Nt at the time of operation and no further myotome has

Nt at the time of operation and no further myotome has developed (n = 16, Figure 1).Cells of the intermediate bmjopen-2016-011824 part of the dermomyotome can not restore the ablated VLLbeen invaded into the somatopleura at HH-19/20 and is covered by a very thick PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/14960617 tissue layer, as described above, it is very difficult to identify the VLL from the lateral mesoderm tissue without the aid of GFP-labelling. So the VLL ablation was carried out under fluorescence light. To minimize the cell toxicity and bleaching, use of UV light was restricted to short periods to localize GFPlabeled cells and to control whether all GFP- labeled cells were removed. The GFP-labeled cells of the VLL were aspirated with a mouth-controlled glass capillary. The surgical ablation was performed only in one somite, so that PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/744568 the neighbouring somite could serve as control for the operated one. One day 2,2,3,3-Tetrafluoropropyl N,N'-diethylcarbamimidothioate trifluoromethanesulfonate after the ablation, we saw the GFP domain of the operated dermomyotome extended less ventrolaterally than that of unoperated dermomyotomes in all embryos (n = 14/14, Figure 2E). These observations indicate that the dermomyotome part located medially to the VLL cannot replace the function of the VLL, with regards to the ventrolateral growth of the dermomyotome. Embryos from the above manipulation were subsequently analysed for Pax3 expression. The Pax3-expression of the VLL of one embryo (n = 5/5) was missing with the exception of the cranial and caudal edge (Figure 2F). The Pax3-expression in the cranial and caudal part of the VLL could be explained due to the incomplete removal of the VLL. Accordingly, the most lateral part of the myotome viewed by the myosin heavy chain antibody (Mf20) was partly disturbed (data not shown).Removal of the intermediate part of the dermomyotome epithelium does not significantly affect the myotome formationIt was not known whether cells of the intermediate dermomyotome can support hypaxial myotome extension in the absence of the VLL. To address this question, we labelled the intermediate part of the dermomyotome with GFP, followed by VLL ablation. A GFP-expressing vector was electroporated into the lateral parts of interlimb epithelial somites at HH-16-17 (Figure 2A). Following a reincubation period of 6 hours, a narrow region of GFP could be observed in the lateral somite at the interlimb level. 24 h later the operated embryo had reached HH-19/20 the GFP-positive domain had broadened (Figure 2C). A craniocaudally cut was made in the middle of the GFP-marked dermomyotome part of a segment. Only GFP-positive cells located laterally to the cut were removed. In this way, the VLL was ablated and remaining GFP-positive part represented the ventrolateral edge of the intermediate part. Since the VLL hasNext we ask whether the intermediate part of the dermomyotome epithelium is essential for the growth of the hypaxial myotome. First we investigated the ventrolateral growth of VLL after ablations of the intermediate dermomyotome at the interlimb level. The VLL primordium of interlimb somites was labelled through GFP N-BOC-3-Fluoro-D-phenylalanine by electroporation at HH-16/17 as described above. Then, the intermediate part of a dermomyotome was removed. After one to two days of reincubation, the GFP-marked VLL of the operated somites extended as far as those of unoperated somites in all analyzed embryos (n = 8/8, Additional file 4: Figure S2). The VLL was completely normal. The Pax3-expression was only partly down-regulated in the intermediate part of the operated dermomyotome. The myotome in the ope.