Jin H et al. (2012), Defining progressive stages in the commitment p...

XB-ART-45250

Genesis 2012 Oct 01;5010:728-40. doi: 10.1002/dvg.22038.

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Defining progressive stages in the commitment process leading to embryonic lens formation.

Jin H , Fisher M , Grainger RM .

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The commitment of regions of the embryo to form particular tissues or organs is a central concept in development, but the mechanisms controlling this process remain elusive. The well-studied model of lens induction is ideal for dissecting key phases of the commitment process. We find in Xenopus tropicalis, at the time of specification of the lens, i.e., when presumptive lens ectoderm (PLE) can be isolated, cultured, and will differentiate into a lens that the PLE is not yet irreversibly committed, or determined, to form a lens. When transplanted into the posterior of a host embryo lens development is prevented at this stage, while ~ 3 h later, using the same assay, determination is complete. Interestingly, we find that specified lens ectoderm, when cultured, acquires the ability to become determined without further tissue interactions. Furthermore, we show that specified PLE has a different gene expression pattern than determined PLE, and that determined PLE can maintain expression of essential regulatory genes (e.g., foxe3, mafB) in an ectopic environment, while specified PLE cannot. These observations set the stage for a detailed mechanistic study of the genes and signals controlling tissue commitment.

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Species referenced: Xenopus tropicalis
Genes referenced: foxe3 maf mafb nrl pax6 sox2 sox3

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	Figure 1. Lens-forming ability of isolated PLE. Explants were taken from staged embryos (Nieuwkoop and Faber,1994) and scored when donor embryos reached Stage 41. Diagrammatic views of the right sides of embryos are shown with dashed boxes indicating the position of the PLE at each stage. Positive cases represent those which showed visible GFP driven by a γ-crystallin promoter. Arrows denote the GFP expression in typical PLE explants shown in the right hand column. Reproduced with permission of the Publisher, John Wiley & Sons.
	Figure 2. Assessment of developmental potential of specified PLE in transplant experiments. Panel a shows a diagram of 12 regions in the side of a Stage 18 embryo into which transplants were placed. b show examples of the different degrees of lens-forming ability in different sites indicated by the level of GFP fluorescence in responding ectoderm. PLEs from Stage 18 embryos were transplanted into different locations of embryos at the same stage. Host embryos were scored at Stage 41. A strong GFP signal (+++) was similar but still less than the level of endogenous gene expression and could be detected in the transplanted PLE when placed into region B1 as shown in panel b. A moderate response (++), clearly discernible, but much weaker than those responses in the anterior of the embryo, is illustrated in c'. Only a weak GFP signal (+) was seen in the transplanted PLE when placed into region C4 as shown in panel d'. Here, the response, though clearly positive, was just discernible. The images shown in this figure, and in other experiments, were photographed with the same exposure, and when transplanted into GFP-labeled hosts, as shown in this figure, more readily compared with host expression to determine relative levels. A summary of responses in all 12 regions is shown in e. In panels b', c', and d', arrows indicate the GFP fluorescence in the transplanted PLE and arrowheads indicate the endogenous GFP in hindbrain and lens of the transgenic embryos. Reproduced with permission of the Publisher, John Wiley & Sons.
	Figure 3. Lens-forming ability of PLE from different stage donors placed in the posterior of Stage 18 embryos. Transplants were made from donor embryos of the γ-crystallin-GFP transgenic line into wild-type hosts. Transplants were scored when hosts reached Stage 41. Diagrammatic views of the right sides of donor and host embryos are shown with boxes indicating the transplanted PLEs. The numbers of positive cases represent those which showed visible GFP driven by the γ-crystallin promoter. Reproduced with permission of the Publisher, John Wiley & Sons.
	Figure 4. Assessment of the developmental potential of Stage 21 PLE in transplant experiments. Stage 21 PLE yielded a positive lens response indicated by fluorescence expression of γ-crystallin-GFP when PLEs were transplanted into any of the 12 regions of Stage 18 embryos. The regional designations of the embryo are the same as those shown in Figure 2. Panels a show results from PLE transplants into different regions. A strong GFP signal (+++) was detected in the transplanted PLE in all sites. A summary of the data from all cases examined in each of the 12 regions is shown in d. In panels a', b', and c', arrows indicate the GFP fluorescence in the transplanted PLE and arrowheads indicate the endogenous GFP in host transgenic embryos.Reproduced with permission of the Publisher, John Wiley & Sons.
	Figure 5. The transition from lens specification to lens determination is tissue autonomous. Panel a shows the procedure used in this experiment, i.e., PLEs from Stage 18 γ-crystallin-GFP3 embryos were removed and cultured alone until the donor embryos reached Stage 21. Then, aged PLE was transplanted into region B4 (b), a posterior region of a Stage 18 host embryo or into region C3 (c), a ventral region. Host embryos were scored at Stage 41. Strong GFP expression could be observed in the transplanted PLE in both regions. In panels b' and c', arrows indicate GFP in the transplanted PLE and arrowheads indicate the endogenous GFP in transgenic host embryos. Reproduced with permission of the Publisher, John Wiley & Sons.
	Figure 6. A comparison of expression levels of lens genes in cultured Stages 14, 18, and 21 PLE. The fold number of gene expression in each group of explants was obtained by comparison of the gene expression level in explants to that in Stage 36 head tissue. All expression levels were normalized to 28S rRNA levels in a given tissue. Expression levels of pax6 (a), foxe3 (b), mafb (c), l-maf (d), sox2 (e), sox3 (f), and γ-crystallin (g) in each group of samples is illustrated. The gene expression level of noncultured Stage 14, Stage 18, and Stage 21 PLE represents the endogenous gene expression in PLE at each corresponding stage. 4 to 36denotes PLE explants excised at Stage 14 and cultured to Stage 36. 8 to 36denotes PLE explants excised at Stage 18 and cultured to Stage 36. 1 to 36denotes PLE explants excised at Stage 21 and cultured to Stage 36. Error bars are standard deviations generated from three independent experiments. Reproduced with permission of the Publisher, John Wiley & Sons.

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Auda-Boucher, Staging of the commitment of murine cardiac cell progenitors. 2000, Pubmed