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and BMP4 Are Not Critical for Chondrogenic Differentiation\nAs noted above, at E12.5, chondrogenic condensation of the digits could be visualized in Bmp2C/C; Bmp4C/C; Prx1::cre limb buds by in situ hybridization with a Sox9 probe. Similar analysis at E10.5 and E11.5 demonstrated a relatively normal process of condensation of the proximal skeletal elements (Figure S1H–S1M), despite the absence of detectable Bmp2 and Bmp4 transcripts. As shown in Figure S1N and S1O, expression of Col II, a chondrocyte marker, is observed in the BMP2, BMP4–deficient cartilage (Figure S1O) as early as E12.5, similar to a wild-type control (Figure S1N). Similarly, at E13.5, the entire limb skeleton could be visualized undergoing chondrogenesis by Alcian blue staining, albeit in a defective pattern, in the BMP2, BMP4–deficient limb buds (Figure 4A and 4B and unpublished data).\nFigure 4 Chondrogenesis Starts and Proceeds Normally Even in the Absence of BMP2 and BMP4\n(A and B) Whole mount skeletons from E13.5 embryos that are stained with alcian blue. (A) Wild-type embryo, (B) Bmp2C/C; Bmp4C/C; Prx1::cre embryo. Black arrow in (B) shows the fusion of the zeugopod and stylopod.\n(C and D) Hematoxylin and eosin–stained sagittal sections of humeri from E13.5 wild-type (C) and Bmp2C/C; Bmp4C/C; Prx1::cre (D) embryos. Thick red arrow in (C) shows the hypertrophic region.\n(E and F) Sagittal sections of wild-type (E) and Bmp2C/C; Bmp4C/C; Prx1::cre (F) humeri from E15.5 embryos were hybridized with digoxigenin-labeled antisense rioprobes for ColX.\n(G and H) Sagittal sections of forelimbs from E13.5 wild-type and BMP2, BMP4–deficient embryos, respectively, stained with radioactive riboprobes for Bmp7 mRNA. (G′) and (H′) show the bright field views of (G) and (H), respectively. As the cartilage elements grow, a wave of hypertrophic differentiation occurs from the center toward the distal ends (reviewed in [39–41]). Histological examination of the humeri at E13.5 showed that while chondrogenic differentiation had taken place in the BMP2, BMP4–deficient humerus, hypertrophic differentiation was delayed (Figure 4C and 4D). However, by E15.5, hypertrophy was evident in these elements as seen by type X collagen expression (Figure 4E and 4F). The hypertrophic cells in BMP2, BMP4–deficient limb also express late hypertrophic markers, such as osteopontin [42] (unpublished data).\nTo examine whether the successful execution of chondrogenesis in the BMP2, BMP4–deficient limbs is a result of compensatory upregulation of other ostegenic BMPs, we carried out in situ hybridization for Bmp6 and Bmp7 in these limbs. At E13.5, we observed no increase in expression of Bmp6 (Figure S1P–S1S) or Bmp7 (compare Figure 4H with Figure 4G) mRNA in the mutant tissues compared to their wild-type counterparts"}

{"project":"craft-ca-core-ex-dev","denotations":[{"id":"T7524","span":{"begin":0,"end":4},"obj":"PR_EXT:000000164"},{"id":"T7525","span":{"begin":9,"end":13},"obj":"PR_EXT:000000165"},{"id":"T7526","span":{"begin":35,"end":47},"obj":"GO:0051216"},{"id":"T7527","span":{"begin":48,"end":63},"obj":"GO_RO_EXT:developmental_differentiation_process"},{"id":"T7528","span":{"begin":90,"end":115},"obj":"GO:0001502"},{"id":"T7529","span":{"begin":123,"end":129},"obj":"UBERON:0002544"},{"id":"T7530","span":{"begin":153,"end":157},"obj":"PR_EXT:000000164"},{"id":"T7531","span":{"begin":162,"end":166},"obj":"PR_EXT:000000165"},{"id":"T7532","span":{"begin":171,"end":175},"obj":"PR_EXT:000013316"},{"id":"T7533","span":{"begin":181,"end":190},"obj":"UBERON:0004347"},{"id":"T7534","span":{"begin":202,"end":215},"obj":"GO:0097617"},{"id":"T7535","span":{"begin":223,"end":227},"obj":"PR_EXT:000015435"},{"id":"T7536","span":{"begin":228,"end":233},"obj":"CHEBI_SO_EXT:molecular_probe"},{"id":"T7537","span":{"begin":344,"end":361},"obj":"UBERON:0004765"},{"id":"T7538","span":{"begin":414,"end":418},"obj":"PR_EXT:000000164"},{"id":"T7539","span":{"begin":423,"end":427},"obj":"PR_EXT:000000165"},{"id":"T7540","span":{"begin":428,"end":439},"obj":"SO_EXT:0000673"},{"id":"T7541","span":{"begin":473,"end":483},"obj":"GO:0010467"},{"id":"T7542","span":{"begin":487,"end":493},"obj":"PR_EXT:000003266"},{"id":"T7543","span":{"begin":497,"end":508},"obj":"CL:0000138"},{"id":"T7544","span":{"begin":509,"end":515},"obj":"CHEBI_SO_EXT:molecular_indicator_or_label_or_marker_or_tag"},{"id":"T7545","span":{"begin":536,"end":540},"obj":"PR_EXT:000000164"},{"id":"T7546","span":{"begin":542,"end":546},"obj":"PR_EXT:000000165"},{"id":"T7547","span":{"begin":557,"end":566},"obj":"UBERON_EXT:cartilage_element_or_tissue"},{"id":"T7548","span":{"begin":612,"end":621},"obj":"SO_EXT:wild_type_entity_or_quality"},{"id":"T7549","span":{"begin":676,"end":689},"obj":"UBERON:0004381"},{"id":"T7550","span":{"begin":721,"end":735},"obj":"GO:0051216"},{"id":"T7551","span":{"begin":799,"end":803},"obj":"PR_EXT:000000164"},{"id":"T7552","span":{"begin":805,"end":809},"obj":"PR_EXT:000000165"},{"id":"T7553","span":{"begin":820,"end":829},"obj":"UBERON:0004347"},{"id":"T7554","span":{"begin":1786,"end":1804},"obj":"UBERON:0007844"},{"id":"T7555","span":{"begin":1805,"end":1809},"obj":"GO:0040007"},{"id":"T7556","span":{"begin":1834,"end":1849},"obj":"GO_RO_EXT:developmental_differentiation_process"},{"id":"T7557","span":{"begin":1951,"end":1957},"obj":"UBERON:0000976"},{"id":"T7558","span":{"begin":1985,"end":1997},"obj":"GO:0051216"},{"id":"T7559","span":{"begin":1998,"end":2013},"obj":"GO_RO_EXT:developmental_differentiation_process"},{"id":"T7560","span":{"begin":2037,"end":2041},"obj":"PR_EXT:000000164"},{"id":"T7561","span":{"begin":2043,"end":2047},"obj":"PR_EXT:000000165"},{"id":"T7562","span":{"begin":2058,"end":2065},"obj":"UBERON:0000976"},{"id":"T7563","span":{"begin":2080,"end":2095},"obj":"GO_RO_EXT:developmental_differentiation_process"},{"id":"T7564","span":{"begin":2200,"end":2215},"obj":"PR_EXT:000005693"},{"id":"T7565","span":{"begin":2207,"end":2215},"obj":"CHEBI_PR_EXT:collagen"},{"id":"T7566","span":{"begin":2216,"end":2226},"obj":"GO:0010467"},{"id":"T7567","span":{"begin":2264,"end":2269},"obj":"CL_GO_EXT:cell"},{"id":"T7568","span":{"begin":2273,"end":2277},"obj":"PR_EXT:000000164"},{"id":"T7569","span":{"begin":2279,"end":2283},"obj":"PR_EXT:000000165"},{"id":"T7570","span":{"begin":2294,"end":2298},"obj":"UBERON:0002101"},{"id":"T7571","span":{"begin":2304,"end":2311},"obj":"GO:0010467"},{"id":"T7572","span":{"begin":2330,"end":2337},"obj":"CHEBI_SO_EXT:molecular_indicator_or_label_or_marker_or_tag"},{"id":"T7573","span":{"begin":2347,"end":2358},"obj":"PR_EXT:000015561"},{"id":"T7574","span":{"begin":2431,"end":2445},"obj":"GO:0051216"},{"id":"T7575","span":{"begin":2453,"end":2457},"obj":"PR_EXT:000000164"},{"id":"T7576","span":{"begin":2459,"end":2463},"obj":"PR_EXT:000000165"},{"id":"T7577","span":{"begin":2474,"end":2479},"obj":"UBERON:0002101"},{"id":"T7578","span":{"begin":2508,"end":2520},"obj":"GO_EXT:positive_regulation"},{"id":"T7579","span":{"begin":2530,"end":2539},"obj":"GO:0001503"},{"id":"T7580","span":{"begin":2540,"end":2544},"obj":"PR_EXT:000000034"},{"id":"T7581","span":{"begin":2569,"end":2582},"obj":"GO:0097617"},{"id":"T7582","span":{"begin":2587,"end":2591},"obj":"PR_EXT:000000167"},{"id":"T7583","span":{"begin":2596,"end":2600},"obj":"PR_EXT:000000168"},{"id":"T7584","span":{"begin":2610,"end":2615},"obj":"UBERON:0002101"},{"id":"T7585","span":{"begin":2654,"end":2664},"obj":"GO:0010467"},{"id":"T7586","span":{"begin":2668,"end":2672},"obj":"PR_EXT:000000167"},{"id":"T7587","span":{"begin":2693,"end":2697},"obj":"PR_EXT:000000168"},{"id":"T7588","span":{"begin":2733,"end":2737},"obj":"CHEBI_SO_EXT:mRNA"},{"id":"T7589","span":{"begin":2745,"end":2751},"obj":"SO_EXT:sequence_altered_entity_or_alteration_process"},{"id":"T7590","span":{"begin":2752,"end":2759},"obj":"UBERON:0000479"},{"id":"T7591","span":{"begin":2778,"end":2787},"obj":"SO_EXT:wild_type_entity_or_quality"}],"text":"BMP2 and BMP4 Are Not Critical for Chondrogenic Differentiation\nAs noted above, at E12.5, chondrogenic condensation of the digits could be visualized in Bmp2C/C; Bmp4C/C; Prx1::cre limb buds by in situ hybridization with a Sox9 probe. Similar analysis at E10.5 and E11.5 demonstrated a relatively normal process of condensation of the proximal skeletal elements (Figure S1H–S1M), despite the absence of detectable Bmp2 and Bmp4 transcripts. As shown in Figure S1N and S1O, expression of Col II, a chondrocyte marker, is observed in the BMP2, BMP4–deficient cartilage (Figure S1O) as early as E12.5, similar to a wild-type control (Figure S1N). Similarly, at E13.5, the entire limb skeleton could be visualized undergoing chondrogenesis by Alcian blue staining, albeit in a defective pattern, in the BMP2, BMP4–deficient limb buds (Figure 4A and 4B and unpublished data).\nFigure 4 Chondrogenesis Starts and Proceeds Normally Even in the Absence of BMP2 and BMP4\n(A and B) Whole mount skeletons from E13.5 embryos that are stained with alcian blue. (A) Wild-type embryo, (B) Bmp2C/C; Bmp4C/C; Prx1::cre embryo. Black arrow in (B) shows the fusion of the zeugopod and stylopod.\n(C and D) Hematoxylin and eosin–stained sagittal sections of humeri from E13.5 wild-type (C) and Bmp2C/C; Bmp4C/C; Prx1::cre (D) embryos. Thick red arrow in (C) shows the hypertrophic region.\n(E and F) Sagittal sections of wild-type (E) and Bmp2C/C; Bmp4C/C; Prx1::cre (F) humeri from E15.5 embryos were hybridized with digoxigenin-labeled antisense rioprobes for ColX.\n(G and H) Sagittal sections of forelimbs from E13.5 wild-type and BMP2, BMP4–deficient embryos, respectively, stained with radioactive riboprobes for Bmp7 mRNA. (G′) and (H′) show the bright field views of (G) and (H), respectively. As the cartilage elements grow, a wave of hypertrophic differentiation occurs from the center toward the distal ends (reviewed in [39–41]). Histological examination of the humeri at E13.5 showed that while chondrogenic differentiation had taken place in the BMP2, BMP4–deficient humerus, hypertrophic differentiation was delayed (Figure 4C and 4D). However, by E15.5, hypertrophy was evident in these elements as seen by type X collagen expression (Figure 4E and 4F). The hypertrophic cells in BMP2, BMP4–deficient limb also express late hypertrophic markers, such as osteopontin [42] (unpublished data).\nTo examine whether the successful execution of chondrogenesis in the BMP2, BMP4–deficient limbs is a result of compensatory upregulation of other ostegenic BMPs, we carried out in situ hybridization for Bmp6 and Bmp7 in these limbs. At E13.5, we observed no increase in expression of Bmp6 (Figure S1P–S1S) or Bmp7 (compare Figure 4H with Figure 4G) mRNA in the mutant tissues compared to their wild-type counterparts"}

{"project":"2_test","denotations":[{"id":"17194222-7859279-85946874","span":{"begin":1910,"end":1912},"obj":"7859279"},{"id":"17194222-1579995-85946874","span":{"begin":1910,"end":1912},"obj":"1579995"},{"id":"17194222-10989939-85946875","span":{"begin":2360,"end":2362},"obj":"10989939"},{"id":"T6417","span":{"begin":1910,"end":1912},"obj":"7859279"},{"id":"T18215","span":{"begin":1910,"end":1912},"obj":"1579995"},{"id":"T64328","span":{"begin":2360,"end":2362},"obj":"10989939"}],"text":"BMP2 and BMP4 Are Not Critical for Chondrogenic Differentiation\nAs noted above, at E12.5, chondrogenic condensation of the digits could be visualized in Bmp2C/C; Bmp4C/C; Prx1::cre limb buds by in situ hybridization with a Sox9 probe. Similar analysis at E10.5 and E11.5 demonstrated a relatively normal process of condensation of the proximal skeletal elements (Figure S1H–S1M), despite the absence of detectable Bmp2 and Bmp4 transcripts. As shown in Figure S1N and S1O, expression of Col II, a chondrocyte marker, is observed in the BMP2, BMP4–deficient cartilage (Figure S1O) as early as E12.5, similar to a wild-type control (Figure S1N). Similarly, at E13.5, the entire limb skeleton could be visualized undergoing chondrogenesis by Alcian blue staining, albeit in a defective pattern, in the BMP2, BMP4–deficient limb buds (Figure 4A and 4B and unpublished data).\nFigure 4 Chondrogenesis Starts and Proceeds Normally Even in the Absence of BMP2 and BMP4\n(A and B) Whole mount skeletons from E13.5 embryos that are stained with alcian blue. (A) Wild-type embryo, (B) Bmp2C/C; Bmp4C/C; Prx1::cre embryo. Black arrow in (B) shows the fusion of the zeugopod and stylopod.\n(C and D) Hematoxylin and eosin–stained sagittal sections of humeri from E13.5 wild-type (C) and Bmp2C/C; Bmp4C/C; Prx1::cre (D) embryos. Thick red arrow in (C) shows the hypertrophic region.\n(E and F) Sagittal sections of wild-type (E) and Bmp2C/C; Bmp4C/C; Prx1::cre (F) humeri from E15.5 embryos were hybridized with digoxigenin-labeled antisense rioprobes for ColX.\n(G and H) Sagittal sections of forelimbs from E13.5 wild-type and BMP2, BMP4–deficient embryos, respectively, stained with radioactive riboprobes for Bmp7 mRNA. (G′) and (H′) show the bright field views of (G) and (H), respectively. As the cartilage elements grow, a wave of hypertrophic differentiation occurs from the center toward the distal ends (reviewed in [39–41]). Histological examination of the humeri at E13.5 showed that while chondrogenic differentiation had taken place in the BMP2, BMP4–deficient humerus, hypertrophic differentiation was delayed (Figure 4C and 4D). However, by E15.5, hypertrophy was evident in these elements as seen by type X collagen expression (Figure 4E and 4F). The hypertrophic cells in BMP2, BMP4–deficient limb also express late hypertrophic markers, such as osteopontin [42] (unpublished data).\nTo examine whether the successful execution of chondrogenesis in the BMP2, BMP4–deficient limbs is a result of compensatory upregulation of other ostegenic BMPs, we carried out in situ hybridization for Bmp6 and Bmp7 in these limbs. At E13.5, we observed no increase in expression of Bmp6 (Figure S1P–S1S) or Bmp7 (compare Figure 4H with Figure 4G) mRNA in the mutant tissues compared to their wild-type counterparts"}

{"project":"craft-ca-core-dev","denotations":[{"id":"T7471","span":{"begin":0,"end":4},"obj":"PR:000000164"},{"id":"T7472","span":{"begin":9,"end":13},"obj":"PR:000000165"},{"id":"T7473","span":{"begin":35,"end":47},"obj":"GO:0051216"},{"id":"T7474","span":{"begin":90,"end":115},"obj":"GO:0001502"},{"id":"T7475","span":{"begin":123,"end":129},"obj":"UBERON:0002544"},{"id":"T7476","span":{"begin":153,"end":157},"obj":"PR:000000164"},{"id":"T7477","span":{"begin":162,"end":166},"obj":"PR:000000165"},{"id":"T7478","span":{"begin":171,"end":175},"obj":"PR:000013316"},{"id":"T7479","span":{"begin":181,"end":190},"obj":"UBERON:0004347"},{"id":"T7480","span":{"begin":202,"end":215},"obj":"GO:0097617"},{"id":"T7481","span":{"begin":223,"end":227},"obj":"PR:000015435"},{"id":"T7482","span":{"begin":344,"end":361},"obj":"UBERON:0004765"},{"id":"T7483","span":{"begin":414,"end":418},"obj":"PR:000000164"},{"id":"T7484","span":{"begin":423,"end":427},"obj":"PR:000000165"},{"id":"T7485","span":{"begin":428,"end":439},"obj":"SO:0000673"},{"id":"T7486","span":{"begin":473,"end":483},"obj":"GO:0010467"},{"id":"T7487","span":{"begin":487,"end":493},"obj":"PR:000003266"},{"id":"T7488","span":{"begin":497,"end":508},"obj":"CL:0000138"},{"id":"T7489","span":{"begin":536,"end":540},"obj":"PR:000000164"},{"id":"T7490","span":{"begin":542,"end":546},"obj":"PR:000000165"},{"id":"T7491","span":{"begin":676,"end":689},"obj":"UBERON:0004381"},{"id":"T7492","span":{"begin":721,"end":735},"obj":"GO:0051216"},{"id":"T7493","span":{"begin":799,"end":803},"obj":"PR:000000164"},{"id":"T7494","span":{"begin":805,"end":809},"obj":"PR:000000165"},{"id":"T7495","span":{"begin":820,"end":829},"obj":"UBERON:0004347"},{"id":"T7496","span":{"begin":1786,"end":1804},"obj":"UBERON:0007844"},{"id":"T7497","span":{"begin":1805,"end":1809},"obj":"GO:0040007"},{"id":"T7498","span":{"begin":1951,"end":1957},"obj":"UBERON:0000976"},{"id":"T7499","span":{"begin":1985,"end":1997},"obj":"GO:0051216"},{"id":"T7500","span":{"begin":2037,"end":2041},"obj":"PR:000000164"},{"id":"T7501","span":{"begin":2043,"end":2047},"obj":"PR:000000165"},{"id":"T7502","span":{"begin":2058,"end":2065},"obj":"UBERON:0000976"},{"id":"T7503","span":{"begin":2200,"end":2215},"obj":"PR:000005693"},{"id":"T7504","span":{"begin":2216,"end":2226},"obj":"GO:0010467"},{"id":"T7505","span":{"begin":2273,"end":2277},"obj":"PR:000000164"},{"id":"T7506","span":{"begin":2279,"end":2283},"obj":"PR:000000165"},{"id":"T7507","span":{"begin":2294,"end":2298},"obj":"UBERON:0002101"},{"id":"T7508","span":{"begin":2304,"end":2311},"obj":"GO:0010467"},{"id":"T7509","span":{"begin":2347,"end":2358},"obj":"PR:000015561"},{"id":"T7510","span":{"begin":2431,"end":2445},"obj":"GO:0051216"},{"id":"T7511","span":{"begin":2453,"end":2457},"obj":"PR:000000164"},{"id":"T7512","span":{"begin":2459,"end":2463},"obj":"PR:000000165"},{"id":"T7513","span":{"begin":2474,"end":2479},"obj":"UBERON:0002101"},{"id":"T7514","span":{"begin":2530,"end":2539},"obj":"GO:0001503"},{"id":"T7515","span":{"begin":2540,"end":2544},"obj":"PR:000000034"},{"id":"T7516","span":{"begin":2569,"end":2582},"obj":"GO:0097617"},{"id":"T7517","span":{"begin":2587,"end":2591},"obj":"PR:000000167"},{"id":"T7518","span":{"begin":2596,"end":2600},"obj":"PR:000000168"},{"id":"T7519","span":{"begin":2610,"end":2615},"obj":"UBERON:0002101"},{"id":"T7520","span":{"begin":2654,"end":2664},"obj":"GO:0010467"},{"id":"T7521","span":{"begin":2668,"end":2672},"obj":"PR:000000167"},{"id":"T7522","span":{"begin":2693,"end":2697},"obj":"PR:000000168"},{"id":"T7523","span":{"begin":2752,"end":2759},"obj":"UBERON:0000479"}],"text":"BMP2 and BMP4 Are Not Critical for Chondrogenic Differentiation\nAs noted above, at E12.5, chondrogenic condensation of the digits could be visualized in Bmp2C/C; Bmp4C/C; Prx1::cre limb buds by in situ hybridization with a Sox9 probe. Similar analysis at E10.5 and E11.5 demonstrated a relatively normal process of condensation of the proximal skeletal elements (Figure S1H–S1M), despite the absence of detectable Bmp2 and Bmp4 transcripts. As shown in Figure S1N and S1O, expression of Col II, a chondrocyte marker, is observed in the BMP2, BMP4–deficient cartilage (Figure S1O) as early as E12.5, similar to a wild-type control (Figure S1N). Similarly, at E13.5, the entire limb skeleton could be visualized undergoing chondrogenesis by Alcian blue staining, albeit in a defective pattern, in the BMP2, BMP4–deficient limb buds (Figure 4A and 4B and unpublished data).\nFigure 4 Chondrogenesis Starts and Proceeds Normally Even in the Absence of BMP2 and BMP4\n(A and B) Whole mount skeletons from E13.5 embryos that are stained with alcian blue. (A) Wild-type embryo, (B) Bmp2C/C; Bmp4C/C; Prx1::cre embryo. Black arrow in (B) shows the fusion of the zeugopod and stylopod.\n(C and D) Hematoxylin and eosin–stained sagittal sections of humeri from E13.5 wild-type (C) and Bmp2C/C; Bmp4C/C; Prx1::cre (D) embryos. Thick red arrow in (C) shows the hypertrophic region.\n(E and F) Sagittal sections of wild-type (E) and Bmp2C/C; Bmp4C/C; Prx1::cre (F) humeri from E15.5 embryos were hybridized with digoxigenin-labeled antisense rioprobes for ColX.\n(G and H) Sagittal sections of forelimbs from E13.5 wild-type and BMP2, BMP4–deficient embryos, respectively, stained with radioactive riboprobes for Bmp7 mRNA. (G′) and (H′) show the bright field views of (G) and (H), respectively. As the cartilage elements grow, a wave of hypertrophic differentiation occurs from the center toward the distal ends (reviewed in [39–41]). Histological examination of the humeri at E13.5 showed that while chondrogenic differentiation had taken place in the BMP2, BMP4–deficient humerus, hypertrophic differentiation was delayed (Figure 4C and 4D). However, by E15.5, hypertrophy was evident in these elements as seen by type X collagen expression (Figure 4E and 4F). The hypertrophic cells in BMP2, BMP4–deficient limb also express late hypertrophic markers, such as osteopontin [42] (unpublished data).\nTo examine whether the successful execution of chondrogenesis in the BMP2, BMP4–deficient limbs is a result of compensatory upregulation of other ostegenic BMPs, we carried out in situ hybridization for Bmp6 and Bmp7 in these limbs. At E13.5, we observed no increase in expression of Bmp6 (Figure S1P–S1S) or Bmp7 (compare Figure 4H with Figure 4G) mRNA in the mutant tissues compared to their wild-type counterparts"}