistochemistry
Embryos were embedded in OCT and sectioned at 10 mm as described previously [23]. For immunohistochemistry, sections were stained with anti-CD44 (BD Biosciences) or with anti-?Np63 (Santa Cruz). A Cy3 conjugated secondary antibody (Jackson Immunoresearch Laboratories) was used. Images were taken using a Leica DMRE microscope (Leica Microsystems Inc.).

Results
Deletion of Floxed Alleles of Bmp2, Bmp4 and Bmp7 in the AER using the Msx2-cre Allele To investigate the role(s) all three AER-expressed Bmp genes play in limb outgrowth and patterning, Bmp2, Bmp4 and Bmp7  were removed from the AER. Triple Bmp2, Bmp4, Bmp7 AER knockout mice were created by crossing floxed animals to males carrying the Msx2-cre allele (see Materials and Methods). Throughout this report, ''triple'' knockouts refer to animals in which Bmp2, Bmp4 and Bmp7 were removed from the AER using the Msx2-cre allele. The Msx2-cre transgene is expressed in the ventral ectoderm and the AER and is expressed earlier in the hindlimb (24 somite stage) than in the forelimb (28 somite stage) [12,14,24]. All three Bmp floxed alleles produce null alleles upon CRE-inducible recombination of the floxed DNA sites [11,25].
To determine if Bmps were removed from the AER in mutant animals, section RNA in situ hybridization on E10.5 embryos were performed using probes that were specific for the deleted region of either Bmp2, Bmp4 or Bmp7 (Figure 1). In mutant hindlimbs, where the Msx2-cre allele is active in the early limb bud ectoderm, Bmp2, Bmp4 and Bmp7 expression was abolished from the AER ( Figure 1A-F). These data indicated that the Msx2-Cre allele successfully deleted all three Bmp genes in the same limb bud.
Msx1 and Msx2 Expression in the Central AER Requires Bmp Expression in the AER BMP proteins are secreted signaling molecules and are expressed both in the limb bud mesenchyme and ectoderm during limb development. To determine if BMP signaling was altered in triple mutants, whole mount RNA in situ hybridization using probes against Msx1 and Msx2 was performed. Both of these transcription factors are known direct targets of the BMP signaling pathway [26]. In triple mutants, Msx1 and Msx2 expression was decreased in the anterior and central AER ( Figure 1G-K). Msx1 and Msx2 expression remained in the posterior AER. In the mesenchyme, Msx1 and Msx2 expression were decreased in the central and anterior limb bud.

Bmp2, Bmp4 and Bmp7 Expression in the AER is Required for Autopod Patterning but not Limb Outgrowth
Previously, we demonstrated that removal of Bmp2 and Bmp4 in the AER resulted in polydactyly, an increase in cell proliferation and a decrease in cell death in limb bud mesenchyme [13]. Bmp7 was still expressed in the AER of these limb buds. In this report, we analyzed triple mutants in which Bmp2, Bmp4 and Bmp7 were removed from the same limb bud AER. In the forelimbs of embryos that contained AER null alleles for all six Bmp alleles (n = 10) or embryos in which only one functional Bmp7 allele remained (n = 15), polydactyly, syndactyly and retention of interdigital tissue was observed in the autopods of all limbs examined (Figure 2 and Figure S1). No defects in limb outgrowth or in the stylopod or zeugopod were observed (Figure 2). In mutant hindlimbs that contained only one allele of Bmp7,  ectrodactyly (n = 9/15) was observed ( Figure 2E and F and Figure  S1). In these limbs, the middle digits were truncated. In triple mutant hindlimbs, ectrodactyly (n = 7/10) and oligodactyly (n = 3/ 10) was observed ( Figure 2G and H and Figure S1). Truncation of the anterior tibia was observed in 3/10 triple mutant hindlimbs ( Figure 2H').

Removal of Bmps in the AER Resulted in an Initial Broadening of Fgf8 Expression Followed by Loss of Fgf8 Expression
To determine whether Bmps regulate the morphology of the AER, Fgf8 expression was analyzed. Fgf8 is expressed in the entire AER and is an excellent marker of AER morphology [27]. In embryos that contained only a single allele of Bmp7 in the AER, Fgf8 expression in the E10.5 hindlimb was broadened ( Figure 3H). In triple mutant mice, AER broadening was seen in both the foreand hindlimbs (Figure 3). By E11.5, a decease in Fgf8 expression was observed in the central AER in hindlimbs of embryos that contained a single functional allele of Bmp7 in the AER. In triple mutants that lacked all six Bmp alleles in the AER, Fgf8 expression was absent throughout the central hindlimb AER and was decreased in both the anterior and posterior AER ( Figure 3L). In the forelimb, a decrease but no gaps in Fgf8 expression was observed throughout the AER of triple mutants ( Figure 3F).

Fgf10 Expression in the Mesenchyme does not Require Bmp Expression in the AER
Fgf10 is expressed throughout the early limb bud [28,29,30]. During later limb development signaling from the AER is required to maintain Fgf10 expression in the underlying limb bud mesenchyme. Mice null for Fgf10 do not form limbs [31,32]. Removal of the BMP receptor Bmpr1a in the AER resulted in initial expression of Fgf10 throughout the limb bud mesenchyme during limb bud initiation but a subsequent loss of Fgf10 during later development [12]. The inability to maintain Fgf10 in Bmpr1a AER knockout mice was due to the lack of normal gene expression within the AER. To determine if expression of Bmp ligands within the AER were required for Fgf10 expression, gene expression was analyzed in triple mutants. In triple mutants, Fgf10 expression was initiated normally and expression was maintained during limb development ( Figure 3M-R).
Bmps Expressed in the AER are Required for Stratification of the AER Analysis of Fgf8 expression in triple mutants indicated that the central region of the AER may be absent by E11.5. To investigate the organization of the AER, two protein markers were examined. The first, p63 plays an important role in stratification of epithelial tissues [33,34]. To determine if the phenotypes observed in Bmp mutant mice were caused by loss of p63, p63 protein expression was examined. A section through the AER of E10.5 and E11.5 triple mutant limb buds showed that removal of Bmp ligands in the AER did not affect p63 expression in the AER (Figure 4A-D). Analysis of p63 revealed that the AER was thinner and less stratified in triple mutants at E10.5 ( Figure 4A, B). By E11.5 only a single p63-positive epithelial cell layer was observed that was indistinguishable from the surrounding epithelium ( Figure 4C, D).
CD44 is a cell adhesion molecule and interacts with extracellular components [35]. Previous studies demonstrated that CD44 was highly expressed in the AER [36]. To determine whether expression of this cell adhesion molecule was altered upon removal of Bmp ligands in the AER, triple mutant embryos were examined using a CD44 antibody. A reduction of CD44 was observed in E10.5 and E11.5 embryos (Figure 4E-H). At E11.5, CD44 was expressed in a single layer of epithelial cell, consistent with observations made using the P63 antibody.

Discussion
Early inactivation of the BMP receptor Bmpr1a in the hindlimb AER results in the absence of an AER and the inability to form a hindlimb [12,37]. This result is in contrast to a previous report in which Noggin, a potent inhibitor of BMP signaling was used to block BMP signaling from the AER [38]. In these mice, limbs were formed but were characterized by syndactyly, postaxial polydactyly and dorsal/ventral patterning defects. It is possible that the differences in the observed defects were due to the timing of inactivation of the BMP signaling pathway; however in both cases the Msx2 promoter was used to remove BMP activity. The phenotypes produced when Bmp2, Bmp4 and Bmp7 were removed from the same AER resembled Msx2-noggin limbs. Limb trunca- tions were never observed (with the exception of loss of part of the tibia in 30% of triple mutant hindlimbs). Our experiments used the same Msx2-cre allele that was used to remove Bmpr1a suggesting that the concurrent removal of all known BMP ligands in the AER is not functional equivalent to removing the receptor that medicates BMP signaling in the AER.
Inactivation of the BMP receptor Bmpr1a in the forelimb resulted in broadening of the tips of distal phalanges [12]. The much less severe forelimb autopod defect is due to an early burst of Bmpr1a activity prior to CRE-mediated gene inactivation in the forelimbs [12]. Removal of Bmp2, Bmp4 and Bmp7 in the forelimb AER produced a more severe patterning defect than what was observed in the AER Bmpr1a receptor knockout. Our in situ expression data suggest that removal of AER-expressed Bmps resulted in a decrease in Msx1/2 expression in the underlying mesenchyme in addition to decreased expression in the AER. It is possible that a decrease in BMP signaling in both the AER and mesenchyme upon inactivation of Bmp2, Bmp4 and Bmp7 in the forelimb AER causes autopod patterning defects that where more severe than what was observed upon removal of only the Bmp signaling pathway in the AER.
Removal of Bmp ligands from the AER were confirmed using RNA in situ probes that were specific for the deleted region of each Bmp allele. In addition, removal of BMP signaling was confirmed by analyzing expression of Msx1 and Msx2, known targets of the BMP signaling pathway [26]. However, we cannot rule out the possibility that BMP activity remained in a few cells in the AER of triple mutant animals and that this potentially very low level of activity was sufficient for limb outgrowth. It is clear from our data that BMP expression in the AER is required for normal digit patterning and to maintain the structure of the central AER.
The BMPR1a receptor can bind additional TGFb ligands besides BMP2, BMP4 and BMP7 [39]. In the limb bud AER a number of additional Tgfb genes are expressed besides Bmps [40,41]. It is possible that complete loss of the hindlimb, which is observed upon removal of Bmpr1a from the AER [12,37], is due to inactivation of the Bmp signaling pathway in addition to other TGFb signaling pathways.
Individuals who contain a mutation in p63 have Split Hand Foot malformations (SHFM) [42]. Mice null for p63 had severe defects in epithelial stratification that affected AER formation, resulting in limb truncations [33,34]. Removal of Bmp2, Bmp4 and Bmp7 from the limb bud AER resulted in defects in the autopod that resembled pattering defects found in SHFM. However, p63 was still expressed throughout the limb bud epithelium including the AER of E10.5 triple mutant embryos. These data suggest that the observed digit defects found in triple Bmp mutants are not caused by loss of P63.
In the central AER removal of Bmp genes resulted in a loss of Fgf8 gene expression and, by E11.5, the AER. These data indicate that BMP expression within the AER is required not for AER formation but rather to maintain this structure during later limb development. Surprisingly, BMP signaling was not lost in the anterior and posterior AER, suggesting that during early stages of development BMP expression in the underlying mesoderm may be sufficient to activate the Bmp signaling pathway in these regions of the limb. All three Bmp genes are highly expressed in posterior limb bud mesenchyme at E10.5 with Bmp7 also expressed in the anterior limb bud mesenchyme at this stage ( Figure S2). Expression in these locations could potentially activate the Bmp signaling pathway in these regions of the limb ( Figure 5). In the future, this hypothesis could be tested by concurrently removing Bmp genes from the AER using the Msx2-cre allele and from the posterior mesenchyme using the Shhcre allele [19]. Our data suggests that during normal limb development, Bmps expressed in the AER are required for digit patterning and BMP proteins produced in both the mesoderm and ectoderm of the limb bud can activate the Bmp signaling pathway in the AER.