Limits...
Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis.

Bandyopadhyay A, Tsuji K, Cox K, Harfe BD, Rosen V, Tabin CJ - PLoS Genet. (2006)

Bottom Line: However, due to complications associated with early embryonic lethality, particularly for Bmp2 and Bmp4, and with functional redundancy among BMP molecules, it has been difficult to decipher the specific roles of these BMP molecules during different stages of limb development.Contrary to earlier suggestions, our results indicate that BMPs neither act as secondary signals downstream of Sonic Hedghog (SHH) in patterning the anteroposterior axis nor as signals from the interdigital mesenchyme in specifying digit identity.In contrast, we find that the loss of both BMP2 and BMP4 results in a severe impairment of osteogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Bone morphogenetic protein (BMP) family members, including BMP2, BMP4, and BMP7, are expressed throughout limb development. BMPs have been implicated in early limb patterning as well as in the process of skeletogenesis. However, due to complications associated with early embryonic lethality, particularly for Bmp2 and Bmp4, and with functional redundancy among BMP molecules, it has been difficult to decipher the specific roles of these BMP molecules during different stages of limb development. To circumvent these issues, we have constructed a series of mouse strains lacking one or more of these BMPs, using conditional alleles in the case of Bmp2 and Bmp4 to remove them specifically from the limb bud mesenchyme. Contrary to earlier suggestions, our results indicate that BMPs neither act as secondary signals downstream of Sonic Hedghog (SHH) in patterning the anteroposterior axis nor as signals from the interdigital mesenchyme in specifying digit identity. We do find that a threshold level of BMP signaling is required for the onset of chondrogenesis, and hence some chondrogenic condensations fail to form in limbs deficient in both BMP2 and BMP4. However, in the condensations that do form, subsequent chondrogenic differentiation proceeds normally even in the absence of BMP2 and BMP7 or BMP2 and BMP4. In contrast, we find that the loss of both BMP2 and BMP4 results in a severe impairment of osteogenesis.

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An Allelic/Nonallelic Series of BMP-Deficient Limbs(A–D) Prx1::cre efficiently recombines Bmp2 and Bmp4 conditional alleles in the limbs. Bmp2 (A and B) and Bmp4 (C and D) whole mount mRNA in situ hybridization in the limb. Wild-type (A) and Bmp2C/C; Prx1::cre (B) are forelimbs from E10.5 mouse embryos. Mesenchymal expression [asterisk (A)] of Bmp2 is abolished in Bmp2C/C; Prx1::cre embryo while the AER expression [black arrow (A and B)] of Bmp2 persists. Note that pink staining in the central region of the limb bud in (B) is nonspecific background. Wild-type (C) and Bmp4C/C; Prx1::cre (D) are forelimbs from E10.5 mouse embryos. Mesenchymal expression [red arrow (C)] of Bmp4 is abolished in Bmp4C/C; Prx1::cre embryo while the AER expression [black arrow (C and D)] of Bmp4 persists.(E–T) Depletion of BMP2 and BMP4 together causes severe limb skeletal defects. (E–T) Whole mount skeletons from newborn animals stained with Alcian blue and Alizarin red. (E–L) Forelimbs, (M–T) hindlimbs. (E and M) Wild-type, (F and N) Bmp2C/C; Prx1::cre, (G and O) Bmp4C/C; Prx1::cre, (H and P) Bmp7 −/−, (I and Q) Bmp2C/C; Bmp4+/C; Prx1::cre, (J and R) Bmp2+/C; Bmp4C/C; Prx1::cre, (K and S) Bmp2C/C; Bmp4C/C; Prx1::cre, (L and T) Bmp2C/C, Bmp7 −/−; Prx1::cre. Thin red arrow in (F), (I), and (L), defective scapula; thick red arrow in (T), failure of fibula to articulate with knee, and thick black arrow in (L) and (T), missing phalanx in digit III.
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pgen-0020216-g001: An Allelic/Nonallelic Series of BMP-Deficient Limbs(A–D) Prx1::cre efficiently recombines Bmp2 and Bmp4 conditional alleles in the limbs. Bmp2 (A and B) and Bmp4 (C and D) whole mount mRNA in situ hybridization in the limb. Wild-type (A) and Bmp2C/C; Prx1::cre (B) are forelimbs from E10.5 mouse embryos. Mesenchymal expression [asterisk (A)] of Bmp2 is abolished in Bmp2C/C; Prx1::cre embryo while the AER expression [black arrow (A and B)] of Bmp2 persists. Note that pink staining in the central region of the limb bud in (B) is nonspecific background. Wild-type (C) and Bmp4C/C; Prx1::cre (D) are forelimbs from E10.5 mouse embryos. Mesenchymal expression [red arrow (C)] of Bmp4 is abolished in Bmp4C/C; Prx1::cre embryo while the AER expression [black arrow (C and D)] of Bmp4 persists.(E–T) Depletion of BMP2 and BMP4 together causes severe limb skeletal defects. (E–T) Whole mount skeletons from newborn animals stained with Alcian blue and Alizarin red. (E–L) Forelimbs, (M–T) hindlimbs. (E and M) Wild-type, (F and N) Bmp2C/C; Prx1::cre, (G and O) Bmp4C/C; Prx1::cre, (H and P) Bmp7 −/−, (I and Q) Bmp2C/C; Bmp4+/C; Prx1::cre, (J and R) Bmp2+/C; Bmp4C/C; Prx1::cre, (K and S) Bmp2C/C; Bmp4C/C; Prx1::cre, (L and T) Bmp2C/C, Bmp7 −/−; Prx1::cre. Thin red arrow in (F), (I), and (L), defective scapula; thick red arrow in (T), failure of fibula to articulate with knee, and thick black arrow in (L) and (T), missing phalanx in digit III.

Mentions: To investigate the roles of BMP signaling at various stages of limb patterning and skeletogenesis, we constructed a series of mice deficient singly or in combination in the ability to produce BMP2, BMP4, and BMP7. BMP7-deficient mice (kindly provided by Dr. Liz Robertson) survive until birth. However, BMP2 and BMP4 are both required for viability early in embryonic development [30,31]. We therefore constructed a conditional allele of Bmp2, introducing loxP sites flanking exon 3. We obtained a conditional allele of Bmp4, in which exon 4 is flanked by loxP sites, from Dr. Holger Kulessa and Dr. Brigid Hogan [16]. Both of these alleles would be expected to result in alleles following recombination (see Materials and Methods for details). To conditionally inactivate Bmp2 and Bmp4 in the limb, we used a well-characterized transgene in which cre-recombinase is expressed under the control of the Prx1 limb enhancer [32]. This transgene expresses cre very early in limb development, resulting in complete recombination of floxed alleles at early limb bud stages. We verified the ability of Prx1::cre to recombine the conditional Bmp2 and Bmp4 alleles at earliest limb bud stages using in situ hybridization. Bmp2 is first expressed in the limb mesenchyme at embryonic day (E)10.5 in the mouse (Figure 1A, asterisk). By the time its expression is first detectable, the floxed Bmp2 allele appears to be completely recombined in the limb mesenchyme, as whole mount in situ hybridization does not detect any mesenchymal Bmp2 transcription (Figure 1B). At E10.5 Bmp4 is expressed in the mouse limb mesenchyme in two stripes at the anterior and posterior margins (Figure 1C, red arrows). These expression domains are completely lost by E10.5 in the presence of the Prx1::cre transgene (Figure 1D). Bmp2 and Bmp4 are also expressed in the AER, where Prx1::cre is inactive, and these domains of expression are not affected (Figure 1A–1D, black arrows).


Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis.

Bandyopadhyay A, Tsuji K, Cox K, Harfe BD, Rosen V, Tabin CJ - PLoS Genet. (2006)

An Allelic/Nonallelic Series of BMP-Deficient Limbs(A–D) Prx1::cre efficiently recombines Bmp2 and Bmp4 conditional alleles in the limbs. Bmp2 (A and B) and Bmp4 (C and D) whole mount mRNA in situ hybridization in the limb. Wild-type (A) and Bmp2C/C; Prx1::cre (B) are forelimbs from E10.5 mouse embryos. Mesenchymal expression [asterisk (A)] of Bmp2 is abolished in Bmp2C/C; Prx1::cre embryo while the AER expression [black arrow (A and B)] of Bmp2 persists. Note that pink staining in the central region of the limb bud in (B) is nonspecific background. Wild-type (C) and Bmp4C/C; Prx1::cre (D) are forelimbs from E10.5 mouse embryos. Mesenchymal expression [red arrow (C)] of Bmp4 is abolished in Bmp4C/C; Prx1::cre embryo while the AER expression [black arrow (C and D)] of Bmp4 persists.(E–T) Depletion of BMP2 and BMP4 together causes severe limb skeletal defects. (E–T) Whole mount skeletons from newborn animals stained with Alcian blue and Alizarin red. (E–L) Forelimbs, (M–T) hindlimbs. (E and M) Wild-type, (F and N) Bmp2C/C; Prx1::cre, (G and O) Bmp4C/C; Prx1::cre, (H and P) Bmp7 −/−, (I and Q) Bmp2C/C; Bmp4+/C; Prx1::cre, (J and R) Bmp2+/C; Bmp4C/C; Prx1::cre, (K and S) Bmp2C/C; Bmp4C/C; Prx1::cre, (L and T) Bmp2C/C, Bmp7 −/−; Prx1::cre. Thin red arrow in (F), (I), and (L), defective scapula; thick red arrow in (T), failure of fibula to articulate with knee, and thick black arrow in (L) and (T), missing phalanx in digit III.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1713256&req=5

pgen-0020216-g001: An Allelic/Nonallelic Series of BMP-Deficient Limbs(A–D) Prx1::cre efficiently recombines Bmp2 and Bmp4 conditional alleles in the limbs. Bmp2 (A and B) and Bmp4 (C and D) whole mount mRNA in situ hybridization in the limb. Wild-type (A) and Bmp2C/C; Prx1::cre (B) are forelimbs from E10.5 mouse embryos. Mesenchymal expression [asterisk (A)] of Bmp2 is abolished in Bmp2C/C; Prx1::cre embryo while the AER expression [black arrow (A and B)] of Bmp2 persists. Note that pink staining in the central region of the limb bud in (B) is nonspecific background. Wild-type (C) and Bmp4C/C; Prx1::cre (D) are forelimbs from E10.5 mouse embryos. Mesenchymal expression [red arrow (C)] of Bmp4 is abolished in Bmp4C/C; Prx1::cre embryo while the AER expression [black arrow (C and D)] of Bmp4 persists.(E–T) Depletion of BMP2 and BMP4 together causes severe limb skeletal defects. (E–T) Whole mount skeletons from newborn animals stained with Alcian blue and Alizarin red. (E–L) Forelimbs, (M–T) hindlimbs. (E and M) Wild-type, (F and N) Bmp2C/C; Prx1::cre, (G and O) Bmp4C/C; Prx1::cre, (H and P) Bmp7 −/−, (I and Q) Bmp2C/C; Bmp4+/C; Prx1::cre, (J and R) Bmp2+/C; Bmp4C/C; Prx1::cre, (K and S) Bmp2C/C; Bmp4C/C; Prx1::cre, (L and T) Bmp2C/C, Bmp7 −/−; Prx1::cre. Thin red arrow in (F), (I), and (L), defective scapula; thick red arrow in (T), failure of fibula to articulate with knee, and thick black arrow in (L) and (T), missing phalanx in digit III.
Mentions: To investigate the roles of BMP signaling at various stages of limb patterning and skeletogenesis, we constructed a series of mice deficient singly or in combination in the ability to produce BMP2, BMP4, and BMP7. BMP7-deficient mice (kindly provided by Dr. Liz Robertson) survive until birth. However, BMP2 and BMP4 are both required for viability early in embryonic development [30,31]. We therefore constructed a conditional allele of Bmp2, introducing loxP sites flanking exon 3. We obtained a conditional allele of Bmp4, in which exon 4 is flanked by loxP sites, from Dr. Holger Kulessa and Dr. Brigid Hogan [16]. Both of these alleles would be expected to result in alleles following recombination (see Materials and Methods for details). To conditionally inactivate Bmp2 and Bmp4 in the limb, we used a well-characterized transgene in which cre-recombinase is expressed under the control of the Prx1 limb enhancer [32]. This transgene expresses cre very early in limb development, resulting in complete recombination of floxed alleles at early limb bud stages. We verified the ability of Prx1::cre to recombine the conditional Bmp2 and Bmp4 alleles at earliest limb bud stages using in situ hybridization. Bmp2 is first expressed in the limb mesenchyme at embryonic day (E)10.5 in the mouse (Figure 1A, asterisk). By the time its expression is first detectable, the floxed Bmp2 allele appears to be completely recombined in the limb mesenchyme, as whole mount in situ hybridization does not detect any mesenchymal Bmp2 transcription (Figure 1B). At E10.5 Bmp4 is expressed in the mouse limb mesenchyme in two stripes at the anterior and posterior margins (Figure 1C, red arrows). These expression domains are completely lost by E10.5 in the presence of the Prx1::cre transgene (Figure 1D). Bmp2 and Bmp4 are also expressed in the AER, where Prx1::cre is inactive, and these domains of expression are not affected (Figure 1A–1D, black arrows).

Bottom Line: However, due to complications associated with early embryonic lethality, particularly for Bmp2 and Bmp4, and with functional redundancy among BMP molecules, it has been difficult to decipher the specific roles of these BMP molecules during different stages of limb development.Contrary to earlier suggestions, our results indicate that BMPs neither act as secondary signals downstream of Sonic Hedghog (SHH) in patterning the anteroposterior axis nor as signals from the interdigital mesenchyme in specifying digit identity.In contrast, we find that the loss of both BMP2 and BMP4 results in a severe impairment of osteogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Bone morphogenetic protein (BMP) family members, including BMP2, BMP4, and BMP7, are expressed throughout limb development. BMPs have been implicated in early limb patterning as well as in the process of skeletogenesis. However, due to complications associated with early embryonic lethality, particularly for Bmp2 and Bmp4, and with functional redundancy among BMP molecules, it has been difficult to decipher the specific roles of these BMP molecules during different stages of limb development. To circumvent these issues, we have constructed a series of mouse strains lacking one or more of these BMPs, using conditional alleles in the case of Bmp2 and Bmp4 to remove them specifically from the limb bud mesenchyme. Contrary to earlier suggestions, our results indicate that BMPs neither act as secondary signals downstream of Sonic Hedghog (SHH) in patterning the anteroposterior axis nor as signals from the interdigital mesenchyme in specifying digit identity. We do find that a threshold level of BMP signaling is required for the onset of chondrogenesis, and hence some chondrogenic condensations fail to form in limbs deficient in both BMP2 and BMP4. However, in the condensations that do form, subsequent chondrogenic differentiation proceeds normally even in the absence of BMP2 and BMP7 or BMP2 and BMP4. In contrast, we find that the loss of both BMP2 and BMP4 results in a severe impairment of osteogenesis.

Show MeSH
Related in: MedlinePlus