Appendix G: In Situ Hybridization: An Improved Whole-Mount Method for Xenopus Embryos

The expression of Brachyury (Bra) during development of Colostethus machalilla was analyzed with a polyclonal antibody. The observed molecular mass of Bra was of 48 kDa, as in Xenopus laevis. During cleavage, low levels of Bra were expressed. In contrast, in the blastula Bra became up-regulated, and Bra protein was present in a wide ring of surface cells. The surface expression of Bra disappeared in the gastrula, and a new ring of Bra-positive nuclei was detected in deep cells around the closing blastopore. The C. machalilla external and internal rings of Bra-positive nuclei apparently mark the prospective mesoderm in the blastula and gastrula, respectively. The two Bra expression rings were dissociated in time in the fairly slow developing embryos of this frog. Brachyury expression in the notochord became visible only after the blastopore closed, in contrast with X. laevis. In addition, Bra expression in the notochord indicated that dorsal convergence and extension occurred after blastopore closure. The C. machalilla Bra-positive notochord was originally exposed on the gastrocoel roof, in agreement with a superficial component of the prospective mesoderm.

Section: Methodsmentioning

confidence: 99%

Expression of Brachyury during development of the dendrobatid frog Colostethus machalilla

Benítez

Pino

2002

“…Whole-mount in situ hybridization was performed according to standard protocol using antisense digoxigeninlabeled probes and BM purple revelation (Roche; Harland, 1991). For serial sections, embryos were post-fixed in MEMFA for 1 hr at room temperature, and embedded in Paraplast.…”

Section: Whole-mount In Situ Hybridizationmentioning

confidence: 99%

Induction and modulation of smooth muscle differentiation in Xenopus embryonic cells

Barillot

Tréguer

Faucheux

et al. 2008

By comparison with skeletal or cardiac developmental programs, little is known regarding the specific factors that promote specification and differentiation of smooth muscle cells from pluripotent cells. We have analyzed the developmental expression of a subset of smooth muscle genes during Xenopus early development and showed that similar to mammals and avians, Xenopus smooth muscle myosin heavy chain (SM-MHC) is a highly specific marker of smooth muscle differentiation. Embryonic cells from animal pole explants of Xenopus blastula can be induced by basic fibroblast growth factor, Wnt, and bone morphogenetic protein signals to adopt the smooth muscle pathway. Explants from early embryos that contain neural crest cells can also differentiate into cells expressing smooth muscle genes. We examined the interplay of several transcription factors, that is SRF, myocardin, and GATA6, that induce the expression of SM-MHC in animal cap cells and found that myocardin-dependent expression of smooth muscle genes in animal cap cells is synergized by SRF but is strongly antagonized by GATA6. Developmental Dynamics 237: 3373-3386, 2008.

“…Dexamethasone was changed daily when embryos were cultured longer than 24 hr. At appropriate stages, embryos were fixed in MEMFA and processed for in situ hybridization with digoxigenin-coupled cRNA probes (as described in Harland, 1991). …”

Section: Embryos and Microinjectionmentioning

confidence: 99%

Isolation and characterization of Xenopus Hey‐1: A downstream mediator of Notch signaling

Rones

Woda

Mercola

et al. 2002

Regulation of Notch signaling likely occurs, at least in part, at the level of basic helix-loop-helix (bHLH) transcription factors that function downstream of Suppressor of Hairless (Su(H)) in the Notch pathway. To begin to characterize modulation of Notch signaling during organogenesis, we examined the bHLH transcription factor, XHey-1 (hairy related-1) in early Xenopus laevis embryos. XHey-1 is expressed in numerous tissues during early development including the somites, head, embryonic kidneys, and heart. Importantly, the expression of XHey-1 was significantly altered in response to perturbation of Notch signaling by means of inducible constructs that served to either activate or suppress Notch signaling through Su(H) in a temporally controlled manner.