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Review articles Control of retinal growth and axon divergence at the chiasm: lessons from Xenopus Fanny Mann and Christine E. Holt* Summary Metamorphosis in frogs is a critical developmental process through which a tadpole changes into an adult froglet. Metamorphic changes include external morphological transformations as well as important changes in the wiring of sensory organs and central nervous system. This review aims to provide an overview on the events that occur in the visual system of metamorphosing amphibians and to discuss recent studies that provide new insight into the molecular mechanisms that control changes in the retinal growth pattern as well as the formation of new axonal pathways in the central nervous system. BioEssays 23:319±326, 2001. ß 2001 John Wiley & Sons, Inc. Introduction The visual system of amphibians undergoes profound remodeling during metamorphosis. In tadpoles, the two eyes are placed laterally with no binocular overlap. As the skull changes shape during metamorphosis, laterally positioned eyes migrate dorsofrontally, leading to a substantial degree of binocular overlap.(1) During embryonic development, retinal ganglion cell axons from the two eyes project to the contralateral side of the brain.(2) Concomitant with the metamorphic change in eye position, however, a new pattern of retinal projections begins to develop, connecting the retina to several nuclei in the ipsilateral thalamus.(3±5) These uncrossed retinothalamic projections subserve the new acquisition of binocular vision appropriate to the predatory lifestyle of the adult frogs (Fig. 1). Another marked change occurring in the metamorphosing visual system is found in the pattern of cell production in the retina. In amphibians, retinal growth results from the proliferation of stem cells located at the periphery of Department of Anatomy, University of Cambridge, Cambridge, UK. *Correspondence to: Christine E. Holt, University of Cambridge, Department of Anatomy, Downing Street, Cambridge CB2 3DY, UK. E-mail: [email protected] Abbreviations: CMZ, ciliary marginal zone; D3, type III deiodinase; IOPA, iopanoic acid; PTU, goitrogen propylthiouracil; RXR, retinoid X receptor; T2, diiodothyronine; T3, triiodothyronine; TH, thyroid hormone; TR, thyroid hormone receptor. BioEssays 23:319±326, ß 2001 John Wiley & Sons, Inc. the retina, in the ciliary marginal zone (CMZ).Whereas in tadpoles the retina grows by proliferating symmetrically, a sudden shift to an asymmetrical growth pattern is observed when metamorphosis begins, with more cells being added at the ventral and temporal margin than at the dorsal and nasal ciliary margin.(6±8) At metamorphic climax, the ratio of dividing cells in dorsal and ventral CMZ reaches 1:10.(8) This is attributed to an increased number of progenitor cells in the ventral compared to the dorsal margin.(9) It has been proposed that the extensive proliferation in the ventral margin serves the production of neurons in the portion of the retina that views the new binocular visual field.(8) The asymmetric retinal growth may also compensate for the changes in eye position throughout metamorphosis.(1,8) Because the retinal position relative to the body is changing, visuomotor readjustments are required, but these are minimized by the fact that the asymmetrical growth pattern allows the retina produced before metamorphosis to view constant regions of the body-centered visual space during ocular migration. Thyroid hormone and the control of retinal growth Metamorphosis in amphibians is under the control of thyroid hormone (TH), which acts directly on tissue to induce metamorphic transformations.(10) In the amphibian Xenopus laevis, the shift in histogenetic pattern from symmetric to asymmetric growth in the developing retina occurs in late tadpoles beginning at stage 54,(6±8) about 30 days postfertilization, when circulating levels of TH begin to rise.(11) Asymmetric growth persists through metamorphic climax (stages 58±64) and declines thereafter. There are several lines of evidence that TH can trigger the change in cell production by a direct action on the eye. Kaltenbach and Hobbs first showed in Rana pipiens that implanting an intraocular TH-releasing pellet in tadpoles causes an increase in retinal cell proliferation.(12) Similarly, in Xenopus laevis, slow release of TH into the eye of premetamorphic tadpoles stimulates cell proliferation restricted to the treated eye.(9) In the latter case, although cells in both dorsal and ventral retina are capable of responding to exogenous TH, a greater proliferation response occurs in the ventral retina, giving rise prematurely to an asymmetric pattern of retinal proliferation.(9) BioEssays 23.4 319 Review articles periphery of the retina.(14,15) Taken together, the results confirm that changing levels of endogenous thyroxine at the onset of metamorphosis initiates the change in retinal growth pattern by inducing an asymmetric ventral but not dorsal increase in retinal cell production (Fig. 2A). Figure 1. Eye position and pattern of retinal projections in premetamorphic and postmetamorphic amphibians. A: In tadpole, the two eyes are placed laterally and most or all of the retinal ganglion cells (orange) project contralaterally to the optic tectum. B: During metamorphosis, the two eyes migrate dorsofrontally leading to the emergence of a binocular field, and a novel pattern of retinal projections to the ipsilateral thalamus develops. These ipsilateral projecting fibers arise from newly generated retinal ganglion cells (blue) that sit in the ventrotemporal portion of the retina viewing the binocular visual space. Reciprocally, the metamorphic increase in ventral retina proliferation can be inhibited by treatment with the goitrogen propylthiouracil (PTU), a chemical that blocks the production of thyroxine in the thyroid gland by preventing the iodination of tyrosine.(13) Xenopus tadpoles reared in the presence of PTU stay permanently premetamorphic as they stop their morphological development at about stage 54. PTU-treated animals remain viable, increase in size and their eyes continue to grow by addition of neurons symmetrically at the dorsal and ventral 320 BioEssays 23.4 Figure 2. The type III deiodinase (D3) enzyme controls the asymmetric responsiveness of retinal progenitors to thyroid hormone (TH) during metamorphosis. A: During normal metamorphosis, as levels of circulating TH increase, retinal growth changes from a symmetric to an asymmetric pattern of proliferation, with more cells being generated in ventral retina compared to dorsal retina. Increased proliferation in ventral retina is directly stimulated by TH and produces a dorsal displacement of the optic nerve head (onh) within the retina. B: D3 enzyme, which degrades TH, is expressed in dorsal (black) but not ventral (white) retinal progenitor cells. In tadpoles treated with iopanoic acid (IOPA), an inhibitor of D3 activity, dorsal progenitor cells are able to respond to exogenous TH by showing an increased rate of proliferation as ventral progenitors normally do. C: In tadpoles carrying the D3 transgene, the ventral retina appears resistant to the proliferating changes that can be induced by exogenous TH. Review articles TH is known to act on target tissues through two thyroid hormone receptors (TR alpha and TR beta) and their heteromeric partner, the retinoid X receptor (RXR).(16,17) TRs can enter the cell nuclei and function as hormone-dependent transcription factors to regulate gene expression.(17) In Xenopus laevis, about 35 genes that are transcriptionally upregulated during tadpole tail resorption initiated by TH have been isolated and sequenced.(18,19) The known TH response genes encode proteins that range from thyroid hormone receptors themselves to proteolytic enzymes, transcription factors and extracellular matrix components. Interestingly, TH-inducible genes encoding the transcription factors xBTEB, TH/bZip and FRA-2 are most actively upregulated in tissues that grow at metamorphosis, including cartilage, muscles, brain and spinal cord, than in structures that undergo cell death and resorption.(20) This suggests a general role for these TH response genes in controlling the programmed metamorphic growth and cell proliferation throughout the entire body of the tadpole, and possibly in the ventral retina. However, the mechanism by which TH preferentially influences cell proliferation in the ventral retina versus dorsal retina has long remained unsolved. After rotating the eye of embryos between stages 26 and 35/36, Beach and Jacobson found that dorsal and ventral retina are intrinsically specified to grow differentially at metamorphosis and that these differences are determined early during embryogenesis.(21) Theoretically, an asymmetrical distribution of thyroid hormone receptors in the ventral and dorsal retina could explain the greater growth of the ventral retina as the result of TH stimulation. This is unlikely to be the case because a study of TR transcripts did not detect an asymmetrical distribution in the retina.(22) The recent understanding of the mechanism controlling asymmetric growth in the retina comes from the study of a TH-inducible gene product, the type III deiodinase (D3), an enzyme that degrades TH by converting the active triiodothyronine (T3) hormone into an inactive diiodothyronine (T2) molecule.(23) First isolated from the Xenopus tadpole tail,(18,23) D3 is also expressed in other tissues of the metamorphosing animal, predominantly in those structures that present a reduced response to TH,(24) and is upregulated in tissues after completing metamorphic changes.(20,25) Transgenic tadpoles that overexpress D3 usually undergo spontaneous metamorphosis, but often arrest their development at the climax of metamorphosis and only rarely complete the late morphologic transformations, such as tail resorption, that require high levels of circulating TH.(26) Moreover, transgenic tadpoles fail to undergo the gross metamorphic changes that can be prematurely induced by treatment with exogenous TH.(26) The results indicate a function of D3 in protecting the tissues from the effect of TH, and suggest a role for D3 in controlling the differential proliferation response of dorsal and ventral retina to TH stimulation. In a recent study, Marsh-Armstrong, Brown and colleagues examined the pattern of D3 expression in the retina during metamorphosis and found an asymmetric distribution, with D3 mRNA expression predominant in the dorsal CMZ, the area of least growth at metamorphosis.(27) D3 mRNA colocalizes with X-Notch-1 mRNA, a marker for retinal progenitor cells.(28) This asymmetric pattern was established as early as stage 35/36 well before the development of the thyroid gland, suggesting that the differential D3 expression in the metamorphosing retina is not a secondary effect of TH induction.(27) Treatment of premetamorphic tadpoles with iopanoic acid (IOPA), an inhibitor of D3 activity,(29) caused exogenous TH to stimulate proliferation in the dorsal retinal margin to the same level as the proliferation induced by TH in the ventral retina (Fig. 2B) .(27) In contrast, tadpoles expressing the D3 transgene are resistant to the increased cell proliferation normally observed in the ventral retina as the result of exogenous TH treatment (Fig. 2C) .(27) Moreover, as spontaneous metamorphosis proceeds in D3 transgenic animal, the ventral retina shows reduced proliferation that parallels a decrease in the number of X-Notch-1 mRNA-positive progenitors in the ventral CMZ.(27) These results provide evidence for a novel mechanism regarding how different cells respond differentially to the same level of a circulating hormone; asymmetric D3 expression in the eye controls the retinal growth pattern at metamorphosis by inactivating TH in the dorsal retina and thus preventing dorsal CMZ progenitors from responding to TH. Development of ipsilateral retinothalamic projections During embryonic development, virtually all retinal ganglion cell axons from the two optic nerves cross each other at the ventral midline of the diencephalon, forming the optic chiasm, and project into the contralateral side of the brain. As metamorphosis proceeds, a novel pattern of retinal projections develops that connect the retina to the ipsilateral thalamus.(3) These projections first appear at the beginning of metamorphosis at stage 54 in Xenopus laevis,(5) about three weeks after crossed retinal projections started to develop in the embryo.(2) Cells giving rise to the ipsilateral retinothalamic projections lie in the ventral and temporal periphery of the adult retina,(4,30) in the regions produced by the differential growth induced by TH.(5) Little is known, however, about the regulatory events that control the delayed appearance of a new retinal pathway in metamorphosing amphibians. Because PTU-reared tadpoles fail to develop ipsilateral retinothalamic projections,(15) it has been proposed that TH controls the production of ipsilateral retinal connections. Hoskins and Grobstein showed that injecting TH into the eye of PTU-reared tadpoles induces the formation of projections to the ipsilateral thalamus, without influencing the pattern of projection from the uninjected eye.(14,15) The authors concluded that TH-induced changes restricted to the retina are sufficient to cause the development of a new pattern of retinal BioEssays 23.4 321 Review articles projections, whereas additional changes along the central retinal pathway seem unnecessary for normal axon guidance. Hoskins proposed that TH triggers the production of a new population of retinal ganglion cells, which differs from the other populations by expressing a cytochemical tag that allows them to follow a preexisting ``ipsilateral route blueprint''.(31) According to this view, the ipsilateral labeled path would already be present in premetamorphic tadpoles but, in the absence of TH induction, retinal ganglion cell axons would ignore it.(31) This hypothesis does not fit, however, with the results from other experiments reporting that tadpoles fail to develop precociously uncrossed retinothalamic projections after intraocular implantation of a TH pellet(4) or when reared in the presence of exogenous TH.(32) One possible explanation is that the retina is not ``competent'' to respond to a TH signal and to produce ipsilaterally projecting ganglion cells until tadpoles reach metamorphosis. To address this issue, Nakagawa, Holt and colleagues used a transplantation approach in which marginal zone tissue harvested from the ventrotemporal retina of metamorphosing frog, supposedly competent to the TH signal, was transplanted into the eye of young tadpoles.(32) Axons from the transplant projected to the tectum in the contralateral side of the brain, either in the presence or the absence of exogenous TH, indicating that retinal ganglion cells, which normally form ipsilateral projections during metamorphosis, do not project ipsilaterally when placed into a younger environment.(32) It is unlikely that the host environment has respecified the transplant as it retained a topographic specificity that coincides with its ventral origin even when placed into dorsal host retina.(32) The heterochronic graft experiments strongly suggest that molecular cues to guide axons ipsilaterally are not expressed in the young embryo. Finally, the finding that D3 transgenic Xenopus do form small uncrossed projections at metamorphosis(27) provides additional support for the idea that changes other than those induced by TH in the retina are needed for the formation of a new uncrossed pathway. The role of Eph family molecules in guiding ipsilateral retinothalamic projections The optic chiasm is the choice point where contralateral and ipsilateral fibers diverge during development of visual projections. In rodents, in vitro experiments suggest that inhibitory cues expressed in the developing optic chiasm differentially affect crossed and uncrossed axons and may play an important role in selectively guiding uncrossed retinal axons away from the ventral midline into the ipsilateral optic tract.(33±35) The molecular nature of these guiding cues is unknown, however. The search for candidate molecules recently led to focus on the Eph family of molecules. The Eph receptors form the largest family of receptor tyrosine kinases with the 14 known members falling into two subclasses: EphA and EphB. With few exceptions, these interact respectively with A-type 322 BioEssays 23.4 (GPI-anchored) and B-type (transmembrane) ephrin ligands. The inhibitory interactions between Eph receptors and ephrins have been found to play important roles in axon guidance and formation of topographic projections.(36) There is also an increasing body of evidence that suggests a role for Eph family molecules in regulating axon growth at the midline of the central nervous system. For example, genetic analysis of mice lacking Eph receptors revealed defects in the formation of commissures in the forebrain,(37,38) guidance of contralaterally projecting tectal axons,(39) and decussation of the corticospinal tract.(40) Recent investigations that examined the molecular nature of axon guidance at the optic chiasm in rodents have involved ephrin-A ligands. Using receptor affinity probe in situ, Marcus, Mason and colleagues found that A-type ephrins are expressed along the ventral aspect of the optic chiasm.(41) In vitro, the addition of EphA-Fc chimeric protein, a competitive inhibitor of Eph/ephrin interactions, reduced the inhibition of axon growth observed when retinal neurites contact aggregates of ventral diencephalic cells.(41) EphA-Fc equally influenced the behavior of ipsilaterally and contralaterally projecting axons, however.(41) These results suggest that ephrin-A ligands act to repel retinal axon growth from inappropriate regions of the ventral diencephalon during the formation of the optic chiasm, but a role in regulating the divergence of crossed and uncrossed axons seems unlikely. Nakagawa et al. studied the expression pattern of Eph receptors in the eye of metamorphosing Xenopus laevis using probes consisting of the ephrin-binding domains fused to alkaline phosphatase, a technique introduced by Cheng and Flanagan.(42) As in other species previously examined,(43±48) Eph receptors in Xenopus were found more highly concentrated in the ventral and temporal regions of the developing retina than in the rest of the retina, with EphB receptors distributed in the ventralmost region of the retina and EphA receptors abundant in the temporal retina.(32) The ventral expression of EphB receptors is established well before metamorphosis begins and could be detected early during embryonic development.(32) This striking coincidence between the expression patterns of Eph tyrosine kinase receptors and the region of the retina giving rise to uncrossed projections suggests a function for the Eph family of molecules in guiding ipsilaterally projecting axons in Xenopus laevis. According to this idea, the delayed appearance of a new ipsilateral pathway at metamorphosis would result from concomitant changes in the expression pattern of Eph receptor ligands, presumably at the chiasmatic region. Thus Nakagawa and collaborators studied the expression of ephrin ligands in the tadpole and frog optic chiasm. Whereas A-type ephrin ligands could not be detected in the chiasm of tadpoles, ephrin-B ligand protein expression was found to be upregulated in the optic chiasm at stage 55 when metamorphosis begins, and expression persists throughout metamorphosis(32) (Fig. 3A,B). To address whether B-type ephrins can guide axons ipsilaterally, the Review articles placing a crystal of the lipophilic agent DiI into the ventral but not the dorsal retina, thus indicating that ectopic ephrin-B2 specifically influenced the behavior of retinal cells carrying EphB receptors.(32) Taken together, these results provided direct evidence for a role of ephrin-Bs in directing the divergence of ipsilaterally and contralaterally projecting axons at the optic chiasm of metamorphosing frogs (Fig. 4). In normal animals, however, ipsilateral projections arise from regions of the retina that express EphB receptors as well as EphA Figure 3. Distribution of transmembrane ephrin-B ligands in the developing optic chiasm revealed by in situ binding of EphB4 ectodomain fused to alkaline phosphatase. A: Frontal section through the chiasm of stage 54 Xenopus laevis tadpole. B: Frontal section through the chiasm of stage 60 Xenopus laevis tadpole. C: Horizontal section through the ventral diencephalon of an E16.5 mouse embryo. The arrowheads indicate the position of the chiasm. On, optic nerve. Scale bar, 100 mm (A,B) and 200 mm (C). Adapted from Nakagawa et al. 2000.(32) zebrafish ephrin-B2 gene was introduced into the chiasm of stage 19±20 tadpoles, long before the onset of normal ephrinB ligand expression here.(32) Precocious expression of ephrinB2 induced an increased number of retinal projections to the ipsilateral side of the brain at stage 41, when retinal projections are normally almost completely crossed. Anterograde labeling of precocious uncrossed projections could be performed by Figure 4. Model for a role of EphB receptors and ephrin-B in regulating retinal fibers crossing at the optic chiasm. A: In premetamorphic tadpole, cells in a small region of the ventral retina express EphB receptors (yellow), but corresponding ephrin-B ligands are not detected along the retinal pathway in the central nervous system. Retinal fibers arising from the entire retina enter the brain, cross the optic chiasm and project to the contralateral side of the brain. B: The asymmetric pattern of retinal growth induced by thyroid hormone at metamorphosis gives rise to an increased number of new retinal ganglion cells in the ventral retina, most of them expressing EphB receptors (yellow). Simultaneously, ephrin-B ligands are upregulated in the chiasmatic region (red). Repulsive interactions between Eph receptors and their ligands may guide growing axons from ventral retina (green) away from the optic chiasm into the ipsilateral optic tract. BioEssays 23.4 323 Review articles receptors. A role of ephrin-B ligands in guiding EphA receptor expressing axons ipsilaterally is made possible by the fact that one of the EphA receptors, EphA4, has been found to interact significantly with some of the transmembrane ephrinB ligands.(49) These results indicate a new role for ephrin-Bs at the ventral midline of the frog diencephalon. Interestingly, ephrinB ligands have been found to be also expressed in the ventral midline of the developing hindbrain(50) and spinal cord(51±55) in rodents. In the spinal cord, cognate receptors are expressed on ipsilaterally projecting axons and are upregulated on commissural axons after crossing the midline.(55) Moreover, in vitro experiments indicated that B-type ephrins are able to induce the collapse of commissural growth cones.(55) These observations suggest a conserved role of Eph/ephrin interactions in sorting out ipsilaterally and contralaterally projecting axons at the ventral midline of the nervous system. How can these results be reconciled with those of Hoskins and Grobstein, which showed a prominent role of TH on the retina itself for the induction of ipsilateral retinothalamic projections?(14,15) One possibility is that, in Hoskins and Grobstein's experiments, TH injected into the retina of PTUreared tadpole leaked out of the eye and also affected the chiasmatic region, inducing metamorphic-like changes such as ephrin-B upregulation. Although TH alone does not appear to be able to stimulate ephrin-B protein expression in early stage tadpoles, it does so in tadpoles near metamorphosis.(32) Alternatively, in Hoskins and Grobstein's experiments, signals to guide ipsilateral projections could have been upregulated in a TH-independent manner in the chiasm of PTU-reared tadpoles during the 3 weeks delay between the ocular injection of TH and the analysis of the retinal projection patterns. The neotenic Ambystoma mexicanum, which does not undergo metamorphosis due to the absence of endogenous TH, develops delayed ipsilateral retinal projections,(4) suggesting that the molecules that normally guide axons ipsilaterally are regulated in the absence of TH. This alone, however, can not explain why the Hoskins and Grobstein's studies found that projections from the uninjected eye were not affected and continue to project contralaterally even in the possible presence of cues to project ipsilaterally. This could be accounted for by the mitogenic activity that TH has on the retinal margins.(9,12,32) In the injected eye, TH induced the proliferation and the production of Eph-expressing retinal ganglion cells that are guided along the ipsilateral retinal pathway, whereas in the non-injected eye of PTU-reared animal, the reduced proliferation rate was not sufficient to give rise to a significant number of ipsilaterally projecting ganglion cells. Future perspectives Several points remain to be clarified to fully understand how ipsilateral retinothalamic projections form at metamorphosis. 324 BioEssays 23.4 First, it is known that ipsilaterally projecting cells represent only a subpopulation (about 10%) of the ganglion cells generated during TH-induced growth of the retina.(30) A higher proportion of cells in the ventrotemporal region of the retina appear to express Eph receptors, however. Why do only few of them respond to the ephrin-B signal at the chiasmatic region? Co-expression of ephrin ligands and Eph receptors on retinal ganglion cells has been found to regulate axon sensitivity to ephrin ligands in vitro.(56) Such a mechanism could explain the differential response of ganglion cells to ephrin-Bs at the optic chiasm. It is also possible that different levels of Eph receptor expression or a specific combination of Eph receptors on individual retinal ganglion cells could modulate how axons respond to ephrin-B signal. Second, in the experiments by Nakagawa, Holt and colleagues, ipsilateral retinal projections, prematurely induced by ephrin-B2 expression in the chiasm of tadpoles, target the optic tectum whereas ipsilaterally projections formed normally during spontaneous metamorphosis terminate in the thalamus. This difference may be due to the absence of a labeled pathway to the thalamus in early embryos, as crossed retinothalamic projections normally develop later around stage 49.(5) The signals that selectively guide retinal ganglion cell axons towards the tectum or the thalamus are so far unknown, however. Finally, a long-standing question remaining to be answered is how retinal axon divergence at the chiasm is regulated in mammals. Do ephrin-Bs play a role during development of the mammalian visual pathway similar to that found in metamorphosing Xenopus laevis? Several lines of evidences argue in favor of this idea. In the mouse, for example, ipsilaterally projecting axons arise from the ventrotemporal portion of the retina,(57,58) the region of highest expression of both A-type and B-type Eph receptors.(59) Furthermore, ephrin-B expression has been recently reported in the optic chiasm of embryonic mice at the time of major retinal axon outgrowth(32,41) (Fig. 3C). Although there is no direct evidence implicating Eph family of molecules in directing crossed and uncrossed projections in rodents, it is tempting to speculate that ephrin-Bs are at play. It will certainly be interesting to investigate whether aberrant crossing occurs at the chiasm of EphB/ephrin-B mutant mice. References 1. Grant S, Keating MJ. Ocular migration and the metamorphic and postmetamorphic maturation of the retinotectal system in Xenopus laevis: an autoradiographic and morphometric study. J Embryol Exp Morph 1986;92:43±69. 2. Holt CE, Harris WA. 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