Why? What is the specific question or hypothesis being tested for this figure? H
ID: 258689 • Letter: W
Question
Why? What is the specific question or hypothesis being tested for this figure?
How? Methods: Describe how the experiment was performed.
What? Results. Summarize the experimental results, highlighting the relevant controls and main conclusions that can be made only from the data in this figure.
So what? Conclusions. Summarize the conclusions from this figure (and only this figure!)
Fig. 1. cdh6 is expressed dynamically in the hindbrain and NCCs. (A) Embryonic brain divisions. Gray box shows region displayed in B-G. (B-G) Dorsal views (anterior left) of cdh6 and FB MB HB 14 hpf C 8 hof SOx10 in situ hybridizations at 14 hpf (B,E). 16 hpf (C,F) and 18 hpf (D,G). Red box marks approximate area shown in H. (H) Confocal images (dorsal views, anterior left, individual z-planes) of fluorescent in situ hybridizations for cdh6 (i, ii) cdh6 and sox10 (ii, ii) at 14 hpf. Yellow dashed lines mark basal cdh6 14 hpf F cdh6 neuroepithelial surfaces and white dashed lines mark apical midlines. Cells between the yellow dashed lines are neuroepithelial cells or premigratory NCCs. Cells outside the yellow dashed lines are mesenchymal cells or delaminated sox10 14 hpf sox10 4 hpt OXO NCCs. Closed yellow arrowheads mark premigratory NCCs sox 14 hpf expressing cdh6 and sox10. Open yellow arrowheads mark neuroepithelial cells expressing only cdh6. Open white arrowheads mark NCCs expressing only sox10. Closed white arrowheads mark NCCs outside the neuroepithelium expressing only sox10. FB, forebrain; MB, midbrain; HB, hindbrain. Scale bars: 10 um. cdh6 sox10 sox1Explanation / Answer
a. Neural crest cells (NCC) undergo epithelial-mesenchymal transition (EMT) during normal development. Cadherin expression is altered during this development. Cadherin 6 levels are increased during EMT in NCC development in chick dorsal neural tube and decrease after EMT. In zebrafish (Danio rerio), cdh6 is expressed in neural keel when NCCs develop. However, the expression of cadherin 6 (Cdh6) relative to neural crest markers has not been investigated in different stages of EMT in zebrafish. The experiments investigated the expression pattern of Cdh6 and NCC marker SOX10 during different stages of EMT in different regions of the Zebrafish brain.
b. Embryos were obtained from zebrafish wild-type lines (AB or Tuebigen), and Tg(-4.9sox10:EGFP or ncadtm101 mutant that are transgenic for GFP linked to NCC. Embryos were grown at 23-28°C and staged for development in hours post fertilization or hpf.
Two techniques are used in this experiment: In situ hybridization and fluorescent in situ hybridization for mRNA expression of Cdh6 and Sox10.
In situ hybridization: In situ probe is prepared by PCR amplification of the Cdh6 gene/Sox10 and insertion into pCS2 vector. In vitro transcription for Digoxygenin-UTP- or fluorescein-labeled riboprobes for cdh6 or sox10 was performed. This was followed by limited alkaline hydrolysis to obtain a probe with a length of ?300 bases. Whole-mount in situ hybridization (entire embryo) was performed with the two probes separately. Colorimetric digoxygenin-UTP probed samples were detected by NBT-BCIP detection. A Nikon TE3000 microscope with a 20× objective using MetaMorph software detected the colorimetric signal. The mRNA transcripts in fluorescent in situ hybridization were detected by tyramide signal amplification. The images were photographed by Nikon FV1000 laser scanning confocal microscope (oil immersion).
c. Results:
A: This part is a cartoon of the three regions of the brain in zebrafish embryos.
B, C and D indicate Cdh6 expression at 14, 16, and 18 hpf. E, F, G indicates Sox10 expression at 14, 16, and 18 hpf. B indicates that cdh6 is broadly expressed only in hindbrain but absent in midbrain at 14 hpf. At this stage many NCCs begin EMT. Sox10 shows a similar expression but it is showing higher intensity (E). At 16 hrs, most NCCs actively undergo EMT. At this stage, cdh6 expression was slightly stronger had a more restricted pattern (C). Cdh6 had highest expression in the boundaries between the midbrain-hindbrain and near rhombomere. However, sox10 was markedly decreased throughout the neuroepithelium (F) in these boundaries. Controls are embryos that are not treated with the probe, which are not shown in the figure. The Sox10 expression (E-G) is also a type of positive control to indicate the presence of NCCs in regions of the brain.
Most NCCs complete EMT by 18 hrs. At this stage, cdh6 expression is very strong and broad throughout the hindbrain (D. However, limited SOX10 expression was seen d in the neuroepithelium (G). Thus, SOX10 expression overlapped with cdh6 expression only at 14 hpf (early EMT) in hindbrain.
Co-expression of SOX10 and cdh6 was analysed by dual color fluorescent in situ hybridization at 14 hpf. There are three types of cells (H): those that co-expressed Sox10 and Cdh6 (yellow closed arrowheads; Hiii), only expressed Cdh6 (open yellow arrowheads; Hi) and those that only expressed Sox10 (white open arrowheads; Hii). Outside the neuroepithelium, cells express only sox10 (Hi-iii, closed white arrowheads).
Conclusion: The colorimetric studies indicate that the timing and level of Cdh6 expression is associated with different functions of NCCs and neuroepithelial cells. Low levels of Cdh6 is seen in NCCs before and during EMT. Cells in neuroepithelium not undergoing EMT show high Cdh6 expression after delamination of NCCs (post EMT). This indicates a tight spatiotemporal regulation of cdh6 expression during EMT in hindbrain NCC. Thus, moderate levels of cdh6 are associated with NCC EMT.
Broad Conclusion: Cdh6 expression is absent in midbrain but is present in hindbrain. Hence, it has different functions in both areas of brain. It is only expressed in hindbrain during early EMT of NCCs after which it is lost. Thus, Cdh6 has is required only for early EMT. Neuroepithelium NCC cells that do not undergo EMT exhibit expression of cdh6. Hence, cdh6 protein cannot on its own induce EMT. It requires other factors to induce EMT in NCCs. This low level expression of Cdh6 may be maintained by transcription factor snail2, which is known to be expressed in zebrafish neuroepithelium.
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