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Dev Dyn
2002 Jun 01;2242:168-85. doi: 10.1002/dvdy.10097.
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Characterizing gene expression during lens formation in Xenopus laevis: evaluating the model for embryonic lens induction.
Henry JJ
,
Carinato ME
,
Schaefer JJ
,
Wolfe AD
,
Walter BE
,
Perry KJ
,
Elbl TN
.
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Few directed searches have been undertaken to identify the genes involved in vertebrate lens formation. In the frog Xenopus, the larval cornea can undergo a process of transdifferentiation to form a new lens once the original lens is removed. Based on preliminary evidence, we have shown that this process shares many elements of a common molecular/genetic pathway to that involved in embryonic lens development. A subtracted cDNA library, enriched for genes expressed during cornea-lens transdifferentiation, was prepared. The similarities/identities of specific clones isolated from the subtracted cDNA library define an expression profile of cells undergoing cornea-lens transdifferentiation ("lens regeneration") and corneal wound healing (the latter representing a consequence of the surgery required to trigger transdifferentiation). Screens were undertaken to search for genes expressed during both transdifferentiation and embryonic lens development. Significantly, new genes were recovered that are also expressed during embryonic lens development. The expression of these genes, as well as others known to be expressed during embryonic development in Xenopus, can be correlated with different periods of embryonic lens induction and development, in an attempt to define these events in a molecular context. This information is considered in light of our current working model of embryonic lens induction, in which specific tissue properties and phases of induction have been previously defined in an experimental context. Expression data reveal the existence of further levels of complexity in this process and suggests that individual phases of lens induction and specific tissue properties are not strictly characterized or defined by expression of individual genes.
Figure 3. Expression patterns of genes isolated from the subtracted cDNA library. RNA whole-mount in situ hybridization revealing the localization of six different mRNAs in Xenopus embryos. All whole-mount early larval stages are shown as rightlateral views, with the anterior ends located to the right of the figure. All whole-mount embryos are shown as left dorsolateral views, with the anterior end directed toward the left side of the figure. A: Expression of B66 (Xmmp-9) at stage 35. Notice that expression is detected in several isolated mesenchymal cells, which lie just beneath the ectoderm. Most of these cells are located along the trunk and in the tail, and very few are seen in the head. B: Expression of B87 (Xenopus CRSP34) at stage 30. Note intense expression in the eye and lens (ey), as well as the otic vesicle (ot), pharyngeal arches (pa), spinal cord (sc), pronephros (pn), and axial musculature. The black arrowhead points to expression in the pineal gland. C: Expression of B99 (identity unknown) at stage 27. Note intense expression in the eye and lens, as well as one of the pharyngeal arches. Some weaker expression may also be seen in the otic vesicle. D: Expression of B105 (identity unknown) at stage 14. Expression is seen to the placodal ectoderm surrounding the anterior neural plate (np). The most intense expression is localized in the presumptive lensectoderm (white arrowhead), whereas some expression is also detected in the area of the presumptive otic region (black arrowhead). Much weaker expression extends anteriorly into the region of the presumptive olfactory ectoderm. Thin lines of expression are also seen in the anterior neural folds (nf). E: Transverse section through the anterior neural plate showing expression in the presumptive lensectoderm (ple) and cells in the neural folds. Relative plane of section is indicated by dotted white line shown in D. F: Expression of B105 (identity unknown) at stage 19. G: Expression of B105 at an even later stage of development (stage 34). Note intense expression in the eye and lens, as well as the otic vesicle. Asterisks mark the locations of segmental expression in the brain. Black arrowhead points to expression in the pineal gland. H: Cross-section through the eye and lens of a stage 30 larva showing expression in the lens (ln). rt, retina. I: Representative negative control showing no hybridization of sense D43 probe (D43s) to a stage 32 embryo. J: Expression of D43 at stage 15. Expression is seen in regions similar to those expressing B105. K: Expression of D43 at stage 32. Expression is detected in the head in structures including the eye, lens, and otic vesicle. Other expression is also seen in the spinal cord, the pronephros and pronephric duct, and in cells of the cloaca (cl). L: Expression of E7. Specimens shown in B and C have been cleared, whereas the others have not. Scale bars = 25 mu m in E, 30 mu m in H, 250 mu m in J (applies to D,F,J), 500 mu m in L (applies to A-C,G,I,K,L).
Fig. 1. Diagrams illustrating the model of embryonic lens induction and development in Xenopus laevis. The various graphs represent dif- ferent tissue properties revealed by tissue transplantation and explant culture experiments (Henry and Grainger, 1987, 1989; Servetnick and Grainger, 1991). The X axes indicate developmental stages (according to Nieuwkoop and Faber, 1956). The Y axis represents the relative intensity of that property. A: Embryonic lens formation appears to be directed by two principal phases of induction, which include essential âearly,â as well as âlateâ tissue interactions. Some slight overlap of these phases of induction occurs around stage 19 when the neural folds undergo fusion, which eventually separates the neural plate from the adjacent placodal ectoderm (terminating planar signals), and the optic vesicles begin to protrude from the developing forebrain and contact the presumptive lens ectoderm. B: Embryonic ectoderm gains an autonomous window of âcompetenceâ (A.C. in Fig. 4) to respond to lens inductive interactions, which is tied to its ability to respond to these interactions in the appro-
priate context. This property reaches a maximum at approximately stage 11.5, as indicated. Presumably competence to respond to these signals is sustained while inductive interactions continue to take place during later stages. C: When competent tissues receive these inductive signals, they gain an increasing lens-forming âbiasâ or propensity to form lens cells. After an initial period of âspecification,â sufficient inductive signals reach the responding tissue such that when it can then be cultured in isolation to produce a lens, the tissue is then considered to be âspecifiedâ (stage 19). Ultimately, the tissue becomes âcommittedâ to the lens cell fate, when it can no longer respond to other signals (definitions according to Slack, 1991; Grainger, 1992, 1996). Presumably this occurs by stage 26, when the lens undergoes cellular differentiation, after a phase of âcommitment.â D: Hypothetical (temporal) patterns of gene expression that might be correlated with and establish each of the different properties described above. See text for further details.
Fig. 2. Polymerase chain reaction (PCR) -based screens to determine whether specific clones recovered from the subtracted cDNA library are present in the transdifferentiating and control cornea cDNA libraries used to construct the subtracted library. The presence of Pax-6 was also examined. One nanogram of each clone’s plasmid DNA was used in the
positive (ô°ƒ) control reactions, and 10 ng each of the transdifferentiating (T) and control (C) cornea library cDNA were included in the other PCR reactions shown here. The blank, negative control reactions are not shown to conserve space. See text for further details.
Fig. 4. Summary of temporal patterns of gene expression within embryonic lensectoderm in Xenopus laevis. Windows of expression are correlated with specific stages of development and to different phases of lens induction, tissue competence, and lens forming-bias as defined in the text and Figure 1. A: Different embryonic stages are represented on a time line of early development. B: Temporal windows of lens forming bias, competence, and induction are mapped onto the time line of early development. C: Temporal windows of gene expression within the de- veloping lensectoderm are mapped onto the time line of early develop- ment. Data are based on the results of this study, as well as that of previous studies (Pannese et al., 1995; Kablar et al., 1996; Hirsch and Harris, 1997; Penzel et al., 1997; Zygar et al., 1998; Schaefer et al., 1999; Kenyon et al., 1999; Hollemann and Pieler, 1999; Zhou et al., 2000; Ishibashi and Yasuda, 2001; Pommereit et al., 2001).
