Our findings demonstrate that Sall3 plays a crucial role in regulating the development of both cone photoreceptors and horizontal interneurons. Our model of Sall3 action is summarized in . Sall3 is necessary for the expression of a range of cone-specific genes, with only a very small number of Sop-positive and Arr3-positive cells detectable in Sall3−/− retinas. Microarray experiments revealed that many other cone-enriched transcripts are selectively downregulated in Sall3−/− retinas, including Pde6c, Pde6h and Crb1, whereas some, such as Gnat2 and Gnb3, show little or no change. Strikingly, overexpression of Sall3 by electroporation was sufficient to induce the expression of both Sop and Arr3, not only in photoreceptors but also in a subset of cells in the INL, without affecting expression of Mop. Rod photoreceptors overexpressing Sall3 in electroporated retinas were
morphologically indistinguishable from those of controls. These rod photoreceptors that overexpressed Sall3 retained their normal morphology, laminar position and pattern of expression of cell-specific markers.
Fig. 11. Role of Sall3 in horizontal cell and S-cone photoreceptor development. (A) Sall3 and Lhx1 expression is activated in postmitotic horizontal cell precursors, which express Ptf1a and Prox1. Expression of Sall3 maintains Lhx1 expression in differentiating (more ...)
These data imply that Sall3 is activated in Sop-expressing photoreceptors undergoing terminal differentiation, and that Sall3 itself might directly activate a subset of cone-specific genes in a coordinated manner, analogous to the orphan nuclear hormone receptor Errβ (Esrrb) in rod photoreceptors (Onishi et al., 2010b
). Several different nuclear hormone receptors, along with the transcriptional co-regulator Pias3, have been shown to be required for medium wavelength-sensitive cones to activate the expression of Mop while simultaneously repressing the expression of Sop. Sall3, however, represents the first cone-expressed transcription factor that selectively actives the expression of Sop.
The strong activation of S-cone-specific transcripts by Sall3 implies a surprising functional homology with the Spalt gene complex of Drosophila
in the regulation of photoreceptor differentiation. In Drosophila
, Spalt genes are necessary for specification of the inner R7 and R8 photoreceptors, which are responsible for color discrimination and in many respects form a cone-like photoreceptor class in the compound eye. Strikingly, loss of function of Spalt genes results in the ectopic expression of Rh1 opsin (NinaE), which is normally expressed in the outer R1-R6 photoreceptors, in the R7 and R8 cells, whereas expression of Rh3-Rh6 is lost (Domingos et al., 2004
; Mollereau et al., 2001
). However, axonal projections of inner photoreceptors are unaltered in Drosophila sal
mutants. This partial shift in photoreceptor identity resembles that seen following Sall3 overexpression in rod photoreceptors. These cells appear morphologically normal and continue to express rhodopsin, but now robustly express cone-specific genes. Notably, Spalt genes are selectively expressed in blue-sensitive Rh5-positive R8 photoreceptors and are required for expression of Rh5 opsin (Sprecher et al., 2007
In mice, we observe that Sall3 is both necessary and sufficient for the expression of blue-sensitive cone opsin but not green-sensitive cone opsin. Such a direct conservation of gene function in photoreceptor development is unusual, and even more surprising because the blue-sensitive visual opsins of insects and vertebrates evolved independently (Shichida and Matsuyama, 2009
). Although this observation might represent evolutionary convergence, it could alternatively imply that ancestral bilateria possessed a dedicated short wavelength-sensitive photoreceptor cell type, the differentiation of which was guided by a Spalt family gene, with the blue-sensitive opsin gene expressed by this cell having changed in different lineages; photoreceptors might, at one point, have coexpressed both blue-sensitive ciliary and rhabdomeric opsins. This possibility is not without precedent, as both vertebrate and invertebrate photoreceptors are known to coexpress different opsin genes with similar spectral sensitivities (Mazzoni et al., 2008
; Porter et al., 2009
). In some cases, opsin genes with similar spectral sensitivity but which are nonetheless highly divergent at the molecular level are coexpressed, such as the blue-sensitive melanopsin and retinal cone opsins of the chick retina (Bailey and Cassone, 2005
; Bellingham et al., 2006
). Analysis of Spalt family gene expression in invertebrates from multiple phyla with well-characterized color vision should further clarify this finding.
Sall3 also plays a pivotal role in regulating the differentiation of horizontal cells, one of two cell types directly postsynaptic to cone photoreceptors, and might do so in part through maintenance of Lhx1 expression. We did not observe defects in the expression of other previously reported horizontal cell-expressed transcription factors in Sall3−/−
retinas, including Pax6, Foxn4 and Ptf1a, although cell counts did reveal a reduction in the number of Prox1-positive cells in the dorsal retina at P0. However, the final laminar position of Sall3−/−
horizontal cells closely resembles that seen in Lhx1
mutants. Horizontal cells of Chx10-Cre; Lhx1lox/lox
mutants fail to undergo initial outward radial migration, resulting in ectopic localization to the inner INL and extension of their dendritic arbor within the IPL (Poche et al., 2007
; Poche et al., 2008
). Horizontal cells of Sall3−/−
retinas appear to initiate Lhx1 expression and undergo outward radial migration normally. However, by P0, Lhx1 expression is drastically reduced, and at later ages the majority of horizontal cell bodies are found at the scleral border of the IPL, eventually taking up residence with amacrine cells and extending dendrites into the IPL, phenocopying the Lhx1
mutants. The phenomenon of ectopic inner nuclear/plexiform layer-associated horizontal cells was also seen in Sall3 overexpression experiments in which Sall3 appeared to be sufficient to at least partially specify horizontal cells, including activating Prox1 and NF165. The lack of coexpression with markers specific to AII amacrine cells indicated that the Prox1-expressing wide-field cells generated in Sall3 electroporation experiments were not a dysmorphic AII population. This does not exclude the possibility that Sall3 overexpression results in the generation of a rare Sall3+
wide-field amacrine subclass. Notably, Spalt genes also regulate prospero
expression in developing Drosophila
photoreceptors, which then acts to guide differentiation of the R7 photoreceptor subtype (Cook et al., 2003
; Domingos et al., 2004
). Overexpression of Sall3 in neonatal retinas was not sufficient to induce expression of Lhx1, and, consequently, the horizontal-like cells demonstrated the same ectopic positioning seen in Lhx1
The crucial role of Sall3 in regulating Lhx1 expression is further underlined by our microarray analysis, which indicates that Lhx1 is one of the most strongly downregulated genes in Sall3−/− retinas. Taken together, these data suggest that sustained Lhx1 expression might regulate multiple stages of horizontal cell migration, and that Sall3 is necessary to maintain Lhx1 expression during the postnatal differentiation of horizontal cells. A limited number of Sall3+ NF165+ and Sall3+ Prox1+ horizontal cells are present in Six3-Cre; Lhx1lox/lox retinas, implying that Sall3 regulates aspects of horizontal cell development at least in part through an Lhx1-independent pathway and that expression of Sall3 itself might not require Lhx1.
A subset of bipolar interneurons selectively expressed Sall3. Dedicated S-cone-selective bipolar cells in the mouse retina that are analogous to primate S-cone-selective midget bipolar cells have been identified (Haverkamp et al., 2005
). The tantalizing possibility exists that Sall3 might regulate the differentiation of S-cones and their dedicated bipolar interneuron. However, the microarray analysis did not reveal any significant changes in known mouse bipolar-expressed genes, and the role of Sall3 in bipolar interneuron development remains to be resolved.