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and genotyping\nThe Hdhex4/5 mice carrying a pGKneo insertion/replacement inactivating mutation in the mouse HD gene homologue have been described previously [10]. The experiments were conducted in accordance with an IACUC approved protocol, through the MGH Subcommittee on Animal Research. Mutant Hdhex4/5/Hdhex4/5 and normal littermates were obtained in timed pregnancies from mating of Hdhex4/5/Hdh+ heterozygotes, genotyped by PCR assay, as described [10]. The day of plug was taken to be E0.5. Embryos that were morphologically normal were pooled separately from morphologically mutant embryos for analysis. Nodal expression was determined in embryos from matings of Hdhex4/5/Hdh+; NdllacZ/Ndl+ compound heterozygotes genotyped by PCR assay as described in [29].\n\nWhole mount and section in situ hybridization and β-gal staining\nAfter dissection in PBS, embryos were fixed overnight in 4% paraformaldehyde at 4°C. For sections, decidua fixed in 4% paraformaldehyde, were embedded in paraffin and sectioned at 7 microns. RNA in situ hybridizations were performed as described previously [55]. Nodal.lacZ expression was assessed by β-galactosidase staining as reported [29], on embryos post fixed in 4% paraformaldehyde. Embryos were mounted in 80% glycerol before being photographed.\nThe huntingtin deficient phenotype is fully penetrant at each of the stages that were assessed [10]. Three to six embryos were evaluated for each marker, with every embryo exhibiting the same mutant phenotype in each case.\n\nAlkaline phosphatase staining of Primordial Germ Cells (PCGs)\nAfter dissections, embryos were fixed in 4% paraformaldehyde briefly and washed and stored in 1 × PBS/0.1% TX-100 at 4°C. Embryos were washed once with Tris-Maleate Buffer (25 mM Tris-Maleate, pH = 9.0, 0.8 mM MgCl2) and were subsequently incubated in alkaline phosphatase staining solution (25 mM Tris-Maleate, pH = 9.0, 0.8 mM MgCl2, 0.4 mg/ml alpha-naphthyl phosphate, 1 mg/ml Fast-Red). Stained embryos were washed in 1 × PBS/0.1% TX-100.\n\nWhole embryo culture\nEmbryos were dissected at E7.5 and washed in DMEM. Embryos were then cultured individually in 1 ml of culture media (75% immediately centrifuged rat serum and 25% DMEM [56]) for 24 hours while rotating in a 37°C incubator in 5% CO2. Embryos were then fixed in 4% paraformaldehyde for analysis."}

{"project":"craft-ca-core-ex-dev","denotations":[{"id":"T7693","span":{"begin":9,"end":13},"obj":"NCBITaxon:10088"},{"id":"T7694","span":{"begin":33,"end":36},"obj":"PR_EXT:000008840"},{"id":"T7695","span":{"begin":42,"end":46},"obj":"NCBITaxon:10088"},{"id":"T7696","span":{"begin":65,"end":74},"obj":"SO_EXT:sequence_insertion_entity_or_process"},{"id":"T7697","span":{"begin":75,"end":86},"obj":"SO_EXT:sequence_substitution_entity_or_process"},{"id":"T7698","span":{"begin":100,"end":108},"obj":"SO_EXT:sequence_alteration_entity_or_process"},{"id":"T7699","span":{"begin":116,"end":121},"obj":"NCBITaxon:10088"},{"id":"T7700","span":{"begin":122,"end":124},"obj":"PR_EXT:000008840"},{"id":"T7701","span":{"begin":125,"end":129},"obj":"SO_EXT:0000704"},{"id":"T7702","span":{"begin":130,"end":139},"obj":"SO_EXT:0000853"},{"id":"T7703","span":{"begin":287,"end":293},"obj":"NCBITaxon:33208"},{"id":"T7704","span":{"begin":304,"end":310},"obj":"SO_EXT:sequence_altered_entity_or_alteration_process"},{"id":"T7705","span":{"begin":311,"end":314},"obj":"PR_EXT:000008840"},{"id":"T7706","span":{"begin":320,"end":323},"obj":"PR_EXT:000008840"},{"id":"T7707","span":{"begin":375,"end":386},"obj":"GO:0007565"},{"id":"T7708","span":{"begin":392,"end":398},"obj":"GO:0007618"},{"id":"T7709","span":{"begin":402,"end":405},"obj":"PR_EXT:000008840"},{"id":"T7710","span":{"begin":411,"end":414},"obj":"PR_EXT:000008840"},{"id":"T7711","span":{"begin":414,"end":415},"obj":"SO_EXT:normal_or_wild_type_or_present"},{"id":"T7712","span":{"begin":485,"end":489},"obj":"UBERON:0010148"},{"id":"T7713","span":{"begin":512,"end":519},"obj":"UBERON:0000922"},{"id":"T7714","span":{"begin":597,"end":603},"obj":"SO_EXT:sequence_altered_entity_or_alteration_process"},{"id":"T7715","span":{"begin":604,"end":611},"obj":"UBERON:0000922"},{"id":"T7716","span":{"begin":626,"end":631},"obj":"PR_EXT:000000105"},{"id":"T7717","span":{"begin":632,"end":642},"obj":"GO:0010467"},{"id":"T7718","span":{"begin":661,"end":668},"obj":"UBERON:0000922"},{"id":"T7719","span":{"begin":674,"end":681},"obj":"GO:0007618"},{"id":"T7720","span":{"begin":685,"end":688},"obj":"PR_EXT:000008840"},{"id":"T7721","span":{"begin":694,"end":697},"obj":"PR_EXT:000008840"},{"id":"T7722","span":{"begin":697,"end":698},"obj":"SO_EXT:normal_or_wild_type_or_present"},{"id":"T7723","span":{"begin":708,"end":711},"obj":"PR_EXT:000000105"},{"id":"T7724","span":{"begin":711,"end":712},"obj":"SO_EXT:normal_or_wild_type_or_present"},{"id":"T7902","span":{"begin":814,"end":827},"obj":"GO:0097617"},{"id":"T7903","span":{"begin":832,"end":837},"obj":"GO_EXT:0004565"},{"id":"T7904","span":{"begin":872,"end":879},"obj":"UBERON:0000922"},{"id":"T7905","span":{"begin":907,"end":923},"obj":"CHEBI_EXT:paraformaldehye"},{"id":"T7906","span":{"begin":946,"end":953},"obj":"UBERON:0002450"},{"id":"T7907","span":{"begin":966,"end":982},"obj":"CHEBI_EXT:paraformaldehye"},{"id":"T7908","span":{"begin":1038,"end":1041},"obj":"CHEBI_SO_EXT:RNA"},{"id":"T7909","span":{"begin":1050,"end":1064},"obj":"GO:0097617"},{"id":"T7910","span":{"begin":1110,"end":1115},"obj":"PR_EXT:000000105"},{"id":"T7911","span":{"begin":1116,"end":1120},"obj":"PR_EXT:000033987"},{"id":"T7912","span":{"begin":1121,"end":1131},"obj":"GO:0010467"},{"id":"T7913","span":{"begin":1148,"end":1163},"obj":"GO_EXT:0004565"},{"id":"T7914","span":{"begin":1194,"end":1201},"obj":"UBERON:0000922"},{"id":"T7915","span":{"begin":1219,"end":1235},"obj":"CHEBI_EXT:paraformaldehye"},{"id":"T7916","span":{"begin":1237,"end":1244},"obj":"UBERON:0000922"},{"id":"T7917","span":{"begin":1265,"end":1273},"obj":"CHEBI:17754"},{"id":"T7918","span":{"begin":1305,"end":1315},"obj":"PR_EXT:000008840"},{"id":"T7919","span":{"begin":1415,"end":1422},"obj":"UBERON:0000922"},{"id":"T7920","span":{"begin":1447,"end":1453},"obj":"CHEBI_SO_EXT:molecular_indicator_or_label_or_marker_or_tag"},{"id":"T7921","span":{"begin":1466,"end":1472},"obj":"UBERON:0000922"},{"id":"T7922","span":{"begin":1493,"end":1499},"obj":"SO_EXT:sequence_altered_entity_or_alteration_process"},{"id":"T8108","span":{"begin":1525,"end":1545},"obj":"GO_PR_EXT:alkaline_phosphatase"},{"id":"T8109","span":{"begin":1558,"end":1579},"obj":"CL:0000670"},{"id":"T8110","span":{"begin":1574,"end":1579},"obj":"CL_GO_EXT:cell"},{"id":"T8111","span":{"begin":1581,"end":1585},"obj":"CL:0000670"},{"id":"T8112","span":{"begin":1606,"end":1613},"obj":"UBERON:0000922"},{"id":"T8113","span":{"begin":1631,"end":1647},"obj":"CHEBI_EXT:paraformaldehye"},{"id":"T8114","span":{"begin":1694,"end":1700},"obj":"CHEBI:9750"},{"id":"T8115","span":{"begin":1709,"end":1716},"obj":"UBERON:0000922"},{"id":"T8116","span":{"begin":1739,"end":1751},"obj":"CHEBI:67100"},{"id":"T8117","span":{"begin":1752,"end":1758},"obj":"CHEBI_CHMO_EXT:buffer_solution"},{"id":"T8118","span":{"begin":1766,"end":1778},"obj":"CHEBI:67100"},{"id":"T8119","span":{"begin":1797,"end":1802},"obj":"CHEBI:6636"},{"id":"T8120","span":{"begin":1839,"end":1859},"ob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and genotyping\nThe Hdhex4/5 mice carrying a pGKneo insertion/replacement inactivating mutation in the mouse HD gene homologue have been described previously [10]. The experiments were conducted in accordance with an IACUC approved protocol, through the MGH Subcommittee on Animal Research. Mutant Hdhex4/5/Hdhex4/5 and normal littermates were obtained in timed pregnancies from mating of Hdhex4/5/Hdh+ heterozygotes, genotyped by PCR assay, as described [10]. The day of plug was taken to be E0.5. Embryos that were morphologically normal were pooled separately from morphologically mutant embryos for analysis. Nodal expression was determined in embryos from matings of Hdhex4/5/Hdh+; NdllacZ/Ndl+ compound heterozygotes genotyped by PCR assay as described in [29].\n\nWhole mount and section in situ hybridization and β-gal staining\nAfter dissection in PBS, embryos were fixed overnight in 4% paraformaldehyde at 4°C. For sections, decidua fixed in 4% paraformaldehyde, were embedded in paraffin and sectioned at 7 microns. RNA in situ hybridizations were performed as described previously [55]. Nodal.lacZ expression was assessed by β-galactosidase staining as reported [29], on embryos post fixed in 4% paraformaldehyde. Embryos were mounted in 80% glycerol before being photographed.\nThe huntingtin deficient phenotype is fully penetrant at each of the stages that were assessed [10]. Three to six embryos were evaluated for each marker, with every embryo exhibiting the same mutant phenotype in each case.\n\nAlkaline phosphatase staining of Primordial Germ Cells (PCGs)\nAfter dissections, embryos were fixed in 4% paraformaldehyde briefly and washed and stored in 1 × PBS/0.1% TX-100 at 4°C. Embryos were washed once with Tris-Maleate Buffer (25 mM Tris-Maleate, pH = 9.0, 0.8 mM MgCl2) and were subsequently incubated in alkaline phosphatase staining solution (25 mM Tris-Maleate, pH = 9.0, 0.8 mM MgCl2, 0.4 mg/ml alpha-naphthyl phosphate, 1 mg/ml Fast-Red). Stained embryos were washed in 1 × PBS/0.1% TX-100.\n\nWhole embryo culture\nEmbryos were dissected at E7.5 and washed in DMEM. Embryos were then cultured individually in 1 ml of culture media (75% immediately centrifuged rat serum and 25% DMEM [56]) for 24 hours while rotating in a 37°C incubator in 5% CO2. Embryos were then fixed in 4% paraformaldehyde for analysis."}

{"project":"craft-ca-core-dev","denotations":[{"id":"T7897","span":{"begin":1237,"end":1244},"obj":"UBERON:0000922"},{"id":"T7898","span":{"begin":1265,"end":1273},"obj":"CHEBI:17754"},{"id":"T7899","span":{"begin":1305,"end":1315},"obj":"PR:000008840"},{"id":"T7900","span":{"begin":1415,"end":1422},"obj":"UBERON:0000922"},{"id":"T7901","span":{"begin":1466,"end":1472},"obj":"UBERON:0000922"},{"id":"T8095","span":{"begin":1558,"end":1579},"obj":"CL:0000670"},{"id":"T8096","span":{"begin":1581,"end":1585},"obj":"CL:0000670"},{"id":"T8097","span":{"begin":1606,"end":1613},"obj":"UBERON:0000922"},{"id":"T8098","span":{"begin":1694,"end":1700},"obj":"CHEBI:9750"},{"id":"T8099","span":{"begin":1709,"end":1716},"obj":"UBERON:0000922"},{"id":"T8100","span":{"begin":1739,"end":1751},"obj":"CHEBI:67100"},{"id":"T8101","span":{"begin":1766,"end":1778},"obj":"CHEBI:67100"},{"id":"T8102","span":{"begin":1797,"end":1802},"obj":"CHEBI:6636"},{"id":"T8103","span":{"begin":1869,"end":1877},"obj":"CHEBI:75958"},{"id":"T8104","span":{"begin":1885,"end":1897},"obj":"CHEBI:67100"},{"id":"T8105","span":{"begin":1916,"end":1921},"obj":"CHEBI:6636"},{"id":"T8106","span":{"begin":1986,"end":1993},"obj":"UBERON:0000922"},{"id":"T8107","span":{"begin":2022,"end":2028},"obj":"CHEBI:9750"},{"id":"T8251","span":{"begin":2037,"end":2043},"obj":"UBERON:0000922"},{"id":"T8252","span":{"begin":2052,"end":2059},"obj":"UBERON:0000922"},{"id":"T8253","span":{"begin":2103,"end":2110},"obj":"UBERON:0000922"},{"id":"T8254","span":{"begin":2197,"end":2200},"obj":"NCBITaxon:10114"},{"id":"T8255","span":{"begin":2201,"end":2206},"obj":"UBERON:0001977"},{"id":"T8256","span":{"begin":2280,"end":2283},"obj":"CHEBI:16526"},{"id":"T8257","span":{"begin":2285,"end":2292},"obj":"UBERON:0000922"},{"id":"T7669","span":{"begin":9,"end":13},"obj":"NCBITaxon:10088"},{"id":"T7670","span":{"begin":33,"end":36},"obj":"PR:000008840"},{"id":"T7671","span":{"begin":42,"end":46},"obj":"NCBITaxon:10088"},{"id":"T7672","span":{"begin":116,"end":121},"obj":"NCBITaxon:10088"},{"id":"T7673","span":{"begin":122,"end":124},"obj":"PR:000008840"},{"id":"T7674","span":{"begin":125,"end":129},"obj":"SO:0000704"},{"id":"T7675","span":{"begin":130,"end":139},"obj":"SO:0000853"},{"id":"T7676","span":{"begin":287,"end":293},"obj":"NCBITaxon:33208"},{"id":"T7677","span":{"begin":311,"end":314},"obj":"PR:000008840"},{"id":"T7678","span":{"begin":320,"end":323},"obj":"PR:000008840"},{"id":"T7679","span":{"begin":375,"end":386},"obj":"GO:0007565"},{"id":"T7680","span":{"begin":392,"end":398},"obj":"GO:0007618"},{"id":"T7681","span":{"begin":402,"end":405},"obj":"PR:000008840"},{"id":"T7682","span":{"begin":411,"end":414},"obj":"PR:000008840"},{"id":"T7683","span":{"begin":485,"end":489},"obj":"UBERON:0010148"},{"id":"T7684","span":{"begin":512,"end":519},"obj":"UBERON:0000922"},{"id":"T7685","span":{"begin":604,"end":611},"obj":"UBERON:0000922"},{"id":"T7686","span":{"begin":626,"end":631},"obj":"PR:000000105"},{"id":"T7687","span":{"begin":632,"end":642},"obj":"GO:0010467"},{"id":"T7688","span":{"begin":661,"end":668},"obj":"UBERON:0000922"},{"id":"T7689","span":{"begin":674,"end":681},"obj":"GO:0007618"},{"id":"T7690","span":{"begin":685,"end":688},"obj":"PR:000008840"},{"id":"T7691","span":{"begin":694,"end":697},"obj":"PR:000008840"},{"id":"T7692","span":{"begin":708,"end":711},"obj":"PR:000000105"},{"id":"T7889","span":{"begin":814,"end":827},"obj":"GO:0097617"},{"id":"T7890","span":{"begin":872,"end":879},"obj":"UBERON:0000922"},{"id":"T7891","span":{"begin":946,"end":953},"obj":"UBERON:0002450"},{"id":"T7892","span":{"begin":1050,"end":1064},"obj":"GO:0097617"},{"id":"T7893","span":{"begin":1110,"end":1115},"obj":"PR:000000105"},{"id":"T7894","span":{"begin":1116,"end":1120},"obj":"PR:000033987"},{"id":"T7895","span":{"begin":1121,"end":1131},"obj":"GO:0010467"},{"id":"T7896","span":{"begin":1194,"end":1201},"obj":"UBERON:0000922"}],"text":"Methods\n\nMice and genotyping\nThe Hdhex4/5 mice carrying a pGKneo insertion/replacement inactivating mutation in the mouse HD gene homologue have been described previously [10]. The experiments were conducted in accordance with an IACUC approved protocol, through the MGH Subcommittee on Animal Research. Mutant Hdhex4/5/Hdhex4/5 and normal littermates were obtained in timed pregnancies from mating of Hdhex4/5/Hdh+ heterozygotes, genotyped by PCR assay, as described [10]. The day of plug was taken to be E0.5. Embryos that were morphologically normal were pooled separately from morphologically mutant embryos for analysis. Nodal expression was determined in embryos from matings of Hdhex4/5/Hdh+; NdllacZ/Ndl+ compound heterozygotes genotyped by PCR assay as described in [29].\n\nWhole mount and section in situ hybridization and β-gal staining\nAfter dissection in PBS, embryos were fixed overnight in 4% paraformaldehyde at 4°C. For sections, decidua fixed in 4% paraformaldehyde, were embedded in paraffin and sectioned at 7 microns. RNA in situ hybridizations were performed as described previously [55]. Nodal.lacZ expression was assessed by β-galactosidase staining as reported [29], on embryos post fixed in 4% paraformaldehyde. Embryos were mounted in 80% glycerol before being photographed.\nThe huntingtin deficient phenotype is fully penetrant at each of the stages that were assessed [10]. Three to six embryos were evaluated for each marker, with every embryo exhibiting the same mutant phenotype in each case.\n\nAlkaline phosphatase staining of Primordial Germ Cells (PCGs)\nAfter dissections, embryos were fixed in 4% paraformaldehyde briefly and washed and stored in 1 × PBS/0.1% TX-100 at 4°C. Embryos were washed once with Tris-Maleate Buffer (25 mM Tris-Maleate, pH = 9.0, 0.8 mM MgCl2) and were subsequently incubated in alkaline phosphatase staining solution (25 mM Tris-Maleate, pH = 9.0, 0.8 mM MgCl2, 0.4 mg/ml alpha-naphthyl phosphate, 1 mg/ml Fast-Red). Stained embryos were washed in 1 × PBS/0.1% TX-100.\n\nWhole embryo culture\nEmbryos were dissected at E7.5 and washed in DMEM. Embryos were then cultured individually in 1 ml of culture media (75% immediately centrifuged rat serum and 25% DMEM [56]) for 24 hours while rotating in a 37°C incubator in 5% CO2. Embryos were then fixed in 4% paraformaldehyde for analysis."}