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. Author manuscript; available in PMC: 2016 Jul 18.
Published in final edited form as: Development. 2015 Jan 23;142(4):743–752. doi: 10.1242/dev.114629

Cavefish eye loss in response to an early block of retinal differentiation progression

Manuel Stemmer 1,#, Laura-Nadine Schuhmacher 1,4,#, Nicholas S Foulkes 1,3, Cristiano Bertolucci 2, Joachim Wittbrodt 1,6
PMCID: PMC4948675  EMSID: EMS63744  PMID: 25617433

Summary

The troglomorphic phenotype shared by diverse cave-dwelling animals is regarded as one of the classical examples of convergent evolution. One unresolved question relates to whether the characteristic eye loss in diverse cave species is based on interference with the same genetic program. Phreatichthys andruzzii, a Somalian cavefish, has evolved under constant conditions in complete darkness and shows severe troglomorphic characteristics such as complete loss of eyes, pigments and scales. In the course of early embryonic development, a complete eye is formed that is subsequently lost. In Astyanax mexicanus, another blind cavefish, eye loss has been attributed to interferences during eye field patterning. To address whether similar pathways have been targeted by evolution independently, we investigated the retinal development of P. andruzzii studying the expression of marker genes involved in eye patterning, morphogenesis, differentiation and maintenance. In contrast to Astyanax, patterning of the eye field and evagination of the optic vesicles proceeds without obvious deviation. However, the subsequent differentiation of retinal cell types is arrested during the generation of the first-born cell type, retinal ganglion cells, which also fail to project correctly to the optic tectum. Eye degeneration in both species is driven by progressive apoptosis. However it is retinal apoptosis in Phreatichthys that progresses in a wave-like manner and eliminates progenitor cells that fail to differentiate in contrast to Astyanax, where lens apoptosis appears to serve as a driving force. Thus, evolution has targeted late retinal differentiation events indicating that there are several ways to discontinue the development and maintenance of an eye.

Introduction

Cave inhabitants often share characteristic changes in morphology that are called troglomorphisms (Pipan and Culver, 2012). These changes result from the absence of light and have been commonly separated into regressive and constructive traits (Hecht et al., 1988). Regressive traits are characterized by the loss of an organ or function, while constructive traits lead to an increase in number or performance of an organ. The most prominent regressive traits in cavefish are eye and pigment loss (Hecht et al., 1988; Jeffery, 2001). Cave species often form an embryonic eye that apparently develops normally before it degenerates during juvenile or early adult stages, in most cases without ever being functional (Berti et al., 2001; Besharse and Brandon, 1974; Hecht et al., 1988). One hypothesis is that neutral mutations accumulate in eye-specific genes after loss of the evolutionary pressure to retain visual perception due to the constant dark environment (Pipan and Culver, 2012; Wilkens, 2011). However, it has been recently suggested that in some cases, eye loss might occur by selection of a gene that has positive effects on unrelated features, thus increasing the organism’s fitness via pleiotropic effects (Protas and Jeffery, 2012; Rohner et al., 2013; Yamamoto et al., 2009). Eye degeneration occurs in several cavefish species (Berti et al., 2001; Meng et al., 2013; Wilkens, 2007) of which Astyanax mexicanus is the best studied (Alunni et al., 2007; Hinaux et al., 2011; Jeffery, 2009; Rétaux et al., 2008; Strickler et al., 2007; Yamamoto, 2000). In this species, decisions for later eye degeneration are apparently rendered during patterning of the early eye field (Menuet et al., 2007; Pottin et al., 2011; Yamamoto et al., 2004). A key, unresolved question is whether this mechanism of eye loss is conserved in other, unrelated cavefish species. To address this issue, we have studied in detail eye development of Phreatichthys andruzzii, a cave dwelling cyprinid that exhibits an even more severe troglomorphic phenotype.

The blind cavefish P. andruzzii has recently been introduced as a cavefish model due to its extreme troglomorphism (Cavallari et al., 2011). The wild population, sampled in the Bud-Bud region of Somalia in a sub-Saharan horizontal limestone formation, has been isolated in a cave environment for approximately two million years. The adult fish completely lack pigment and eyes, as well as scales. Conversely the number of neuromast sensory cells is dramatically increased (Berti et al., 2001; Dezfuli et al., 2009a; Dezfuli et al., 2009b). The eyes in Phreatichthys have been shown to initially develop, but then degenerate and are eventually overgrown by orbit tissue and skin (Berti et al., 2001). The eye does not exhibit any functional architecture at any time point.

It has been previously shown that the vertebrate eye develops along an evolutionarily conserved spatiotemporal axis, with respect to morphogenesis and the expression of corresponding key transcription factors (Agathocleous and Harris, 2009; Kay et al., 2005; Loosli et al., 2001; Zuber et al., 2003). Eye formation is initiated by the activity of conserved transcription factors (Six3, Sox2, Pax6, Rx3) with the specification of the eye field in the anterior neuroectoderm. The eye becomes obvious with the lateral evagination of the optic vesicle (Shh/Gli, Rx3) and subsequent optic cup formation (Pax2, Pax6), followed by growth (Rx2, Sox2). These events are all tightly connected to changes in the expression of the above mentioned (transcription) factors.

Growth and morphogenesis are tightly interconnected with the stepwise and highly stereotypic differentiation of the retinal cell types. Initially undifferentiated retinal progenitor cells differentiate into the seven distinct cell types of the adult retina in tightly coordinated temporal progression: Retinal ganglion cells (RGC) are born first, followed by horizontal, amacrine and bipolar cells and the late born rod and cone photoreceptors as well as non-neuronal Mueller glia cells (Centanin and Wittbrodt, 2014). The tip of the retina, an area called the ciliary marginal zone (CMZ) remains undifferentiated and functions as a stem cell niche, contributing to the life-long growth of the eye (Centanin et al., 2011).

We found that in Phreatichthys the establishment of the eye field and the initial steps of retinal morphogenesis appear largely normal as indicated by the expression of the transcription factors involved in the corresponding steps. These genes are expressed in patterns highly reminiscent to those described for other teleosts. Conversely, markers for retinal ganglion cells are expressed only partially and genes involved in the differentiation of subsequently formed cell types were not expressed or only at very low levels. In addition to our in situ hybridization study, we observed disorganization of the retinal architecture and strong activity of the apoptosis gene Caspase3, an indicator of cell death.

Results

In order to investigate the early retinal development in Phreatichthys andruzzii, we documented the expression of key marker genes selected for their involvement in retinal development and performed in situ hybridization to assay and correlate their expression with morphologically apparent alterations. Early eye field specification, stem cells and retinal progenitor cells (RPCs) were visualized by using probes detecting six3a, pax2.1, pax6a, sox2, rx3 and rx2 transcripts. Embryonic midline signalling was analysed with probes against shh, nkx2.1 and fgf8. As specific markers for RPCs at later stages, six3 and nr2e1 were selected.

Differentiated cell types in the retina were identified by the expression of well-established cell type specific transcriptions factors. In particular we analysed atoh7, a transiently expressed transcription factor that marks the genesis of retinal ganglion cells (Kay et al., 2001). Those cells were additionally visualized by pou4f2 and ils1 expression (Moshiri et al., 2008; Mu et al., 2008). Photoreceptors were analysed by the expression of crx, opsin (long-wave sensitive), and rhodopsin. Barhl2 (Jusuf et al., 2012) was employed as an amacrine cell marker and vsx1 (Passini et al., 1997) as a marker for bipolar cells. Since none of these genes had been described for Phreatichthys so far, we cloned and sequence validated all of the respective orthologs from Phreatichthys cDNA.

During development of Phreatichthys, the retina started to degenerate rapidly after 45 hours post fertilization (hpf). Just ten hours later, the eyes were strikingly smaller or barely visible and started to sink into the surrounding tissue. We thus focused our detailed analysis on the first 45 hours post fertilization, divided into six developmental stages, 10 hpf, 19/20 hpf, 31 hpf, 35 hpf, 38 hpf and 45 hpf.

Early patterning and eye morphogenesis

To examine the early phase of eye field patterning, optic vesicle morphogenesis and optic cup formation, we analysed the expression of sox2, six3a, rx3, rx2, pax2.1 and pax6a. To address embryonic midline signalling, we used probes against fgf8, shh and nkx2.1.

The pan-neural marker gene sox2 promotes neural fate, maintains progenitor cells in an undifferentiated state and is additionally active in promoting stemness in adult stem cells (Agathocleous et al., 2009; Ekonomou et al., 2005). In Phreatichthys embryos early expression of sox2 was detected throughout the neuro-ectoderm including the eye field (10 hpf) and optic vesicles (19 hpf) (Fig. 1A,B). Six3a is one of the initiation factors for eye development (Carl et al., 2002; Loosli et al., 1999) and was detected early in the forming eye field (Figure 1C). It was also expressed in the developing forebrain of P. andruzzii at 19 hpf (Figure 1D).

Fig. 1. Early patterning and eye morphogenesis in Phreatichthys andruzzii.

Fig. 1

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In situ hybridization with antisense RNA probes, stained with visible dye NBT/BCIP. Stages 10 hpf and 19/20 hpf show dorsal view of embryos dissected from yolk, anterior to the left. Sox2 staining was present in the entire neuroectoderm at stage 10 hpf (A) and in brain and optic vesicles at stage 19 hpf (B). Six3a was expressed in the eye field at 10 hpf (C) and in the anterior neuroectoderm and optic vesicles at 20 hpf (D). Rx3 expressed in entire anterior neuroectoderm at 10 hpf (E) and the evaginating optic vesicles and forebrain at 19 hpf (F). Fgf8 was detected rostrally in the embryonic midline at 10 hpf (G) extending posteriorly at 20 hpf (H) (arrows) and in MHB (arrowhead). Additional expression in the midbrain/hindbrain boundary (MHB) (G,H). Shh expression along the midline in stage 10 hpf (I) and 19 hpf (J), markedly in the ventral forebrain and midbrain. Nkx2.1 expression in anterior midline, similar to shh at 10 hpf (K) and forebrain at 20 hpf (L). Pax2.1 expressed in MHB at 10 hpf and 20 hpf (M,N) and optic stalk at 20 hpf (N) (arrow). Pax6 was expressed in the eye field, midbrain and hindbrain at stage 10 hpf (O). At stage 19 hpf (P), expression was additionally seen in the evaginating optic vesicles. Scale bars are 50µm.

Retinal homeobox (rx) genes represent a set of key factors involved in vertebrate eye patterning and morphogenesis. Loss of function of some of these genes leads to eyeless phenotypes in mouse and fish (Loosli et al., 2001; Loosli et al., 2003; Mathers et al., 1997). The role of Rx3 is to guide cells through optic vesicle evagination towards a photoreceptor fate in the late gastrula while Rx2 is suggested to function in photoreceptors, and actively proliferating retinal stem and progenitor as well as Müller glia cells.

Transcripts of rx3 were detected in the anterior neuroectoderm (eye field) at 10hpf and the optic vesicles and midbrain at 19 hpf (Fig. 1E,F). At stage 31 hpf, staining for both sox2 and rx2 was strong in the newly formed retina, where it was confined to the periphery (Fig. S1E,H), consistent with their expression in the forming ciliary marginal zone in other teleost species.

In Astyanax an early expansion of midline signalling highlighted by the expression of the secreted signalling factors Shh has been proposed to contribute to the subsequent eye loss (Menuet et al., 2007; Yamamoto et al., 2004). In response, fgf8 is expressed earlier in the Astyanax cavefish than in its surface counterpart (Pottin et al., 2011). We therefore analysed Phreatichthys fgf8 and shh expression. We detected an apparently normal fgf8 expression in Phreatichthys embryos as well as fgf8 transcripts initially in the anterior neuroectoderm and at the midbrain-hindbrain boundary (MHB) (10 hpf, Fig.1G). At subsequent stages we found fgf8 expression in the anterior midline as well as the MHB (20 hpf, Fig.1P). Also the shh expression pattern appeared unaffected and shh was expressed along the embryonic midline at 10 hpf (Fig. 1I), and in the ventral neural tube and notochord at stage 19 hpf (Fig. 1J). Consequently, the expression pattern of the shh downstream target gene nkx2.1 is indistinguishable from the pattern in comparable stages other teleost species (Fig. 1K,L).

Fates along the medio-lateral axis of the neural plate are highly sensitive to Shh signalling. We addressed whether Shh signalling in the ventral midline affects the establishment of proximal or distal retinal fates, indicative of altered Shh signalling activity (Macdonald et al., 1995). We thus compared the complementary expression domains of proximal (Pax2) and distal (Pax6) markers.

In the eye field (10 hpf) we detected pax2 expression in the proximal eye field, adjacent to midline shh (compare Figs. 1I and 1M). As development proceeds, pax2 expression remained confined to the proximal part of the evaginated optic vesicle (Fig. 1N) and was clearly excluded from the distal domain of the forming optic vesicle (arrow in Fig. 1N).

The initial pax6 expression demarcates the early eye field. Here it plays a key role in the formation of the optic primordia and subsequently in the promotion of progenitor cell proliferation (Lagutin et al., 2003; Walther and Gruss, 1991; Zaghloul and Moody, 2007). At 10 hpf (early neurula) pax6 mRNA was detected in the eye field of the anterior neuroectoderm and the forming dorsal diencephalon (Fig. 1O). At 19 hpf (optic vesicle stage), the eye field expression of pax6 is shifted distally to the retinal progenitor cells of the evaginating optic vesicles (Fig. 1P) and at a later stage (31 hpf) its expression has spread throughout the retina (Fig. S1A,B). We also studied the pattern of the distally expressed key retinal gene pax6 as well as markers for the distal most domain of the retina, the ciliary marginal zone at subsequent stages of retinal development. Pax6 expression was detected in a layer close to the lens (Fig. 2A, S1A-D) consistent with its expression in RGCs as reported in other teleosts. Similarly we detected very faint six3 expression in the entire retina (Fig. 2E, Fig. S1L-N) consistent with proper proximo-distal pattering of the early eye field. The expression of rx2 and sox2 in the periphery of the retina hints at the establishment of a CMZ (Centanin et al., 2011), the stem cell domain of anamniotes that contributes to life long growth of their retina (Fig. 2B-D, S1E-K). The formation of an active CMZ is further supported by PCNA immunohistochemistry revealing active proliferation in this domain (Fig. 2G,G’). In contrast to other teleosts however, the nuclear receptor nr2e1 (tlx) was only weakly expressed here (Fig. 2F, Fig. S1).

Fig. 2. Establishing CMZ in Phreatichthys andruzzii.

Fig. 2

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Pax6, sox2 and rx2 were prominently expressed in the retina at 35 hpf to 38 hpf (A-D). Pax6 expressed throughout the retina, slightly stronger closer to the lens (A). Sox2 expression at 35 hpf confined to a dorsal peripheral part of the retina (B). Rx2 expression was strong across the retina (C), a lateral view shows rx2 expression forming a ring around the lens (D). Weak six3a expression at 38 hpf (E). Low level Nr2e1 expression (arrow) in dorsal peripheral part at 38 hpf (F), reminiscent of Sox2. Transverse sections (A-C, E, F) and lateral view (D), dorsal up. Anti-PCNA immunohistochemistry revealed signal in the CMZ region at 45hpf (G; arrows); DAPI channel in G’. Retinae are encircled by dotted lines. Further stages can be found in Fig. S1. Scale bars are 50µm.

Our data indicate that there was no apparent enhancement or absence of the expression of key genes involved in the establishment of the eye field, its subsequent patterning and optic vesicle evagination respectively. Apparently the levels of midline signalling do not repress distal retinal fates in Phreatichthys and a ciliary marginal zone is initially established.

We therefore extended our analysis to later stages and specifically investigated genes involved in the differentiation of retinal cell types and the layering of the retina.

Differentiation and layering of the retina does not proceed

In all vertebrates retinal cell types are born in a stereotypic temporal order resulting in a clear retinal lamination. Retinal ganglion cells (RGCs) are forming first followed by cone photoreceptors, amacrine and horizontal cells and eventually bipolar cells, rod photoreceptors and Müller glia cells (Centanin and Wittbrodt, 2014). In zebrafish the initial wave of retinal differentiation giving rise to retinal ganglion cells preceded by a central to peripheral spreading of atoh7 expression in the retina (Kay et al., 2005). In the absence of Atoh7, retinal ganglion cells (RGCs) cannot differentiate from retinal progenitor cells. We used atoh7 as a marker for the initiation of RGC differentiation and detected its initial expression in a central area of the retina at 31 hpf (Fig. 3A) from where, as in zebrafish, atoh7 expression spreads towards the periphery at 35 hpf as an expanding ring (Fig. 3B) similarly to the expression of rx2 (Fig. 2C,D). Independently of atoh7, isl1 expression marks RGC, however at a differentiated state. It follows atoh7 expression from the centre (35 hpf, Fig. 3F) to the periphery (38 hpf, Fig. 3G) and was expressed in a ring-domain directly adjacent to the lens at 38 hpf (Fig. 3G). In contrast to the expression in zebrafish, its expression was strongly reduced at subsequent stages (Fig. 3H). As a third marker for terminally differentiated RGCs we employed pou4f2. It was faintly expressed from 31 hpf to 38 hpf close to the lens, a position where differentiated RGCs are located in a functional retina (Fig. 3I-K). Similar to isl1, the expression of pou4f2 was barely detectable beyond 40hpf (Fig. 3L). Taken together these results indicate that RGCs are initially formed but not maintained as fully differentiated cells. While the expression of atoh7, a maker for cells in their final mitosis prior to RGC differentiation is maintained beyond 45 hpf, the expression of the terminal differentiation markers isl1 and pou4f2 ceases (Fig. 3D,H,L).

Fig. 3. Expression of differentiation markers in the retina of Phreatichthys andruzzii.

Fig. 3

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Atoh7 (differentiating RGCs) was expressed centrally of the retina (A) at 31 hpf and spread towards the periphery at 45 hpf (B). At 38 hpf (C) and 45 hpf (D) stayed expressed strongly. Adjacent to the lens, Isl1 (differentiated RGCs) follows atoh7 expression pattern from the center (35 hpf, F) to the periphery (38 hpf, G). At 45 hpf (H) isl1 expression was reduced markedly. Pou4f2 (differentiated RGCs) was detected only faintly from 31 hpf to 38 hpf (J,K) close to the lens (RGC region) and not detectable at 31 hpf (I) and 45 hpf (L). The amacrine cell marker barhl2 was not epxpressed at all analysed stages (M-P). Vsx1 (bipolar cell marker) showed ectopic staining at various time points; 31 hpf (Q), 38 hpf (S) and 45 hpf (T). Retinae are encircled by dotted lines. All panels show transverse sections.

Even though present in the genome, no expression of photoreceptor marker genes (crx, opsin, rhodopsin) was detectable neither by whole mount in situ hybridization, nor by RT-PCR (data not shown). To address the presence of the subsequent cell types born in the vertebrate retina we analysed the expression of barhl2 (amacrine cells) and vsx1 (bipolar cells).

Barhl2 was not expressed at any of the stages (31-45 hpf, Fig. 3M-P). This was partially also the case for the expression of the bipolar cell marker vsx1. It showed occasional transient expression in small patches randomly located in the neuroretina (Fig. 3Q-T). In the absence of apparent RGC and photoreceptor differentiation and retinal lamination, a faint staining could be observed at 31 hpf in the ventral retina (Fig. 3Q), at 38 hpf at the dorsal edge of the retina (Fig. 3S) and around a rosette structure indicative of apoptotic centres at 45 hpf (Fig. 3T, arrow).

Taken together these data indicate that retinal ganglion cells are born in the retina and their differentiation is initiated, but not maintained. Strikingly, also the differentiation of any of the subsequently born cell types could not be detected with multiple independent markers.

Ganglion cell projections to the tectum are impaired

To address whether mature retinal ganglion cells with fully extended axons were formed and whether those project to the optic tectum, we visualized the axonal tracts in the nervous system of Phreatichthys. We employed a monoclonal antibody directed against acetylated α-tubulin, which is predominantly found in the axoneme of mature neurons.

Marker gene analysis had indicated that the first differentiating RGCs appear at 35 hpf (Fig. 3F). At that stage the acetylated tubulin staining showed very few axons leaving the eye, with no obvious projections from the eye to the optic tectum (Fig. 4A,A’). In contrast to functional fish retinae, the few forming axons did not exit the retina at a single site (optic disc) but rather individually without any clear direction of their projections (arrows in Fig. 4A,A’). At 38 hpf, the peak of RGC marker expression (differentiation) some RGC axons formed a dispersed optic disc like structure (arrow in Fig. 4B’). However, those axons remained in the area of the eye and did not form any structure resembling a projecting optic nerve. These short axon clusters did not extend beyond the eye and did not form any projections to the tectum at this or subsequent stages.

Fig. 4. Anatomy of the nervous system of Phreatichthys andruzzii.

Fig. 4

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Whole mount immunostainings with anti-acetylated tubulin antibody (green) and DAPI (blue). All embryos are in lateral view with anterior to the left. Pictures show maximum z projections. At stage 35 hpf (A,A’) growing axons connecting hindbrain, midbrain, forebrain and olfactory system become apparent; additionally thin axons exiting the retina were detected (arrows). More extensive staining of the retina was observed at stage 38 hpf (B,B’). Few stained RGC axons bundles likely represent a widened optic disk (arrow). At stage 45 hpf (C,C’) only two neighboring retinal cells show axonal protrusions (arrow), while the neurons of the olfactory system had grown in number. At 86 hpf (D,D’), the eye had degenerated and sunk into the surrounding tissue. Only few residual neurons in the retina showed the tubulin signal of, the olfactory bulb had grown further. D’ is a magnification of D, showing retinal staining and thin axons protruding from the eye and seemingly projecting further (arrow). Retinae are encircled by dotted lines. di, diencephalon; fb, forebrain; hb, hindbrain; mb, midbrain; ob, olfactory bulb (arrowhead); pt, pretectum; tel, telencephalon. Scale bars are 50µm.

RGCs with short axons are initially formed (Fig. 4A-C) and transiently maintained. They vary in length (25-100µm), and stretch only over a small fraction of the distance to the optic tectum. At 86 hpf, the eyes were already degenerated and had sunken into the surrounding tissue. Even though the eye could not be seen from the outside, it was recognizable by condensed DAPI staining. Only very few isolated residual RGCs still showed short axons (arrow in Fig. 4D’). The axonal connections/protrusions observed at 35 hpf, 45 hpf and 86 hpf could only be found occasionally and not in all the embryos analysed (not shown). Interestingly and in contrast to the situation in the retina, the number of neurons within the olfactory bulb increased massively in the first five days of embryogenesis (arrowhead in Fig. 4A-D).

Degeneration of the cavefish eye is accompanied by activated Caspase3 expression

Active cell death by apoptosis has been described to be a key feature of eye degeneration in Astyanax and we observed autolytic vacuoles in the retina of Phreatichthys (Berti et al., 2001). Immunostaining on whole mount preparations and on cryosections with an antibody directed against activated Caspase3 was performed to investigate the timing and distribution of apoptotic cells in Phreatichthys relative to the block in retinal differentiation.

Prior to the onset of RGC differentiation (31 hpf, Fig. 3A) in the eye only a few apoptotic cells were detected, while there was a marked focus of activated Caspase3 positive cells in the olfactory bulb (arrow in Fig. 5A).

Fig. 5. Distribution of apoptotic cells in embryos of Phreatichthys andruzzii.

Fig. 5

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Immunostaining with and antibody directed against activated Caspase3 (red) and DAPI (blue) A-C’ show lateral view of whole mount embryo with anterior to the left and dorsal up. D and E show cryosections. Caspase staining was detected in the embryo at stage 31 hpf (A), most prominently in the olfactory bulb (arrow), only faintly in the retina. At stage 35 hpf (B) prominent apoptosis is detected in the retina and in the olfactory bulb (arrow) (maximum projections in A, B). Optical sections indicate that apotosis is initiated centrally at 35 hpf (C, at the level of the lens). (C’) higher magnification of C showing that retinal apoptosis had not yet reached the periphery of the eye. At stage 45 hpf (D at central level, E at peripheral level), extensive apoptosis in the entire retina is detected. Higher densities of activated Caspase3 positive cells were detected in rosettes surrounding the autolytic vesicles (arrows). Whole mount images of TUNEL stained eyes (F,G) at 38 hpf. Staining resembled activated Caspase3 pattern. Scale bars are 50µm.

With the onset of RGC terminal differentiation at 35 hpf, a marked increase in retinal apoptosis was detected (Fig. 5B). The increase became obvious in comparison to the olfactory bulb (arrow in Fig. 5A). Concomitant with the progression of atoh7 expression preceding RGC differentiation we observed an increase in retinal apoptosis, seemingly spreading from the centre of the retina to its margins (Fig. 5C-E). This is well represented in the serial sections (Fig. 5D,E) where active Caspase3 is preferentially detected in the periphery (Fig. 5E). Here, autolytic vacuoles form (Fig. 5E, arrows) that consist of dead cell material only. The predominant localization of apoptotic activity to the neural retinal was independently confirmed by TUNEL staining (Fig. 5F,G). Similar to active Caspase3, DNA fragmentation as hallmark of apoptosis was detected in the central region of the retina (compare Fig. 5C’ and Figs. 5F,G). Interestingly and in contrast to Astyanax, only very few apoptotic cells were encountered in the lens at any stage analysed.

Discussion

We have studied eye development and degeneration in the Somalian cavefish Phreatichthys to address whether evolution has targeted a similar “developmental soft spot” in distantly related cavefish, or alternatively, if independent steps in eye development and maintenance were targeted. We reveal that, differently from Astyanax, the eye primordium was morphologically unaltered and key factors involved in early eye development were expressed in patterns highly reminiscent of the pattern of their orthologs in eyed teleosts. However, the key step of retina formation, the differentiation of neuronal cell types failed to proceed beyond the birth of the first retinal neurons, the retinal ganglion cells. These neither fully established nor maintained (even partial) functionality. Concomitant with the birth of retinal ganglion cells, we detected a wave of apoptosis following the wave of RGC initiation. Consistently, only few retinal neurons extending short axons formed in the prospective ganglion cell layer of the retina. Even though an optic disk-like structure was established transiently, the few surviving retinal ganglion cells failed to establish connections with the optic tectum that, similar to the retina, was severely affected by apoptosis.

The early eye field is established and patterned in Phreatichthys andruzzii

It has been suggested that in Astyanax the patterning of the eye field is already affected (Yamamoto et al., 2004). Prior to the evagination of the optic vesicles, the eye field is defined at the anterior end of the neuroectoderm. It is patterned in a proximo-distal direction by signals emanating from the ventral midline. In particular the secreted signalling molecule Shh has been demonstrated to promote proximal cell fates (indicated by pax2 expression) and at the same time repress distal fates, in particular pax6 expression (Macdonald et al., 1995). This aspect of early pattering seems affected in Astyanax, where enhanced expression of midline shh (Yamamoto et al., 2004) results in the repression of Pax6 in the distal eye field leading to a reduced size of the optic primordia from early stages onward (Rétaux et al., 2008; Soares et al., 2004).

Our analyses in Phreatichthys andruzzii indicate that there is no such enhanced activity of Shh in the midline. We found neither an expanded shh expression domain during the establishment of the eye field nor enhanced shh activity as indicated by an enhanced proximal pax2 expression at the expense of distal pax6 expression or by shifts in the expression of other factors downstream of shh (e. g. nkx2.1, fgf8). In contrast to Astyanax, Phreatichthys pax2 and pax6 are expressed in a pattern comparable to other teleost species. This apparently normal expression of proximal pax2 and distal pax6 serve as internal control for correct early patterning, even in the absence of a comparison with a surface sister species. Thus, evolution has apparently targeted different developmental modules in Astyanax and in Phreatichthys, both resulting in the absence of eyes.

Neuroretinal differentiation is initiated, but does not proceed

Concomitant with optic cup formation, the differentiation of the neural retina proceeds. As in all vertebrates, retinal progenitor cells of teleosts follow a stereotypic order of differentiation into retinal cell types. Differentiation in P. andruzzii is initiated in central progenitors and expands radially. However, it never reaches the periphery where a second reservoir of stem cells in the CMZ contributes to the growing retina. Our analyses indicate that in Phreatichthys, the eyes are not maintained due to a failure of progenitor cells to terminally differentiate and thus to follow the continuous and stereotypic formation of mature retinal cell types. Failing to terminally differentiate, retinal progenitor cells are eliminated by a wave of progressive apoptosis that follows the RGC initiation as indicated by atoh7, one of the first differentiation factors expressed in the retina which marks retinal progenitor cells destined to become RGCs in their final mitosis (Kay et al., 2001; Mu et al., 2005). While the initial step of RGC differentiation is taken, markers for postmitotic, differentiated RGCs (isl1 and pou4f2, (Prasov and Glaser, 2012)) are only transiently expressed. Pou4f2 has been specifically attributed to the establishment of axonal projections from RGCs (Jain et al., 2012). Consistently, only few RGC axons form transiently and fail to project towards the optic tectum.

Our data point to a block in differentiation after the appearance of RGCs in the inner layer of the retina (Fig. 6). The retina develops normally at the onset, but subsequent cell types are not generated and thereby overall eye development arrests.

Fig. 6. Eye development of Phreatichthys andruzzii ceases during RGC differentiation.

Fig. 6

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Description of normal eye development in known fish models (A): The neuroretina is initially undifferentiated, but eventually differentiates from the centre to the periphery in a stereotypic fashion. RGCs are the first-born cells in a layer close to the lens, followed by six more cell types including the last-born Müller Glia cells. Each cell type will be located within its corresponding retinal layer, while the undifferentiated area becomes more restricted with time and eventually forms the CMZ, contributing to growth. By contrast, eye development in P. andruzzii (B) ceases while RGCs become specified and no further cell types and subsequent layering can be detected. Together with the onset of differentiation, apoptosis is initiated and spreads over the neuroretina until full eye degeneration. RGCs, retinal ganglion cells; INL, inner nuclear layer; IPL, inner plexiform layer; OPL, outer plexiform layer; ONL, outer nuclear layer.

Eye regression is more severe in Phreatichthys compared to other cavefish models (Berti et al., 2001; Jeffery, 2009; Meng et al., 2013; Wilkens, 2007), correlating well with the longer separation from its surface ancestors (ca. 2 mya). However, there is no correlation between the time of separation and the developmental module tagged by different cave species as indicated by an early module targeted in Astyanax (young cavefish) while a late differentiation module was affected in ancient cavefish Phreatichthys.

These scenarios are underlined by the fact that atoh7 is normally expressed only transiently in zebrafish and is not found in mature retinal ganglion cells. The role of early Atoh7 expression in zebrafish is to confer on the progenitors, the competence to differentiate towards RGCs. The late expression of atoh7 in Phreatichthys and the absence of later cell types support the hypothesis that RGCs do not differentiate properly and the remaining progenitors are not competent to give rise to other cell types.

Strikingly, none of the cell types that follow on RGCs in the birth order in the vertebrate retina could be detected. Rather than observing a secondary wave of differentiation with markers for the inner nuclear layer, barhl2 (amacrine cells, (Mo et al., 2004; Schuhmacher et al., 2011) and vsx1 (bipolar cells, (Passini et al., 1997)), we revealed a wave of apoptosis following the initiation of RGC differentiation. This is in striking contrast to the situation in Astyanax (Alunni et al., 2007) where marker gene expression indicates a terminal differentiation of different retinal neurons.

It had been suggested that a key player in cavefish eye degeneration is apoptosis (Berti et al., 2001; Jeffery and Martasian, 1998). We show that in Phreatichthys, Caspase3 activity is high in the entire head during early stages. While there was some variability early on, we consistently observed increased apoptosis levels in the retina from 35 hpf onwards.

In contrast to Astyanax, where cell death was first detected in the lens and subsequently in autolytic rosettes in the retina, we could not observe enhanced apoptosis in the lens. It cannot be excluded that the shrinking lens is affecting the retina. Conversely the failure to differentiate the retinal cell types could also impact on the maintenance of the lens. Eye degeneration can also occur lens independent, like in the Chinese cavefish Sinocyclocheilus (Meng et al., 2013).

We hypothesize that in response to the de-regulated differentiation of RPCs and subsequent cell types, retinal apoptosis is initiated to protect the structure against over-proliferation of aberrant cells. Thus, we speculate that a simple differentiation block building on intrinsic control mechanisms elegantly eliminates the Phreatichthys retina.

Conclusion

Our studies in Phreatichthys shed light on an evolutionary strategy to eradicate an organ by building on endogenous check points for differentiation control and their measures to eliminate cells that fail to differentiate according to plan. It is striking to note that evolution has apparently followed different strategies in different species to eventually eliminate an organ. By exploiting these different evolutionary strategies that have eradicated the eye, insight gained from the study of additional cavefish species has the exciting potential to identify the normal developmental building blocks that establish and maintain the retina.

Materials and Methods

Fixation of embryos

Embryos were raised at 28°C as previously described (Berti et al., 2001) and fixed at different stages with 4% paraformaldehyde (PFA, pH7.2) in 1x PTW (Phosphate-buffered saline, PBS, 0.1% Tween20, pH7.3) for 24 hours at 4°C. Afterwards, they were transferred to 100% methanol and stored at -20°C. The animal handling procedures and research protocols were approved by the Institutional Animal Care and Use Committee of the University of Ferrara (Italy) and by the Italian Ministry of Health. All fish housing and care were conformed to the Directive 2010/63/UE on the protection of animals used for scientific purposes.

Cloning of partial cDNAs

Reactions were performed with Quiagen Hot Star Taq PCR Kit. Each reaction of 50µl contained 1x reaction buffer, 1x Q-Solution, 200 µM of dATP, dTTP, dGTP and dCTP, 0.5 µM of each primer, 100 µM MgCl2, 1x DMSO, 50 ng cDNA/genomic DNA and 0.02 U/µl Hot Star Taq DNA Polymerase. The PCR was run under the following cycling conditions: Initial heat activation at 95°C for 15 minutes, ten cycles of 95°C for 30 seconds, 45°C 1 minute +0.5°C/cycle, 72°C for 1 minutes, ten cycles of 95°C for 30 seconds, 52°C for 1 minutes, 72°C for 1 minutes, ten cycles of 95°C for 30 seconds, 55°C for 1 minutes +0.5°C/cycle, 72°C for 1 minute, five cycles of 95°C for 30 seconds, 62°C for 1 minute, 72°C for 1 minutes, final extension of 10 minutes at 72°C and cooling to 4°C for 5 minutes. PCR products were cloned into pGemTeasy vector (Promega) following the instructions of the suppliers. The validity of the respective cDNAs was confirmed by sequence analysis. See supplementary material for primer sequences (Table S1). Sequences were submitted to GenBank (NCBI): PA_atoh7 (KJ867022), PA_barhl2 (KJ867023), PA_pou4f2 (KJ867024), PA_pax6 (KJ867025), PA_rx2 (KJ867026), PA_rx3 (KJ867027), PA_shh (KJ867028), PA_six3a (KJ867029), PA_sox2 (KJ867030), PA_nr2e1 (KJ867031), PA_vsx1 (KJ867032), PA_isl1 (KJ867033), PA_crx (KJ867034), PA_opsin (KJ867035), PA_rhodopsin (KJ867036), PA_fgf8 (KP125521), PA_nkx2.1 (KP125522), PA_pax2.1 (KP125523).

Generation of DIG labeled ribonucleotide-probes

For the generation of DIG labeled ribonucleotide-probes, plasmid DNA containing the respective inserts were linearized and transcribed with SP6 or T7 polymerase in the presence of DIG labeled dUTP according to the instructions of the manufacturer (Roche).

Whole mount in situ hybridization

In situ hybridization (ISH) protocols are based on a protocol for single color ISH in zebrafish (Thisse and Thisse, 2008). ISH were stained with 4-nitroblue tetrazolium chloride (NBT) and 5-Bromo-4-chloro-3-indolylphosphate (BCIP) from Roche.

Vibratome sectioning and imaging

Embryos were embedded in gelatin albumin (PBS containing 0.5% gelatin, 30% albumin from Bovine serum Cohn fraction 5, Sigma) and solidified with 105 µl Glutaraldehyde. 30 µm transversal sections were cut with a VT1000 S Vibrating Blade Microtome (Leica) and mounted on microscope slides with Mowiol (1 g/ml Glycerin, 0.4 g/ml Mowiol 4-88 (Carl Roth), 0.4 M Tris-HCl pH8.5, 25 mg/ml DABCO). Embryos of stages 10 hpf and 19 hpf were dissected from the yolk and mounted in 50% Glycerol/PTW on slides.

Cryosectioning and immunohistochemistry

After overnight fixation at 4°C, embryos were washed 5x5 minutes in PTW and then permeabilized with 100% acetone for 10minutes at -20°C. Embryos were transferred back to PTW, washed and cryoprotected with 30% Sucrose/1x PTW for 24 h at 4°C. Embryos were mounted in polyethylene embedding molds (Polyscience) and sucrose was removed completely. The specimens were embedded with Tissue Tek® O.C.T.™ medium (Sakura). 16-30 µm sections were cut on a Cryostat CM3050S (Leica) and mounted on SuperFrost® Plus Slides (Thermo Scientific).

After at least 2 hours of drying at RT, sections were rehydrated with PTW and blocked with 10% serum blocking buffer (1x BSA, 0.8% Triton-X100, 10% sheep serum) for 1h on a shaker at RT. Blocking buffer was removed and slides were washed 4x 5 minutes with PTW. Primary antibodies were diluted in 1% serum buffer (BSA, 0.8% Triton-X100, 1% sheep serum) and pipetted onto the sections, which were then covered with parafilm and stored in a closed humid box overnight at 4°C. The slides were washed 4x 30 minutes with PTW and then incubated with the secondary antibody diluted in 1% serum buffer overnight at 4°C. For Caspase3 staining, monoclonal anti-rabbit activated Caspase3 antibody (Abcam) was used diluted 1:300. Anti-mouse PCNA antibody (Millipore) was diluted 1:100. Secondary antibodies were Alexa 488 anti-mouse and Alexa 594 anti-rabbit. After antibody staining, slides were incubated with PTW containing 1:1000 DAPI (stock concentration 500 µg/ml) 15 minutes at RT. Afterwards, they were washed with PTW, mounted with 50% glycerol and covered with a coverslip.

Whole mount immunohistochemistry

Embryos were stored in 100% methanol and rehydrated to PTW in successive washings. Embryos were blocked with 10% serum blocking buffer (1x BSA, 0.8% Triton-X100, 10% sheep serum) in 2 ml reaction tubes for 1.5 hours on a shaker at RT. Blocking buffer was removed and embryos were washed 4 x 5 minutes with PTW. Embryos were then transferred to 0.5 ml reaction tubes and 300 µl of antibody solution was added. Primary antibodies were diluted in 1% serum buffer (BSA, 0.8% Triton-X100, 1% sheep serum) and tubes were incubated on a rotator at 4°C over night. After incubation, embryos were washed 4x 30 minutes with PTW in 2 ml tubes and then incubated with the secondary antibody diluted in 1% serum buffer overnight at 4°C in 0.5ml tubes. For Caspase3 staining, monoclonal anti-rabbit activated Caspase3 antibody (Abcam) specific for apoptotic cells was used diluted 1:300. Monoclonal anti-mouse acetylated tubulin antibody was diluted 1:500. Secondary antibodies were Alexa 488 anti-mouse and Alexa 594 anti-rabbit. DAPI was added together with the secondary antibodies, diluted 1:500. Subsequently, embryos were washed with PTW, mounted laterally with 1% low melting agarose in a glass bottom dish and imaged by confocal microscopy.

Whole mount TUNEL staining

Embryos were fixed, stored and proteinase K treated according to the in situ hybridization protocol. TUNEL assay was carried out with In Situ Cell Death Detection Kit, AP (Roche) according to the manufacturer’s instructions.

Imaging

Fluorescent samples were imaged using a Leica SPE confocal microscope, 20x water immersion objective and Leica Application Suite (LAS) software. Alexa488 was excited at 488nm by Argon laser, Alexa594 by the 543nm Helium Neon laser, DAPI by an UV laser at 405nm. Emission was sensed at 500-550nm for Alexa488, 650-700nm for Alexa594, 410-500nm for DAPI. In situ hybridization and TUNEL stained samples were imaged with an Axio Imager M1 (Zeiss), differential interference contrast (DIC) channel and 10x or 20x objective, Zeiss AxioCam camera and AxioVision software. Pictures were processed using Adobe Photoshop CS4 and Adobe Illustrator CS4.

Supplementary Material

Supplementary Information

Acknowledgements

We acknowledge the entire Wittbrodt department for critical discussion during the project and comments on the manuscript. We thank Elena Frigato for the help in the animal care and sampling. M.S. is a member of the HBIGS graduate school for life sciences at Heidelberg University. The project was supported by the German Research Foundation (DFG, J.W.) and the European Research Council (ERC, J.W.).

Footnotes

Author Contributions

L.-N. S. and J.W. initiated the project. L.N.S, M.S. and J.W. planned the experiments in collaboration with C.B. and N.S.F.. L.-N. S. and M.S. performed all experiments and analyzed the data together with J.W.. Staged Phreatichthys embryos were provided by C.B.. M.S., L.-N. S. and J.W. wrote the manuscript with contributions from C.B. and N.S.F..

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Supplementary Materials

Supplementary Information
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