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Volumn 285, Issue 5426, 1999, Pages 403-406

Differences in left-right axis pathways in house and chick: Functions of FGF8 and SHH

Author keywords

[No Author keywords available]

Indexed keywords

SONIC HEDGEHOG PROTEIN;

EID: 0033575364     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5426.403     Document Type: Article
Times cited : (265)

References (37)
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    • Embryos were isolated at the 0 to 2 somite stage (∼E8.0), and a heparin bead (H5263, Sigma) soaked in phosphate-buffered saline (PBS) containing bovine serum albumin (BSA, 1 mg/ml) (control BSA-bead) or in PBS-BSA with FGF8 (1 mg/ml) (b isoform; R&D Systems, Minneapolis, MN) (FGF8-bead) was inserted into the right LPM lateral to the node. After 1 hour in stationary culture, the embryos were incubated for 6 to 8 hours in a rotary culture apparatus, as described by K. Sturm and P. Tam [Methods Enzymol. 225, 164 (1993)]. During the culture period the embryos developed to the 4 to 8 somite stage. After culture, the embryos were washed in PBS, fixed, and assayed for β-Gal activity as described (77). Ectopic nodal expression was detected in tissue near the bead in 11 out of 18 embryos with FGF8-beads, and in 1 out of 7 embryos with control beads. The Nodal expression domain appeared normal in two embryos with FGF8-beads implanted in the left LPM. No ectopic Nodal expression was detected in 22 embryos isolated at the 4 to 8 somite stage, after implantation of an FGF8-bead in the right LPM and culture as described above.
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    • Nodal expression was detected by X-Gal staining for β-Gal activity (77). Lefty and Pitx2 expression were detected by RNA in situ hybridzation (76). The probe that was used to detect Lefty expression (75) hybridizes to Lefty2 RNA, which is detected in the lateral plate mesoderm, and to Leftyl RNA, which is detected in the prospective floor plate (ventral midline). Because Lefty1 RNA was not consistently detected in normal embryos, its absence in the mutant embryos is not considered significant.
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    • note
    • -/+ mice, respectively. We thank M. Kuehn, H. Hamada, and M. Blum for Nodal, Lefty, and Pitx2 probes, respectively, and J. Bristow and D. Goldman for helpful advice. We thank M. Embry and A. Gannon for technical assistance. We are also grateful to S. Strickland and our laboratory colleagues for critical readings of the manuscript. E.N.M. was supported by a Mentored Clinical Scientist Development award HD01216 from the NIH. This work was supported by NIH grant RO1 HD34380 (G.R.M.).


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