In order to preserve the spatial coordinates of visual input, retinal ganglion cell (RGC) axons are topographically organized in the visual processing centres of the midbrain. Retinotopic mapping has been most extensively studied in the optic tectum of fish, amphibians, and chick, and in the superior colliculus of mice. Within both the retina and the tectum, axially restricted expression of the Eph and Ephrin families of axon guidance molecules provides some of the positional information required for retinotectal map formation. Interactions between Eph receptor tyrosine kinases and their cognate Ephrin ligands result in cytoskeletal rearrangements and changes in cell adhesion, thereby eliciting either repulsive or attractive responses. By interpreting the molecular Eph and Ephrin code, RGC axons form a precisely ordered arrangement within the optic tectum that accurately reflects their axial position within the retina [1, 2].
Axial patterning of the retina is required to establish the correct domains of Eph and Ephrin expression. During eye development, retinal patterning occurs along both the dorsal-ventral (DV) and nasal-temporal (NT) axes [3, 4]. The DV axis is established through an antagonistic relationship between the Bone morphogenetic protein (Bmp) and Hedgehog signalling pathways. In the dorsal retina, Smad-dependent Bmp/Growth differentiation factor (Gdf) signalling initiates expression of the dorsal-specific T-box transcription factors tbx5 and tbx2b, which in turn activate ephrinB expression [5–8]. Additionally, Wnt signalling is required to maintain dorsal identity [9, 10]. In the ventral retina, Hedgehog signals from the ventral midline induce the expression of Vax homeodomain transcription factors [11–13], thereby establishing ventral ephB expression [14–17]. Restricted ephrinB and ephB expression along the DV axis is required for normal formation of the retinotectal map [18, 19].
The NT axis is defined by the restricted expression of forkhead transcription factors foxG1 (bf1) and foxD1 (bf2) in the nasal and temporal retina, respectively [20–22]. These factors function antagonistically to promote the expression of ephrinA ligands in the nasal retina and a subset of ephA receptors in the temporal domain. Altering the normal domains of ephrinA and ephA expression causes defects in retinotectal map formation [23–25]. Similarly to the DV axis, secreted signalling proteins are also involved in NT patterning. Fibroblast growth factor (Fgf) signals from the telencephalon and periocular mesenchyme promote nasal (ephrinA) and repress temporal fates (ephA) [26–28]. At this time, it is not clear whether temporal identity represents a retinal ground state or if it is induced by an unidentified factor.
Axial patterning of the tectum/superior colliculus is also a critical component of proper retinotectal map formation. In the midbrain, eph and ephrin genes are expressed in opposing gradients. EphrinA ligands are expressed in a posterior to anterior gradient, while EphA receptors are expressed in an opposing anterior to posterior gradient . Likewise, along the medial-lateral axis, EphrinB ligands tend to be expressed in a medial to lateral gradient while EphB receptors exhibit an opposing lateral to medial gradient . These opposing gradients, together with the repulsive interactions between Eph-Ephrin molecules, suggested a gradient matching model of retinotectal map formation . This model is supported by experiments showing that, for example, EphA3-expressing temporal RGCs tend not to innervate posterior regions of the tectum expressing high levels of EphrinA ligands [31, 32]. However, this model does not explain all facets of retinotectal map formation, and other factors such as attractive Eph-Ephrin interactions, axon competition , and other molecular cues may refine the process . It is clear, however, that the precise topographic mapping of RGCs onto the tectum/superior colliculus is a highly regulated process in which Eph and Ephrin interactions play a key role.
Eph and Ephrin interactions have been well studied in the hindbrain, where they are involved in cell sorting and restricting cell movements between rhombomeres [34–36]. Of particular importance in regulating hindbrain eph and ephrin expression are the TALE-class homeodomain transcription factors Meis/Pknox and Pbx, which act in trimeric complexes with Hox proteins to impart segmental identity to the hindbrain rhombomeres [37–41]. However, Pbx and Meis also perform Hox-independent roles in eye, lens, midbrain, heart and muscle development [42–46].
Meis1 is a particularly attractive candidate for playing an important role in patterning the visual system. meis1 expression in the developing eye and midbrain is conserved across multiple species, and Meis1-deficiency causes microphthalmia in mice, chickens and zebrafish [47–49]. The Drosophila Meis homolog Homothorax (Hth) also plays an important role in insect eye development [50, 51]. Structurally, Meis proteins contain a Pbx-interaction domain in the amino terminus, a DNA-binding homeodomain and a carboxy-terminal activation domain . In addition to the trimeric Meis-Pbx-Hox complexes that regulate hindbrain patterning, Meis proteins can form heterodimeric complexes with Pbx and with a subset of posterior Hox proteins [53, 54]. Meis and its binding partners have been identified as important regulators of eph and ephrin gene expression in the midbrain and hindbrain through both direct and indirect mechanisms [37, 44, 55–59]. However, despite this well-characterized role in hindbrain axial patterning and the regulation of eph and ephrin gene expression, the function of Meis1 in axial patterning of the retina and in the formation of the retinotectal map has not been fully addressed.
In this study, we use morpholino-mediated knockdown of Meis1 protein in zebrafish to determine if Meis1 patterns the retinotectal system. In the DV axis, Meis1 promotes ocular Bmp signalling through the positive regulation of smad1 expression and the negative regulation of follistatin a (fsta). With regard to NT patterning, Meis1 knockdown causes a loss of temporal identity in the retina. This phenotype can be attributed to an increase in retinal Fgf signalling and a decrease in foxd1 expression in the temporal retina. We also demonstrate that Meis1 positively regulates ephrin gene expression in the tectum. Consistent with these patterning defects, Meis1-depleted embryos also exhibit retinotectal mapping defects in both the NT and DV axes. We conclude that Meis1 contributes to retinotectal map formation by specifying positional information in both the retina and tectum.