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Keywords:

  • homeobox genes;
  • proliferation;
  • retinal development

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. NOTE ADDED IN PROOF
  8. REFERENCES

A major event affecting the eye during amphibian metamorphosis is an asymmetrical growth of the ventrotemporal portion of the retina compared with its dorsonasal counterpart. This event is due to an increased proliferation of the precursors of the ventral ciliary marginal zone (CMZ). Here, we analyze the expression patterns of several key homeobox genes implicated in eye development (Xrx1, Xvax2, Xsix3, Xpax6, Xchx10, Xotx2) to understand whether they are active at the time in which the metamorphic changes of the retina occur. We also analyze their expression patterns in the ventral and dorsal CMZ and compare them with bromodeoxyuridine incorporation in the CMZ. Our results suggest that the metamorphic CMZ maintains the functional subdivisions described during embryonic development. Moreover, we find that genes involved in proliferation and cell type determination of the embryonic retina are actively transcribed in the proliferating CMZ, thus indicating a potential regulatory role for these genes in the metamorphic retina. Developmental Dynamics 233:645–651, 2005. © 2005 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. NOTE ADDED IN PROOF
  8. REFERENCES

Amphibian metamorphosis consists of several concomitant processes that involve most tissues and organs of the organism. In particular, the visual system undergoes profound remodeling. Eyes migrate from the lateral position typical of tadpoles to a dorsofrontal one. Concomitant with the metamorphic change in eye position, a new pattern of retinal projections develops, connecting the retina to the ipsilateral thalamus. These uncrossed retinal projections, together with the change in eye position, subserve the new acquisition of binocular vision, appropriate to the predatory lifestyle of the adult frogs (Grant and Keating, 1986). Another marked change concerns the apposition of new cells in the retina. In amphibians, retinal growth continues throughout the life of the organism, and results from the proliferation of stem cells located at the periphery of the retina, in the ciliary marginal zone (CMZ). Whereas in tadpoles retinal growth is symmetrical, a sudden shift to an asymmetrical growth pattern is observed when metamorphosis begins. More cells are added to the ventrotemporal portion of the retina than to the dorsonasal portion. This finding is due to an increased number of progenitors in the ventral compared with the dorsal margin. The extensive proliferation of the ventral CMZ serves the production of neurons in the portion of the retina that views the new binocular visual field. Moreover, it may also compensate for the changes in eye position observed during metamorphosis (Straznicky and Gaze, 1971; Mann and Holt, 2001). The CMZ is spatially ordered with respect to cellular development and differentiation, with the youngest and least-determined stem cells closest to the periphery, the proliferative retinoblasts in the middle, and postmitotic cells at the central edge (Wetts et al., 1989). In past years, the embryonic CMZ has been extensively studied. These studies have shown that many of the genes involved in eye development and in retinogenesis are expressed in the CMZ following this spatial order, which, in turn, reflects the onset of their expression during early eye development and suggests a genetic cascade governing retinogenesis (Dorsky et al., 1995; Perron et al., 1998; Ohnuma et al., 2002). Coupling gene expression studies with bromodeoxyuridine (BrdU) incorporation analyses has allowed the subdivision of the CMZ into different zones (Dorsky et al., 1995; Perron et al., 1998; Ohnuma et al., 2002), corresponding to different phases of retinogenesis. These studies mainly focus on the embryonic CMZ, whereas almost no data are available on the CMZ during metamorphosis and especially on its ventral portion, where a great increase in proliferation occurs. The genes responsible for this proliferation burst have not been characterized yet.

In this study, we characterize the expression patterns during metamorphosis of several transcription factors involved in embryonic eye development. To this end, we compared premetamorphic (stage 54) and metamorphic (stage 63) retinae, particularly focusing on the proliferating CMZ. For this analysis, we selected genes involved in the proliferation of retinoblasts, such as Xrx1 (Mathers et al., 1997; Andreazzoli et al., 2003; Casarosa et al., 2003) and Xsix3 (Loosli et al., 1999; Bernier et al., 2000; Del Bene et al., 2004), genes involved both in early steps of eye development and in the determination of specific retinal cell types, such as Xotx2 (Pannese et al., 1995; Viczian et al., 2003), Xpax6 (Chow et al., 1999; Marquardt et al., 2001), and Xchx10 (Liu et al., 1994; Burmeister et al., 1996; Belecky-Adams et al., 1997), and genes involved in dorsoventral patterning of the retina, such as Xvax2 (Barbieri et al., 1999, 2002; Mui et al., 2002) and Xpax2 (Heller and Brandli, 1997). We found that the expression of these genes in the CMZ correlates with areas displaying different levels of BrdU incorporation, thus defining discrete functional regions in the metamorphic retina.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. NOTE ADDED IN PROOF
  8. REFERENCES

Homeobox Genes Involved in Eye Development Continue To Be Expressed in the Premetamorphic and Metamorphic Retina

To understand whether genes implicated in early eye development and retinogenesis could also be involved in the developmental changes that occur in the retina during metamorphosis, we performed in situ hybridization experiments on premetamorphic (stage 54) and metamorphic (stage 63) retinae. Of the seven genes we analyzed, six of them continue to be expressed during both stages, and their expression patterns closely resemble those found at the end of retinogenesis, when the retina is mature. The only exception we found is the Xpax2 gene, which shows no detectable expression either during or before metamorphosis (data not shown).

To analyze the genes effectively expressed at these stages, we initially focused our attention on the central retina, where the layers are clearly identifiable (Fig. 1 for stage 63, stage 54 not shown). Xrx1 (Fig. 1B″) is expressed in the outer nuclear layer (ONL) and in isolated cells located in the central portion of the inner nuclear layer (INL). Among the analyzed vertebrates, rx genes often display nonoverlapping expression patterns. rx/rax in rat embryos is expressed in Müller glial cells (Furukawa et al., 2000), which have cell bodies located in the central portion of the INL and endfeet terminating in the ONL. Among the fish rx genes, Rx3 displays the developmental pattern of expression most similar to Xrx1. Both in zebrafish and in medaka, Rx3 is expressed in the INL (Chuang et al., 1999; Loosli et al., 2001). The present data do not allow us to discriminate between different cell types; this characterization will require further analysis. Xsix3 transcripts (Fig. 1D″) are found in the INL and in scattered cells of the ganglion cell layer (GCL), as was reported for stage 39 Xenopus embryos (Ghanbari et al., 2001) and for Hamburger and Hamilton stage 39 chick embryos (Bovolenta et al., 1998). At the stages we analyzed, Xotx2 expression (Fig. 1F″) is located in the outer portion of the INL. This is strongly reminiscent of its expression at stage 41 and is likely to correspond to bipolar cells. Indeed, as previously shown, Xotx2 plays a crucial role in bipolar cell differentiation (Viczian et al., 2003). Xpax6 (Fig. 1H″) is expressed in cells of the GCL and of the inner portion of the INL, maintaining the pattern described for earlier stages (Hirsch and Harris, 1997). The INL expression could correspond to amacrine cells, in which mouse Pax6 is localized at the end of retinogenesis (postnatal day 10; Marquardt et al., 2001). The expression of Xchx10 (Fig. 1J″) is found in the central portion of the INL. Because all the chx10 orthologues so far described display a specific expression in bipolar cells (Liu et al., 1994, mouse; Passini et al., 1997, goldfish and zebrafish [vsx1 and 2]; Chen and Cepko, 2000, chick), and chx10 functional inactivation in mouse leads to loss of this cell type (Burmeister et al., 1996), we hypothesize that the Xchx10-expressing cells we observe are bipolar cells. Finally, vax2 was already reported to be expressed in the ventral portion of stage 59/60 Xenopus retinae (Liu et al., 2001), and in the GCL of the ventral part of mouse retinae (Mui et al., 2002). Here, we show that Xvax2 transcripts (Fig. 1L″) are found in many cells of the GCL, and in few cells located in the inner portion of the INL. Due to their morphology and laminar position, these cells could correspond to ganglion and amacrine cells, respectively. For each of the analyzed genes, we observe the same expression patterns in the central retina of stage 54 (premetamorphic) tadpoles (data not shown). These data show that many of the genes implicated in eye development during embryogenesis are also expressed during premetamorphic and metamorphic stages, thus suggesting for them a role in the retina not only during embryonic development but also at later stages.

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Figure 1. Expression patterns of homeobox genes and double staining for bromodeoxyuridine (BrdU) incorporation and gene expression in stage 63 dorsal and ventral ciliary marginal zone (CMZ). In all dorsal CMZ panels (A,A′,C,C′,E,E′,G,G′,I,I′,K,K′), central retina is to the top-left and peripheral to the right. In all ventral CMZ panels (B,B′,D,D′,F,F′,H,H′,J,J′,L,L′), central retina is to the top-right and peripheral to the bottom-left. Expression of the genes is depicted in blue and BrdU in red. Dorsal and ventral CMZ are as indicated. A,B:Xrx1. A′,B′: Double-staining BrdU and Xrx1. C,D:Xsix3. C′,D′: Double-staining BrdU and Xsix3. E,F:Xotx2. E′,F′: Double-staining BrdU and Xotx2. G,H:Xpax6. G′,H′: Double-staining BrdU and Xpax6. I,J:Xchx10. I′,J′: Double-staining BrdU and Xchx10. K,L:Xvax2. K′,L′: Double-staining BrdU and Xvax2. B″,D″,F″,H″,J″, L″: In situ hybridizations on the central region of the retina. Genes are as indicated. Brackets in B′,D′,F′,H′,J′,L′ indicate zones I, II, and III of the CMZ. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

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Expression of Homeobox Genes in the Premetamorphic and Metamorphic CMZ

To find possible connections between the analyzed homeobox genes and the proliferation increase observed during metamorphosis, we coupled in situ hybridization experiments with BrdU incorporation and analyzed both dorsal and ventral CMZs. A similar analysis on the embryonic retina previously led to the subdivision of the embryonic CMZ into different zones, according to the proliferative state of the cells and to the combination of regulatory genes expressed (Perron et al., 1998). Our data indicate a subdivision of the metamorphic CMZ that is comparable to the one observed during embryonic development. The most peripheral part (zone I in Perron et al., 1998) is where stem cells are located. In fact, we see low levels of BrdU incorporation in the most peripheral part of the CMZ (Fig. 1B′,D′, F′,H′,J′,L′), which is in accordance with the slow rate of division observed in stem cells (Doetsch et al., 1999). This zone expresses few of the genes analyzed, namely Xrx1 (Fig. 1B,B′), Xsix3 (Fig. 1D,D′), and Xpax6 (Fig. 1H,H′). These three genes are expressed in all the zones of the metamorphic ventral CMZ, starting from zone I and remaining expressed throughout the CMZ. Xchx10 also shows a weak signal in zone I (Fig. J,J′; and data not shown). Its expression increases in the adjacent zone, where Xvax2 starts being expressed (Fig. 1J,J′,L,L′). In this area, we observe very high levels of BrdU incorporation (see also Fig. 1B′,D′,F′,H′). It could correspond to the zones II and III already reported for the embryonic CMZ, in which highly proliferating retinoblasts are found (Perron et al., 1998). Xchx10 and Xvax2 are both expressed starting from the most peripheral edge of this area (Fig. 1J,J′,L,L′), because the limit of high expression of these two genes corresponds to the limit of high BrdU incorporation. Proceeding toward the central retina, Xvax2 remains expressed throughout the CMZ, as well as in the mature retina, as the other genes. The only exception is Xchx10, which shows a gap of expression, one to two cell layers wide, located approximately at the central end of the highly proliferative region (Fig. 1J,J′, arrowheads). Perron et al. (1998) subdivide the highly proliferative area in two, according to the differential expression of the proneural and neurogenic genes they analyze. The genes we have used do not allow this further subdivision, with the exception of Xotx2. Indeed, similarly to the situation described for the embryonic CMZ, the peripheral margin of Xotx2 expression is located in the middle of the highly proliferative region (Fig. 1F,F′). In fact, this peculiar expression of Xotx2 does suggest a further subdivision of the metamorphic CMZ, as for the embryonic one.

We also analyzed the dorsal CMZ at stage 63. Except Xvax2, all the genes we have tested are expressed in the dorsal CMZ during metamorphosis (Fig. 1A,A′,C,C′,E,E′,G,G′,I,I′,K,K′), with an overall expression pattern comparable to that found in the ventral CMZ. On the other hand, due to the lower number of precursors, the dorsal CMZ is much smaller than its ventral counterpart, as shown by BrdU incorporation (compare in Fig. 1A′ to B′, C′ to D′, and following). This feature renders it very difficult to allow a subdivision in zones as was done for the metamorphic ventral CMZ (this study) and for the embryonic CMZ (Perron et al., 1998).

Similar observations and conclusions can be drawn for both ventral and dorsal CMZ at a time point in which the proliferation burst has not yet occurred, namely at stage 54, which corresponds to a premetamorphic stage (Fig. 2B,B′,D,D′,F,F′,H, H′,J,J′,L,L′ for ventral CMZ; Fig. 2A,A′,C,C′,E,E′,G,G′,I,I′,K,K′ for dorsal CMZ). Indeed, we observe much lower levels of BrdU incorporation and, in general, the ventral CMZ appears smaller than at metamorphic stages. Nevertheless, the overall distribution of the transcripts of the genes studied is quite similar to that observed in the metamorphic CMZs.

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Figure 2. Double staining for bromodeoxyuridine (BrdU) incorporation and homeobox gene expression in stage 54 dorsal and ventral ciliary marginal zone (CMZ). Orientation of panels is as in Figure 1. Expression of the genes is depicted in blue and BrdU in red. A,A′C,C′,E,E′,G,G′,I,I′,K,K′: Dorsal CMZ. B,B′,D,D′,F,F′,H,H′,J,J′,L,L′: Ventral CMZ. A,B:Xrx1. A′,B′: Double-staining BrdU (red) and Xrx1. C,D:Xsix3. C′,D′: Double-staining BrdU (red) and Xsix3. E,F:Xotx2. E′,F′: Double-staining BrdU (red) and Xotx2. G,H:Xpax6. G′,H′: Double-staining BrdU (red) and Xpax6. I,J:Xchx10. I′,J′: Double-staining BrdU (red) and Xchx10. K,L:Xvax2. K′,L′: Double-staining BrdU (red) and Xvax2.

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Based on our present data, the CMZ appears to maintain the functional structure established in the embryo, even during the dramatic changes the retina undergoes during metamorphosis (Fig. 3). This finding suggests that the regulation of the increased growth of the ventral retina during metamorphosis may involve—at least in part—the same genes controlling embryonic retinogenesis.

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Figure 3. Schematic model of the expression of homeobox genes in the ventral ciliary marginal zone (CMZ) of the stage 63 embryo. Cells in zone I, the most peripheral, express only the early genes Xrx1, Xsix3, Xpax6, and probably correspond to stem cells. These cells are characterized by low bromodeoxyuridine (BrdU) incorporation levels. Cells in zone II still express these genes but express in addition Xchx10 and Xvax2. These cells correspond to proliferating retinoblasts, with high BrdU incorporation levels, as cells in zone III. Cells in zone III still express all the previous genes and, in addition, Xotx2. Cells in zone IV express the same combination of genes as cells in zone III, but are postmitotic. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

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All the metamorphic processes in Amphibia are under the control of thyroid hormone (TH), including the proliferation increase observed in the retina. The genetic and molecular mechanisms through which TH works to regulate retinal proliferation are not yet completely understood (see as a review, Mann and Holt, 2001). The present study provides initial data that could help us to further characterize the genetic network underlying TH function in the metamorphic retina.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. NOTE ADDED IN PROOF
  8. REFERENCES

Xenopus embryos were generated and staged as described (Nieuwkoop and Faber, 1967; Newport and Kirschner, 1982). Nonradioactive in situ hybridizations were performed as described (Casarosa et al., 2003). The Xenopus Xchx10 plasmid is a 2,994-bp IMAGE cDNA (accession no. BC044049). The predicted amino acid sequence displays a high similarity to the other vertebrate Chx10 proteins, sharing the highest conservation in the homeodomain. The cloning of full-length Xchx10 and a detailed analysis of its expression pattern during embryogenesis will be described elsewhere. BrdU (Roche) incorporation was performed dissecting the eyes of premetamorphic (stage 54) and metamorphic (stage 63) tadpoles and culturing them for 4 hr in culture medium (90% Leibovitz-10% fetal calf serum, Gibco) containing 150 μg/ml BrdU. Eyes were then fixed, cryoprotected, and cryosectioned as described (Casarosa et al., 2003). BrdU was revealed according to manufacturer's instructions using a Cy3-conjugated secondary antibody (Sigma).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. NOTE ADDED IN PROOF
  8. REFERENCES

We thank M. Fabbri and G. De Matienzo for technical assistance, and S. Di Maria for frog care. We also thank Federico Cremisi for identifying the BC044049 clone.

NOTE ADDED IN PROOF

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. NOTE ADDED IN PROOF
  8. REFERENCES

Sequence analysis of the full length Xchx10 clone revealed that this cDNA is more closely related to chick Chx10-1 and mouse Vsx1 than to chick and mouse Chx10.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. NOTE ADDED IN PROOF
  8. REFERENCES