Day RC and Beck CW (2011), Transdifferentiation from cornea to lens in Xen...

XB-ART-43809

BMC Dev Biol 2011 Jan 26;11:54. doi: 10.1186/1471-213X-11-54.

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Transdifferentiation from cornea to lens in Xenopus laevis depends on BMP signalling and involves upregulation of Wnt signalling.

Day RC , Beck CW .

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Surgical removal of the lens from larval Xenopus laevis results in a rapid transdifferention of central corneal cells to form a new lens. The trigger for this process is understood to be an induction event arising from the unprecedented exposure of the cornea to the vitreous humour that occurs following lens removal. The molecular identity of this trigger is unknown. Here, we have used a functional transgenic approach to show that BMP signalling is required for lens regeneration and a microarray approach to identify genes that are upregulated specifically during this process. Analysis of the array data strongly implicates Wnt signalling and the Pitx family of transcription factors in the process of cornea to lens transdifferentiation. Our analysis also captured several genes associated with congenital cataract in humans. Pluripotency genes, in contrast, were not upregulated, supporting the idea that corneal cells transdifferentiate without returning to a stem cell state. Several genes from the array were expressed in the forming lens during embryogenesis. One of these, Nipsnap1, is a known direct target of BMP signalling. Our results strongly implicate the developmental Wnt and BMP signalling pathways in the process of cornea to lens transdifferentiation (CLT) in Xenopus, and suggest direct transdifferentiation between these two anterior eye tissues.

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Species referenced: Xenopus laevis
Genes referenced: amph cbx1 ccdc124 celf1 creld1 cryaa cryba1 crybb1 crybb3 crygbl.1 dvl2 fgf8 fut6 fzd7 gdf3 glb1 gnai2 h2ac21 hsp70 hsp90ab1 kif19 lin28a lmo4.1 lmo4.2 lrp6 luc7l3 mmp11 mmp14 mta2 mybph nipsnap1 nog pax6 pdik1l pdlim1 pitx1 pitx2 pou5f3 pou5f3.2 pou5f3.3 prox1 six1 sox2 taf1b tcf4 tcf7 tcf7l2 tert tsr1 tsr2 unk wnt2 wnt6 wnt7a zic3

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	Figure 1. BMP signalling is required for lens regeneration. A) Lentectomy of N1 transgenic tadpoles at stage 50. These tadpoles carry the Hsp70:Noggin; γ-Crystallin:GFP transgene and express GFP in the central lens cells, but not the outermost lens epithelial cells. Removal of the lens can be visualised by the lack of GFP in the lentectomised eye. B) Heat shock initiates expression of the BMP inhibitor noggin, attenuating BMP signalling. In around 60-70% of non heat shocked, lentectomised eyes, regeneration of the lens from the overlying cornea occurs and GFP can be seen after 3-5 days and a new lens is formed after 14 days. When the tadpoles are subjected to heat shock, no GFP is expressed and no lens regenerated. Bright field views are to the left with corresponding fluorescent views adjacent. C) Graph showing the effect of heat shock activation of Noggin on lens regeneration as determined by the presence of any detectable GFP in the eye 10 days after lentectomy. ** indicates p < 0.001. (Chi squared analysis).
	Figure 2. Histology of WT and BMP inhibited (N1) tadpole eyes following lentectomy. (A-D) Representative histological sections through the eyes of transgenic N1 tadpoles 1, 3, 5 and 7 days after lentectomy. Inset in A shows arrows pointing to columnar corneal cells (E-H) Representative sections of wild type eyes at 1, 3, 5 and 7 days after lentectomy. Inset in E indicates columnar corneal cells (arrows). Arrowheads in F indicate the forming lentoid moving into the vitreous. Both transgenic and wild type eyes were subjected to heat shock at -3, 24 and 48 hours relative to lentectomy. Abbreviations: c, cornea; h, hypertrophic cells; i, iris (pigmented epithelium); l, lens; r, retina; v, vitreous. Scale bars in D and H corresponds to 50 μm and applies to all panels. Inset scale bar in A and E corresponds to 10 μm.
	Figure 3. Identification of genes that mimic expression of crystallins during CLT. A) Strategy for identifying CLT specific genes by microarray. Lenses were removed from stage 50 tadpoles and used for RNA extraction of samples L1-3 (differentiated lens). The tadpoles that had undergone lentectomy were allowed to recover for three days before harvesting the cornea for samples R1-3 (regenerating). Finally, a second set of tadpoles were subjected to mock surgery, the cornea was cut, lifted and replaced as for lentectomy but the inner cornea and lens were left in situ. After 3 days corneas were recovered from these animals, generating samples S1, 3 and 4 (sham operated). B) Heatmap of Affymetrix array data showing that the pattern of crystallin expression varies among replicate R samples, with R2 consistently lower. An average expression was used to search for similar patterns in the data. C) Heatmap of top 50 significant matches to the crystallin pattern, excluding the crystallin genes used to generate the pattern average. R/S is the ratio of transcript expression in R1-3 vs. S1, 3 and 4 levels. Bold type in C indicates R > 1.5 S.
	Figure 4. Expression of lens specific genes during development. In situ hybridisation of three Crystallin genes and Cugbp1b during development of the lens, indicated by dark blue staining. A-D, Cryaa expression at st. 26 (A), 30 (B) and 38 (C and D). E-H, Expression of Cryba1 throughout the lens at st. 26 (E), 30 (F) and 38 (G and H). I-L, expression of Crygb. I) at st. 26, note that red arrowhead indicates a transient stripe of expression in the hindbrain. Crygb is expressed in the central lens at st. 30 (J) and 38 (K and L). M-P) Cugbp1b transcripts are absent from the forming eye at st 26 (M), detected in the central lens cells at st.30 (N), and in the outer lens cells at st. 38 (O and P). Q) Tsr2 is expressed in epithelial cells including the cells overlying the eye, shown at stage 30. R) Prox1 expression is lens specific at stage 30, as previously described [23]. Sox2 is expressed throughout the eye as well as in the olfactory placode lateral line primordia and branchial arches as previously described [24]. White arrows indicate the position of the eye, scale bars in D, H, L, P, Q, R and S are 200 μm and red dots show the approximate margin of the eye. Scale bars in A, E, I and M apply to all other panels and correspond to 1 mm. Embryos are oriented with anterior to the left and dorsal uppermost.
	Figure 5. Heat map of top 50 genes, where expression is higher in samples undergoing CLT (R) than in stage matched sham operated corneas (S) or lenses (L). Darker colour indicates higher expression. R/S indicates the ratio of transcript expression in R1-3 vs. S1, 3 and 4 levels. Where possible, the genes have been manually annotated with reference to the probe source files. "Transcribed" is used to denote genes where no annotation was possible. "(sim)" indicates high similarity to the named gene.
	Figure 6. Expression of selected transdifferentiation genes during development. In situ hybridisation of five regeneration associated genes (RAG). Dark blue staining marks expression, and a white arrow indicates the position of the lens. A) Nipsnap1 expression appears in lens cells at stage 30 (A) and is also found in the pronephros (pn) and branchial arches (ba) from stage 32 (B). At stage 36, Nipsnap1 expression is seen in the pronephros and pronephritic duct (pnd) and otic vesicle (ov) as well as in the periphery of the lens (C). D) Tbp1b is expressed predominantly in lens, otic vesicle and branchial arches at stage 32. E) Tcf7 is expressed in lens and choroid fissure of the retina (cf), the midbrain-hindbrain junction (m/h), cement gland (cg), otic vesicle and pronephritic duct at at stage 32. F) Pdik1l is expressed in the developing lens and retina (re) and branchial arches at stage 32. G) Dvl2 is expressed in eye tissues but is not specific, with expression extending throughout the embryo including the somites (s), otic vesicle and branchial arches. Embryos are oriented with anterior to the left and dorsal uppermost.
	Figure 7. Heat Map showing that expression of pluripotency associated genes does not change during CLT. Darker colour indicates higher levels of expression. R/S indicates the ratio of transcript expression in R1-3 vs. S1, 3 and 4 levels. Where possible, the genes have been manually annotated with reference to the probe source files.




	cryba1 (crystallin beta A1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.

	prox1 (prospero homeobox 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28/29, lateral view, anterior left, dorsal up.
	celf1 (CUGBP Elav-like family member 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.



	nipsnap1 ((nipsnap homolog 1)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.

	taf1b (TATA-box binding protein associated factor, RNA polymerase I, B) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
	tcf7 (transcription factor 7 (T-cell specific, HMG-box) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
	dvl2 (dishevelled segment polarity protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
	pdik1l (PDLIM1 interacting kinase 1 like) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.

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