Role of Pitx1 upstream of Tbx4 in specification of hindlimb identity

Science

Volumn 283, Issue 5408, 1999, Pages 1736-1739

  Logan, Malcolm ^a   Tabin, Clifford J ^a  

^a HARVARD MEDICAL SCHOOL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

MYOSIN; TRANSCRIPTION FACTOR; TRANSCRIPTION FACTOR PTX1; UNCLASSIFIED DRUG;

ANIMAL EXPERIMENT; ANIMAL TISSUE; ARTICLE; BONE DEVELOPMENT; CHICKEN; CONTROLLED STUDY; ECTODERM; EMBRYO; FORELIMB; GENE EXPRESSION; GENE INDUCTION; GENETIC TRANSCRIPTION; HINDLIMB; IMMUNOHISTOCHEMISTRY; IN SITU HYBRIDIZATION; LIMB DEVELOPMENT; MESODERM; MUSCLE DEVELOPMENT; NONHUMAN; PRIORITY JOURNAL;

ANIMALS; AVIAN PROTEINS; BODY PATTERNING; CELL DEATH; CHICK EMBRYO; ECTODERM; FEATHERS; FOOT; GENE EXPRESSION REGULATION, DEVELOPMENTAL; GENETIC VECTORS; HINDLIMB; HOMEODOMAIN PROTEINS; LIMB BUD; MESODERM; MUSCLE, SKELETAL; PAIRED BOX TRANSCRIPTION FACTORS; RETROVIRIDAE; T-BOX DOMAIN PROTEINS; TRANSCRIPTION FACTORS; WING;

EID: 0033548659     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.283.5408.1736     Document Type: Article

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11. Fertilized White Leghorn chicken eggs were incubated at 37°C in a humidified incubator and the stage of development was determined according to the guidelines of Hamilton and Hamburger (19). We cloned the entire open reading frame of chick Pitx1 into the RCAS plasmid using conventional methods (26). Viral infection was carried out on embryos at stage 10. The presumptive wing and leg territories were targeted as previously described in (26), whole-mount in situ hybridizations were carried out essentially as described in [R. D. Riddle, R. L. Johnson, E. Laufer, C. J. Tabin, Cell 75, 1401 (1993)], and 20-μm-cryosection in situ hybridizations were carried out essentially as described in [Z. Z. Bao and C. L. Cepko, J. Neurosci. 17(4), 1425 (1997)]. Probes for HoxD9, HoxC9, HoxC10, and HoxC11 are described in (18) and those for Tbx4, Tbx5, and Pitx1 are described in (6). A probe template specific for the 3′ untranslated region (UTR) of Pitx1 was generated by polymerase chain reaction with a Pitx1 3′-UTR forward primer TGACACGAAC-CACCTCCG, an M13 universal primer as reverse primer, and a full-length library isolate (OT17) as template. Antisense probes were made with T7 RNA polymerase.
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27. We thank L. Paganessi for technical assistance, G. Kardon for help in analysis of the limb musculature and preparing a schematic of the muscle pattern, D. Smith for assisting in injection experiments, and J. C. Izpisua-Betmonte, M. G. Rosenfeld, J. Drouin, and T. Ogura for communicating results before publication. M.L. was funded by a postdoctoral fellowship from the Human Frontier Science Program Organisation. This work was funded by a grant from the NIH.