Most frogs have a characteristic biphasic life history, with a specialized aquatic larval stage and a typically terrestrial adult. Transformation of the larva into the adult takes place during metamorphosis, a period of dramatic reorganization of the body plan at the end of larval life, which involves the loss of many larval tissues, the establishment of novel adult tissues as well as complex spatial tissue rearrangements under the control of thyroid hormones [1,2]. The phylogenetic distribution of this biphasic life history indicates that it is the ancestral developmental pattern of extant anurans [3-7]. However, in several anuran lineages, the free-living larval stage has been secondarily reduced or lost resulting in direct development [8-12]. While some larval features are recapitulated within the egg in direct developers, other larval features are lost and ontogenetic trajectories are modified and abbreviated. The degree to which larval development is recapitulated or abolished differs between different lineages of direct developing frogs with frogs of the genus Eleutherodactylus (Leptodactylidae) showing the most radical deviations from the ancestral pattern.

In Eleutherodactylus, including the particularly well studied Puerto Rican species E. coqui, many larval structures of the epidermis, the nervous system, the musculoskeletal system, and the inner organs never develop, while an adult-like cranial skeleton together with its associated muscles and nerves develops precociously [9,11,13-21]. Limb buds, which develop in larval stages of biphasically developing frogs, form already in early embryonic stages of E. coqui and this is paralleled by accelerated growth of the spinal cord and precocious development of dorsal root ganglia and lateral motor columns in the spinal cord, which provide the sensory and motor innervation of limbs, respectively [18,19,22-28]. Interestingly, another region of the central nervous system (CNS), the neural retina, also displays accelerated embryonic growth in E. coqui even though this is not accompanied by precocious differentiation of retinal cell layers [29]. As a consequence of these changes in timing (heterochronies) of various developmental events, embryos of E. coqui at any given stage present a complex mosaic of traits, some corresponding to embryonic stages of development of biphasically developing frogs, others corresponding to larval, metamorphic or adult stages (Fig. 1, Tables 1, 2).

Figure 1 Heterochrony plot comparing the timing of retinotectal and brain development (colored symbols, suites of characters I-IV) with development of the spinal cord (grey symbols, suites of characters V-VIII) and other characters (black symbols, suites of characters (more ...)

Table 1 Schedule of retinotectal development in different anurans

Table 2 Schedule of development of brain tracts and commissures in different anurans1 . (Fig. 1: pink triangles)

Despite these ontogenetic modifications, E. coqui retains some sort of cryptic metamorphosis [11,30]. During the last third of development prior to hatching (stages TS 12 – TS 15), its musculoskeletal system and several other tissues are remodeled in a way resembling metamorphic reorganization in biphasic developers. Moreover, these events are thyroid hormone dependent: they occur immediately after the thyroid axis becomes functional in E. coqui (as indicated by maturation of the thyroid gland and upregulation of thyroid hormone receptors TRβ) and can be arrested when thyroid hormone synthesis is blocked [11,30,31].

In both limb and cranial muscle development, precocious development of target structures in E. coqui is accompanied by a correspondingly precocious development of their innervation. The present paper addresses the question, whether the differentiation of the retina in E. coqui also remains closely temporally coordinated with the differentiation of the major central target of retinofugally projecting fibers in frogs, the optic tectum, and how this relates to the differentiation of other parts of the brain. Furthermore, it explores, whether growth of the optic tectum is accelerated like growth of the retina in E. coqui compared to the ancestral pattern of biphasically developing frogs.

In order to address these questions, development of retina and tectum is compared between E. coqui and the biphasically developing frogs Discoglossus pictus (Discoglossidae) and Physalaemus pustulosus (Leptodactylidae). While retinotectal projections have been studied in adult D. pictus [32], its embryonic development has not been previously reported. D. pictus was chosen as a representative of a basal anuran family with biphasic development. P. pustulosus was chosen, because it is relatively closely related to E. coqui (both frogs belong to the Leptodactylidae) but retains biphasic development. Comparison between the biphasic pattern of development in D. pictus, P. pustulosus, and other previously studied biphasically developing anurans (e.g. Xenopus) allows to identify shared features, which likely reflect the ancestral pattern of anuran development, from which direct development in Eleutherodactylus was derived. In addition, the expression of the neuronal differentiation gene NeuroD [33,34] in retina and tectum was compared between E. coqui and Xenopus laevis (Pipidae), since this gene has not been cloned from D. pictus or P. pustulosus.

The present study shows that in E. coqui embryonic growth of the optic tectum is accelerated paralleling rapid growth of the retina, while differentiation of tectal layers and development of the retinotectal projection proceeds according to a much slower schedule in coordination with retinal differentiation and the differentiation of fiber tracts in the brain. Thus, E. coqui hatchlings resemble postmetamorphic froglets of biphasically developing frog species with respect to differentiation of cranial muscles, limbs and spinal cord, and the size of their brains, but resemble tadpoles with respect to differentiation of their retinotectal system. These findings suggest that the schedule of differentiation of directly interconnected parts of the CNS such as retina and tectum remains coordinated during evolution, but can evolve largely decoupled from growth control and from the differentiation of other parts of the CNS (e.g. spinal cord).

In the following, I will frequently refer to E. coqui hatchlings (at stage TS 15) and compare them with various developmental stages in biphasically developing frogs. As a note of clarification, I should point out that this is done without intending to attach any particular significance to the time of hatching itself. The latter is highly variable between species and has little developmental significance. E. coqui hatchlings are rather singled out for comparison because with respect to many characters (e.g., musculoskeletal system, tail regression, limbs, brain size) they have reached a developmental stage, which corresponds to biphasically frogs at the completion of metamorphosis.

Figure 2 Retinal development in E. coqui and D. pictus. (A, B) Morphometric analysis of retinal development (modified from [29]). Cross-sectional area of the central retina (light blue) or of its cellular layer (dark blue) is plotted against developmental age. (more ...)

Figure 3 Tectal development in E. coqui and D. pictus. (A, B) Morphometric analysis of tectal development. Volume of the entire optic tectum (light blue) or of its cellular layer (dark blue) is plotted against developmental age. In addition, a proliferative index (more ...)

The development of the retina in E. coqui and D. pictus has been previously described [29]. Briefly, in E. coqui, outgrowth of the first retinofugal fibers and differentiation of the various cell and fiber layers of the retina during embryonic development is accompanied by rapid retinal growth (Fig. 2A, C). In contrast, in D. pictus, retinofugal fibers grow out and retinal layers form during embryonic development, preceding a period of prolonged growth of the retina during subsequent larval development (Fig. 2B, C). Similar to D. pictus, the formation of different retinal layers in P. pustulosus (data not shown) is also completed during embryonic development (with formation of a layer of photoreceptor outer segments at Go 27) prior to retinal growth during larval stages. At the stage when retinal layer formation is completed, the retinae of D. pictus and P. pustulosus have reached only about one third of their size at metamorphic climax, whereas the retina of E. coqui has reached more than 90 % of its size at hatching.

The development of the optic tectum shows similar differences between species. In the mature anuran tectum, 9 layers can be distinguished, numbered from 1 on the ventricle to 9 on the surface [35]. During early development of E. coqui the optic tectum consists of a single cellular layer, which increases rapidly in size (Fig. 3A, C, 4A–C). A superficial fiber layer, which will subsequently develop into layers 7–9 (two fiber layers with an interspersed layer of scattered cell bodies), is first evident at stage TS 11. At stage TS 13, when the tectum has reached more than 90 % of its size at hatching, a second fiber layer (layer 5) appears within the cellular layer, dividing the latter in a peripheral (layer 6) and ventricular part. Subdivision of this ventricular layer into distinct layers 1–4 (an ependymal layer and two cellular layers with a fiber layer sandwiched in between) becomes, however, only evident six days after hatching.

Figure 4 Cell proliferation during tectal development in E. coqui (A-C) and D. pictus (D-F) as revealed by immunostaining for proliferating cell nuclear antigen (PCNA) in transverse paraffine sections. The border of the optic tectum is indicated on the right side (more ...)

In contrast, the optic tectum remains small throughout embryonic development of D. pictus (Fig. 3B, C, 4D–F)) and P. pustulosus (data not shown). The superficial (layers 7–9) and deep (layer 5) fiber layers appear at early larval stages (Go 29 in D. pictus, Go 27 in P. pustulosus), when the tectum has reached only a small fraction (about one third in D. pictus, less than one fourth in P. pustulosus) of its size at the end of metamorphic climax. Subdivision of the ventricular cell layer into layers 1–4 becomes first apparent during metamorphic climax (Go 41–42) in both species. Thus, at hatching the tectum of E. coqui (Fig. 4C) resembles the tectum of D. pictus at early to mid larval stages (Fig. 4F) with respect to the differentiation of tectal layers.

The time course of PCNA immunostaining in the retina of E. coqui and D. pictus has been previously described [29]. Briefly, in E. coqui PCNA immunopositive cells occur throughout the outer part of the inner nuclear layer of the retina until the latter has almost reached its size at hatching and only then become restricted to the ciliary margin (Fig. 2A, C). In contrast, in D. pictus (Fig. 2B, C) and P. pustulosus (data not shown), PCNA positive cells become restricted to the ciliary margin already at midembryonic stages (GH 23–27 in both species), preceding the period of larval retinal growth.

In the optic tectum of early embryos of E. coqui (Figs. 3A, C, 4A), D. pictus (Figs. 3B, C, 4D), and P. pustulosus (data not shown), PCNA immunopositive cells occupy the entire cellular layer. The first PCNA immunonegative and presumably postmitotic cells are seen in the lateral part of the cellular layer at stages TS 9 in E. coqui (Fig. 4B) and at stages GH 23–27 in D. pictus (Fig. 4E) and P. pustulosus. PCNA immunostaining is maintained along the entire ventricle (Fig. 4C, F) at least until 6 days after hatching in E. coqui and until the end of metamorphic climax (Go 45) in D. pictus and P. pustulosus.

Figure 5 Neurogenesis in the retina and tectum of E. coqui (A-C, G-I) and X. laevis (D-F, J-L) as revealed by in situ hybridization for NeuroD in transverse paraffine sections. A-F: At early embryonic stages, NeuroD begins to be expressed in scattered cells throughout (more ...)

In the optic tectum of E. coqui, NeuroD was never strongly expressed at any stage of development, although expression was evident in other regions of the midbrain and elsewhere in the brain (Fig. 5G–I). In contrast, while the embryonic tectum of X. laevis also does not express NeuroD, tectal NeuroD expression is upregulated from early tadpole stages (NF 46) on (Fig. 5J–L).

Figure 6 Contralateral retinotectal projections in E. coqui (A-C) and D. pictus (D-F) as revealed by biocytin tracing. At two days before hatching, the optic projections of E. coqui (A, B) resemble early larvae of D. pictus (E, F). After crossing in the optic (more ...)

In E. coqui, the first neurites of retinal ganglion cells grow out at stage TS 6. These fibers cross to the contralateral side of the diencephalon in the optic chiasm at stage TS 7. The majority of labeled fibers there form the marginal optic tract, which courses dorsad. The first fibers reach the contralateral optic tectum already at stage TS 7. By stage TS 11 retinofugal fibers are sparsely distributed over the lateral part of approximately the rostral half of the tectal surface (layer 9) (Fig. 6A, B), while the caudal tip is reached only 12 days after hatching. However, even 12 days after hatching the medial part of the tectum is still free of retinofugal fibers (Fig. 6C). Terminal fields in the contralateral thalamus (rostral visual nucleus, neuropil of Bellonci, corpus geniculatum thalamicum) and pretectum (uncinate field) develop from stage TS 13 on. A much smaller number of fibers courses caudad in the contralateral basal optic tract from stage TS 11 on and terminates in the basal optic neuropil in the tegmentum. In addition to the contralateral projections, a few retinofugal fibers are evident in the ipsilateral marginal optic tract from stage TS 9 on, but no prominent ipsilateral retinothalamic projections have developed even at 12 days after hatching.

In D. pictus, outgrowth of the first neurites of retinal ganglion cells occurs at stage GH 22 and these fibers cross in the optic chiasm, reach the contralateral marginal optic tract and extend towards the optic tectum by stage GH 23. At early larval stages (Go 30), the surface of the lateral part of the rostral half of the tectum is densely covered by retinofugal fibers (Fig. 6E, F). Retinofugal fibers reach the caudal limit of the tectum at stage Go 40 and extend towards the midline along the entire rostrocaudal extent of the tectum at the end of metamorphic climax (Go 45) (Fig. 6G). Terminal fields in the contralateral thalamus and pretectum and a small basal optic tract are evident from early larval stages (stage Go 30, possibly already GH 30) on. A few fibers in the ipsilateral marginal optic tract are also observed from stage Go 30 on.

Applications of biocytin to the optic tectum of various stages of E. coqui and D. pictus allowed to determine that the first tectofugal fibers develop at stage GH 22 in D. pictus and at stage TS 5 in E. coqui (data not shown), prior to the ingrowth of retinofugal fibers into the tectum.

Figure 7 Development of early fiber tracts in the brain of E. coqui (A-C) and D. pictus (D-F) as revealed by immunohistochemistry for acetylated tubulin. Asterisks indicate the optic stalk in all panels. A, D: The tract of the postoptic commissure and its associated (more ...)

In E. coqui and D. pictus, three phases of fiber tract development can be recognized (commissures often form slightly later than the associated fiber tracts). In P. pustulosus, fiber tract development follows a similar time course, but the earliest phase of brain tract development could not be observed, because no embryos earlier than stage GH 20 were investigated. In the first phase of development (TS 4–5 in E. coqui; GH 18 in D. pictus; ≤GH 20 in P. pustulosus), the tract of the postoptic commissure (TPOC) develops in the forebrain and continues caudad as the ventral longitudinal tract (VLT) (Fig. 7A, B). In the hindbrain, the descending tracts of the trigeminal and vestibulocochlear nerves develop. In the second phase of development (TS 6–7 in E. coqui; GH 20–22 in D. pictus and P. pustulosus), several additional fiber tracts develop in the forebrain and midbrain (tract of anterior commissure, supraoptic tract, tract of posterior commissure, tract of commissure of posterior tuberculum, tract of ventral tegmental commissure, tract of cerebellar commissure) and join the pioneering fibers of the TPOC and VLT. In addition, a discrete dorsoventral diencephalic tract (DVDT) emanating from the epiphysis as a well defined bundle of fibers forms in D. pictus and P. pustulosus at these stages. In E. coqui, no such distinct bundle of fibers was observed, but fibers running ventrad throughout the thalamus appeared at stage TS 6, a subset of which may correspond to the DVDT. In the third phase of development (≥ TS 8 in E. coqui; ≥GH 23–24 in D. pictus and P. pustulosus), the tract of the habenular commissure joins the axonal scaffold and the intertectal commissure develops. However, at stage TS 9 in E. coqui and GH 22 in D. pictus only single fibers crossing between the two tectal hemispheres could be observed, indicating that the bulk of the intertectal commissure develops later.

The development of the retina and tectum in D. pictus and P. pustulosus as documented here has many similarities with retinotectal development in other biphasically developing frogs including Rana [48-51]), Limnodynastes [52,53], and Xenopus, which is particularly well studied [40,54-68]. Since this pattern of retinotectal development is shared between neobatrachian frogs (Rana, Limnodynastes, Physalaemus), including the leptodactylid frog Physalaemus and more basal "archaeobatrachian" lineages such as discoglossids (Discoglossus) and pipids (Xenopus), it probably represents the primitive condition for extant anurans. Thus, the differences in retinotectal development observed in Eleutherodactylus are derived within this direct developing clade of leptodactylid frogs.

In the various biphasically developing species, the different retinal layers develop during embryonic development, when the retina is still small and has reached only a fraction (about one third) of its cross-sectional area at the completion of metamorphosis. The first retinotectal connections are established during early embryonic development, shortly after the major axon tracts have developed in the brain (Tables 1, 2). The expression of NeuroD (only described for Xenopus), which plays a role in regulating neuronal differentiation and the formation of particular neuronal subtypes such as photoreceptors and amacrine cells in the retina [33,34,37,69-75] becomes restricted to the ciliary margin, the outer part of the inner nuclear layer, and the outer nuclear layer as soon as the different retinal layers develop. Proliferation becomes largely restricted to the ciliary margin at the end of embryonic development, from which the retina then grows during larval stages by addition of cells to all cellular layers.

When the first retinofugal fibers reach the contralateral tectum at late embryonic stages, the latter is still very small and consists predominantly of proliferating cells. Retinal fibers cover the tectum in a rostrocaudal direction during larval stages, as the optic tectum continues to grow by proliferation from the ventricular layer, which is most pronounced caudomedially. At early larval stages, the first tectal layers (layer 7–9 and layer 5) differentiate and retinal fibers cover approximately half of the lateral part of the tectum leaving its midline free of fibers. At this stage the tectum has reached only a fraction (about one third) of its size at the completion of metamorphosis. At the end of metamorphic climax, the entire surface of the tectum is covered by retinofugal fibers. In addition to the main, contralateral projection to thalamus, pretectum, and tectum, an ipsilateral projection to thalamus and pretectum, which subserves important functions for binocular vision in postmetamorphic frogs, begins to develop at late larval stages in Xenopus [66,76]. In D. pictus, a sparse ipsilateral projection develops already at early larval stages.

Retinotectal development in E. coqui has been modified in a number of respects from this ancestral anuran pattern. While the spatiotemporal order of differentiation of retinal and tectal layers and the formation of the retinotectal projection in E. coqui have been conserved, patterns of growth and proliferation of retina and tectum have been modified. Both retina and optic tectum grow rapidly during initial formation of the retinotectal projection in embryonic stages so that at a stage when formation of retinal layers and tectal layers 5–9 are completed, they have already reached around 90 % of their size at hatching. While retinal growth in biphasically developing frogs is mostly due to addition of cells from the proliferative ciliary margin, the retina of E. coqui grows by proliferation of cells throughout the outer part of the inner nuclear layer of the retina until it has almost reached its size at hatching [29]. Thus, although NeuroD expression in the ciliary margin, the outer part of the inner nuclear layer, and the outer nuclear layer resembles the condition in Xenopus, many NeuroD expressing cells in the inner nuclear layer of E. coqui retinae are probably proliferating progenitor cells in contrast to Xenopus. NeuroD expression in some retinal ganglion cells and absence of NeuroD expression in the tectum of E. coqui also differ from X. laevis, suggesting that some of the functions of NeuroD have changed during evolution of E. coqui. A sparse ipsilateral retinothalamic projection develops already at embryonic stage TS 9, prior to completion of retinal layer formation and, thus, significantly earlier than in biphasically developing frogs. This may represent an adaptive heterochronic shift related to precocious adoption of postmetamorphic head shape and eye position in E. coqui, allowing onset of binocular vision already at hatching stages when the optic tectum is still in a relatively immature, larval-like state (note dissociation between suites XI/XII indicating cranial remodeling and suite II indicating retinotectal differentiation in Fig. 1).

In biphasically developing anurans such as Xenopus, thyroid hormones have been implicated in promoting increased proliferation in the retina during mid-larval stages after the thyroid gland develops and thyroid hormone levels rise [77,78]. However, the precocious growth of retina and tectum in E. coqui is unlikely to be due to the precocious action of thyroid hormones, because acceleration of retinal and tectal growth is observed from early embryonic stages on, long before the thyroid gland matures and the thyroid axis becomes functional around stage TS 10 in E. coqui [30,31]. Although the mechanisms underlying the increase in proliferation in E. coqui remain at present obscure, the conserved schedule of retinotectal differentiation implies that increased proliferation is not simply due to a general delay in neural differentiation. Changes in the probability of cell cycle exit or the length of cell cycles are better compatible with the pattern observed.

As a consequence of the rapid growth of retina and tectum during the development of the retinotectal projection, retinofugal fibers in E. coqui encounter a much larger target territory as they enter and distribute over the tectum than in biphasically developing frogs. This raises the question, whether the formation of a topographic map of retinal fibers on the tectal surface, as known from other vertebrates, may be compromised in E. coqui. However, the formation of retinotopic maps, which is now known to involve gradients of Eph receptors and their ephrin ligands in retina and tectum, is very plastic and can expand or contract in response to drastic experimental reductions of retinal or tectal size, respectively [79,80]. In many fishes and amphibians plastic mechanisms of map formation are important to maintain an ordered retinotopic map throughout development, because their tectum grows in a rostrocaudal direction, while new retinal neurons are added in a radial direction [57,65,81]. This plasticity probably permitted the drastic acceleration of retinotectal growth in Eleutherodactylus without compromising the ability to form a well ordered retinotopic map.

Increased proliferation and accelerated growth in E. coqui is not confined to the retina and tectum, but is also evident in other parts of the CNS such as the spinal cord [28] and the brain stem (Schlosser, unpublished observation) (compare suites III, IV, and VIII in Fig. 1). This suggests that altered growth patterns in E. coqui may be due to systemic regulatory changes affecting proliferation of neural progenitors throughout the CNS, although independent regulatory changes in different parts of the CNS cannot be ruled out.

Although schedules of retinotectal differentiation and the formation of early brain tracts remain tightly coordinated with each other in E. coqui, they have become dissociated from differentiation in other parts of the central and peripheral nervous system. For example, the lateral motor columns (LMCs) in the spinal cord develop precociously in E. coqui paralleling precocious development of the limbs, which they innervate [22-25,27,28]. As a consequence, in E. coqui LMCs develop simultaneous with the retinotectal system, while in biphasically developing frogs they develop in larval stages after differentiation of most retinal and tectal layers is completed (compare suites VII and II in Fig. 1). Similarly, adult-like cranial skeletal structures and muscles together with their motor innervation precociously differentiate in E. coqui in stages [9,13,16,18,19,21], when the retinotectal system is still in an immature, larva-like condition (compare suites XI/XII and II in Fig. 1).

Taken together, this indicates that during evolution of Eleutherodactylus, the schedule of differentiation in CNS areas remained coordinated with the schedule of differentiation of those regions in the CNS or in the periphery, with which they are directly connected (retina-tectum-brain tracts, LMC-limbs, cranial motor neurons-cranial muscles), whereas profound temporal dissociations have taken place between structures that are not or only indirectly connected.

Previous studies have shown that limbs, spinal cord, and the cranial musculoskeletal system develop precociously in direct developing frogs of the genus Eleutherodactylus. The present study indicates that retinotectal development was also modified during evolution of Eleutherodactlyus. Cell proliferation and growth in retina and tectum have been greatly accelerated in Eleutherodactylus, while differentiation of retina, tectum, and fiber tracts in the embryonic brain, which remain temporally coordinated with each other, proceed along a much slower schedule. Consequently, Eleutherodactylus hatchlings appear like postmetamorphic froglets with respect to limb, spinal cord, and cranial development and have a correspondingly large brain, while their retinotectal system is still immature and tadpole-like. This suggests that differentiation events in directly interconnected parts of the CNS such as retina and tectum remain coordinated during evolution, while they may be dissociated from proliferation control and from differentiation events in other parts of the CNS probably reflecting the modular character of CNS development.

The authors declare that they have no competing interests.

I wish to thank Gerhard Roth, R. Glenn Northcutt, and Chris Kintner for providing lab space. This work was in part supported by research scholarship SCHL 450/1-1 of the German Science Foundation.