micrornas in retinal development

VS04CH02_Hindges ARI 14 August 2018 11:4

Annual Review of Vision Science

MicroRNAs in RetinalDevelopmentThomas A. Reh1 and Robert Hindges2

1Department of Biological Structure, University of Washington, Seattle, Washington 98195,USA; email: [email protected] for Developmental Neurobiology, MRC Centre for Neurodevelopmental Disorders,King’s College London, London SE1 1UL, United Kingdom; email: [email protected]

Annu. Rev. Vis. Sci. 2018. 4:25–44

First published as a Review in Advance onJune 11, 2018

The Annual Review of Vision Science is online atvision.annualreviews.org

https://doi.org/10.1146/annurev-vision-091517-034357

Copyright c© 2018 by Annual Reviews.All rights reserved

Keywords

retina, microRNA, Dicer, development, retinal cells, cell differentiation,axon guidance

Abstract

The small RNA regulatory molecules called microRNAs (miRNAs) play keyroles in the development of most organisms. The expression of many dif-ferent miRNAs has been described in the developing and mature vertebrateretina. The ability of miRNAs to regulate a diversity of messenger RNA tar-gets allows them to have effects on many different developmental processes,but the functions of only a few miRNAs have been documented to date. De-velopmental transitions between cell states appear to be particularly sensitiveto miRNA loss of function, as evidenced by specific miRNA knockdowns orfrom global perturbations in miRNA levels (e.g., Dicer deletion). However,we are still in only the very early stages of understanding the range of cellularfunctions miRNAs control during development.

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https://www.annualreviews.org/doi/full/10.1146/annurev-vision-091517-034357


1. BACKGROUND

MicroRNAs (miRNAs) are small noncoding RNA molecules that control gene expression throughposttranscriptional regulation. They recognize complementary sequences on messenger RNAmolecules (mRNAs), which then leads to either (a) destabilization of the mRNA and accelerationof its destruction or (b) interference with the mRNA translation machinery (Bartel 2004). MostmiRNAs are transcribed by RNA polymerase II to generate primary miRNAs, or pri-miRNAs,which may contain multiple miRNAs (Figure 1). These long transcripts are then processed while

Cap AAAAAA

Pri-miRNA

Protein-coding gene

Cap AAAAA

Splicing

mRNA

Drosha

DGCR8

Editing

Cap AAAA

Translational repression

mRNA degradation

DicerTRBP

AgoRISC

x x

x

Cytoplasm

NucleusmiRNA gene

RNA Pol II

Pre-miRNA

Exportin-5

miRNA*degradation

Mature mRNA

Exon 2

Exon 1 Exon 2

Exon 1

Figure 1Biogenesis of microRNAs (miRNAs). Canonical pathway (left): miRNA genes are transcribed by RNApolymerase II (Pol II) to give rise to long primary transcripts [primary miRNAs (pri-miRNAs)], which formhairpin structures. This step is subject to regulation by transcriptional activators or inhibitors (not shown).Still in the nucleus, pri-miRNAs are then processed by a protein complex containing the ribonuclease IIIDrosha and DGCR8, resulting in a 60–70-nucleotide stem/loop structure [precursor miRNAs(pre-miRNAs)]. Furthermore, in some cases, pre-miRNA sequences can be subject to base modifications byspecific RNA editing. The pre-miRNAs are then exported to the cytosol with the help of Exportin-5through a GTP-dependent process. There, the pre-miRNAs are further processed by the Dicer-TRBPcomplex to yield a 21–24-nucleotide-long miRNA:miRNA∗ duplex molecule. Finally, after destruction ofone strand [most of the time, the passenger strand, denoted with an asterisk (∗)], mature miRNAs are loadedinto an RNA-induced silencing complex (RISC) that includes Argonaute (Ago). The miRNA then binds tothe 3′ untranslated regions of specific messenger RNA (mRNA) targets and, depending on its seed sequence,leads either to translational repression or to the cleavage and degradation of the target. Mirtron pathway(right): Pre-miRNAs can also be generated through an alternative, Drosha-independent pathway. Here,miRNA sequences are located in introns of primary mRNA transcripts generated by RNA Pol II. Theirexpression pattern is therefore exactly following the mirtron-harboring gene. The miRNA sequence issubsequently spliced out from the primary transcript by the spliceosome and then enters the canonicalpathway. Additional abbreviation: DGCR8, DiGeorge syndrome critical region 8.

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still in the nucleus by the DiGeorge syndrome critical region 8 (DGCR8) protein and the ribonu-clease Drosha to form shorter, about 70-nucleotide-long precursor miRNAs (pre-miRNAs) thatare subsequently exported to the cytoplasm by Exportin-5. The pre-miRNAs are further reducedto 21–23-nucleotide double-stranded mature miRNAs by the endoribonuclease Dicer, complexedto TRBP. The mature miRNA becomes part of the RNA-induced silencing complex (RISC), con-taining Argonaute (Ago) as a key component, to effect gene silencing of mRNA targets throughbinding to complementary sequences in their 3′ untranslated region (UTR) (Figure 1). In addi-tion to this canonical pathway described, some alternative mechanisms to generate miRNAs exist,bypassing, for example, Drosha or Dicer processing (Ha & Kim 2014).

Although initially considered primarily as translational repressors (Olsen & Ambros 1999),miRNAs were also shown to cause mRNA degradation (Bagga et al. 2005, Lim et al. 2005), andmore recent evidence indicates that in mammalian cells, mRNA degradation accounts for mostof the repression mediated by miRNA, with a smaller contribution from translation repression(Eichhorn et al. 2014). A given miRNA recognizes its targets by base pairing via a short sequence,known as the seed sequence (6–8 nucleotides), at the 5′ end. Early target prediction programswere largely based on the complementation of this short seed region with a potential targetmRNA. These target prediction databases typically report thousands of targets for a particularmiRNA. The vast majority of these predicted targets have not been confirmed by biological andbiochemical experiments, and many may not be actual targets; however, they serve as a source ofpotential targets for confirmatory studies.

To experimentally determine mRNA targets, researchers can use antibodies to Ago to precipi-tate the complex with miRNAs and their targets [CLIP (crosslinking and immunoprecipitation)],and this has provided additional insight into the mechanisms of miRNA-mediated gene silenc-ing (Chi et al. 2009). Directly sequencing the miRNA:mRNA complexes, using, for example,AGO-CLIP, PAR (photoactivatable ribonucleoside–enhanced)-CLIP, CLEAR (covalent ligationof endogenous Ago-bound RNAs)-CLIP, and CLASH (crosslinking, ligation, and sequencing ofhybrids), has shown that most mRNAs (a) are typically paired with two or more miRNAs, makingcombinatorial regulation likely, and (b) are frequently paired in a noncanonical manner, suggest-ing that the potential targets available for an miRNA may be substantially different than thoseidentified by seed matches (Chi et al. 2009, Hafner et al. 2010, Helwak et al. 2013, Moore et al.2015). For example, one AGO-CLIP study found 7-mer and 8-mer seeds in only about half theAgo-bound sites.

There are several levels of redundancy in miRNA:target regulation. First, each miRNA cantarget many mRNAs. Second, many mRNAs can be targeted by several miRNAs. Third, miRNAsare frequently in families of related molecules, and so several family members can target the samesite on an mRNA. These considerations make it sometimes difficult to discern the roles of spe-cific miRNAs in a given biological process. Knockout studies that delete all members of a givenmiRNA family are rare, though miRNA sponges (see Section 3) can be used to reduce several fam-ily members simultaneously. However, since many mRNAs are best targeted by a combinationof different miRNAs, loss-of-function studies typically require antagonism of multiple miRNAfamily members of multiple miRNA families. Focused overexpression studies of miRNAs are alsocomplicated by the diversity of targets and the additional effects on several biological processesthat are outside the focus of the specific study. These considerations have made making progressin the field more difficult than doing so in the analysis of protein-coding genes, which is a morewell-established discipline; nevertheless, significant advancement has been made in the identifi-cation of miRNAs expressed in the retina during development, in mature retina, and in retinaldisease.

www.annualreviews.org • MicroRNAs in Retinal Development 27

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2. CHARACTERIZING miRNA EXPRESSION IN DEVELOPING RETINA

Several studies have characterized miRNA expression in the developing retina in a variety ofspecies, including fish, frogs, and mice, using a variety of methods, such as microarrays, reversetranscription polymerase chain reaction (RT-PCR), in situ hybridization, NanoString technology,and miRNA sensors (Arora et al. 2010, Damiani et al. 2008, Decembrini et al. 2009, Deo et al.2006, Georgi & Reh 2010, Hackler et al. 2010, Karali et al. 2010, La Torre et al. 2013, Wohl & Reh2016, Xu et al. 2007). Although there are many differences among species and across the methodsof analyses, in the particular miRNAs that have been reported, many of the most highly expressedmiRNAs are common to both the diverse species and the particular methods used. For example, ina microarray study of the developing frog retina, the most highly expressed miRNAs include let-7c, miR-183, and miR-125b, and these same miRNAs are highly expressed in developing mouseretina as well (Decembrini et al. 2009, Georgi & Reh 2010, Hackler et al. 2010, La Torre et al.2013). Table 1 lists some of the most commonly reported miRNAs found in developing retina.

Table 1 Highly expressed microRNAs in developing retina

MicroRNA References

hsa-miR-18a Georgi & Reh (2010), Hackler et al. (2010)

mmu-let-7a Georgi & Reh (2010), Hackler et al. (2010), La Torre et al. (2013), Shen et al. (2008), Wohl & Reh (2016),Xu et al. (2007)

mmu-let-7c Georgi & Reh (2010), Hackler et al. (2010), La Torre et al. (2013), Shen et al. (2008), Wohl & Reh (2016),Xu et al. (2007)

mmu-let-7e Hackler et al. (2010), Georgi & Reh (2010), Loscher et al. (2007), Shen et al. (2008), Wohl & Reh (2016),Xu et al. (2007)

mmu-let-7f Georgi & Reh (2010), Hackler et al. (2010), La Torre et al. (2013), Shen et al. (2008), Xu et al. (2007)

mmu-miR-101a Georgi & Reh (2010), Shen et al. (2008)

mmu-miR-106b Georgi & Reh (2010), La Torre et al. (2013), Loscher et al. (2007), Shen et al. (2008), Xu et al. (2007)

mmu-miR-124 Arora et al. (2007), Georgi & Reh (2010), Hackler et al. (2010), Kapsimali et al. (2007), Karali et al. (2007), LaTorre et al. (2013), Loscher et al. (2007), Makarev et al. (2006), Ryan et al. (2006), Shen et al. (2008), Wohl& Reh (2016), Xu et al. (2007)

mmu-miR-125a-5p Georgi & Reh (2010), Hackler et al. (2010), Loscher et al. (2007), Shen et al. (2008), Wohl & Reh (2016), Xuet al. (2007)

mmu-miR-125b Georgi & Reh (2010), Hackler et al. (2010), Kapsimali et al. (2007), La Torre et al. (2013), Loscher et al.(2007), Makarev et al. (2006), Ryan et al. (2006), Xu et al. (2007), Shen et al. (2008), Wohl & Reh (2016)

mmu-miR-136 Georgi & Reh (2010), Hackler et al. (2010), Loscher et al. (2007)

mmu-miR-16 Georgi & Reh (2010), Hackler et al. (2010), La Torre et al. (2013), Shen et al. (2008), Wohl & Reh (2016), Xuet al. (2007)

mmu-miR-17 Georgi & Reh (2010), Hackler et al. (2010), Shen et al. (2008)

mmu-miR-182 Georgi & Reh (2010), Hackler et al. (2010), Jin et al. (2009), Kapsimali et al. (2007), Karali et al. (2007), LaTorre et al. (2013), Loscher et al. (2007), Loscher et al. (2008), Ryan et al. (2006), Shen et al. (2008), Wohl &Reh (2016), Xu et al. (2007)

mmu-miR-183 Georgi & Reh (2010), Hackler et al. (2010), Jin et al. (2009), Kapsimali et al. (2007), La Torre et al. (2013),Loscher et al. (2007), Loscher et al. (2008), Ryan et al. (2006), Shen et al. (2008), Wohl & Reh (2016), Xuet al. (2007)

mmu-miR-183∗ Georgi & Reh (2010), Hackler et al. (2010)

mmu-miR-191 Georgi & Reh (2010), Hackler et al. (2010), Loscher et al. (2007), Shen et al. (2008), Xu et al. (2007)

(Continued)

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Table 1 (Continued)

MicroRNA References

mmu-miR-200b∗ Georgi & Reh (2010), Hackler et al. (2010)

mmu-miR-26a Georgi & Reh (2010), Hackler et al. (2010), Ryan et al. (2006), Shen et al. (2008), Shi et al. (2009), Xu et al.(2007)

mmu-miR-204 Georgi & Reh (2010), Hackler et al. (2010), Wohl & Reh (2016)

mmu-miR-26b Georgi & Reh (2010), Loscher et al. (2007), Shen et al. (2008), Xu et al. (2007)

mmu-miR-30a Hackler et al. (2010), Xu et al. (2007)

mmu-miR-690 Georgi & Reh (2010), Hackler et al. (2010)

mmu-miR-691 Georgi & Reh (2010), Hackler et al. (2010), Loscher et al. (2007)


mmu-miR-720 Georgi & Reh (2010), Wohl & Reh (2016)

mmu-miR-7a Arora et al. (2007), Georgi & Reh (2010), Hackler et al. (2010), Li & Carthew (2005), Xu et al. (2007)

mmu-miR-7b Arora et al. (2007), Georgi & Reh (2010), Li & Carthew (2005)

mmu-miR-9 Georgi & Reh (2010), Hackler et al. (2010), Kapsimali et al. (2007), Karali et al. (2007), La Torre et al. (2013),Shen et al. (2008), Wohl & Reh (2016), Xu et al. (2007)

mmu-miR-9∗ Georgi & Reh (2010), Hackler et al. (2010), Loscher et al. (2007), Shen et al. (2008), Xu et al. (2007)


mmu-miR-96 Georgi & Reh (2010), Hackler et al. (2010), Jin et al. (2009), Kapsimali et al. (2007), Loscher et al. (2007),Loscher et al. (2008), Shen et al. (2008), Wohl & Reh (2016), Xu et al. (2007)

mmu-miR-99b Xu et al. (2007), Hackler et al. (2010), Georgi & Reh (2010), Wohl & Reh (2016)

∗Denotes passenger strand.

Most of these studies have assayed miRNAs from total retinal samples, and so the miRNAs thatare reported are likely expressed either in an abundant cell type, like photoreceptors or progenitorcells, or in multiple cell types. Some of the studies have documented differences between speciesthat might be real developmental differences or, alternatively, may reflect relative differences incell numbers. For example, mice have a relative abundance of photoreceptors compared withfrogs, and so the miRNAs from the photoreceptor-expressed miR-182/183/96 cluster are moreabundant in mouse retinas. Some of the most commonly reported miRNAs in developing retinaare also present in other regions of the developing central nervous system (CNS), like let-7 familymembers, miR-9, and miR-125, but there are many miRNAs specifically expressed in developingretina (Figure 2). In addition, in several studies, it has been noted that miRNAs derived from acommon genomic cluster tend to be coexpressed with similar developmental profiles. An exampleis the mouse miRNA cluster containing miR-106a, miR-130b, miR-17, miR-18a, miR-19a, miR-20a, miR-20b, and miR-93; these were all reported to be expressed in embryonic mouse retinaand coordinately regulated (Hackler et al. 2010).

Localization of miRNAs to specific cell types has been evaluated using several different ap-proaches. In situ hybridization has been carried out for some of the most abundant miRNAs andprovides cellular resolution (Deo et al. 2006, Xu et al. 2007). A complementary approach usingmiRNA sensors has also been made for several miRNAs and again provides cellular resolution(La Torre et al. 2013). Both approaches show miR-183 expression in photoreceptors, for exam-ple. Laser capture microdissection (LCM) and fluorescence-activated cell sorting (FACS) havealso been used to localize miRNA expression to specific cell types. LCM was used to localizemiR-204 to the inner nuclear layer (Hackler et al. 2010), and this finding was confirmed andrefined to show specific expression in the Muller glia by FACS and NanoString analysis (Wohl &


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miR-34amiR-132miR-29cmiR-29amiR-24miR-23bmiR-23amiR-22miR-29bmiR-27bmiR-129-3pmiR-27amiR-143miR-338-3pmiR-138miR-127miR-151-5pmiR-193let-7emiR-98let-7dlet-7clet-7blet-7amiR-136let-7imiR-124miR-26amiR-195miR-148amiR-140miR-181amiR-183*miR-183miR-182miR-96miR-181bmiR-210miR-204miR-452miR-713miR-33miR-181a-1*miR-542miR-292-5pmiR-744miR-689miR-290-5pmiR-15amiR-423-5pmiR-320miR-677miR-301amiR-130bmiR-374miR-32

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miRNAs commonto brain and retina

miRNAs specificto mature retina

miRNAs expressedin developing retina

mmu-miR-103

mmu-miR-106a

mmu-miR-106b

mmu-miR-130a

mmu-miR-130b

mmu-miR-135a

mmu-miR-146b

mmu-miR-153

mmu-miR-15b

mmu-miR-17

mmu-miR-18a

mmu-miR-19a

mmu-miR-19b

mmu-miR-207

mmu-miR-20a

mmu-miR-20b

mmu-miR-212

mmu-miR-25

mmu-miR-301a

mmu-miR-32

mmu-miR-489

mmu-miR-551b

mmu-miR-689

mmu-miR-700

mmu-miR-92b

mmu-miR-93

Figure 2(a) Comparison of brain and retinal miRNAs during development. Microarray study of the retina and brain to show common andunique microRNAs (miRNAs). Some miRNAs are present in neurons in both brain and retina and show increases from embryonictissues to adult neural tissues. Many members of the let-7 family fall into this class. However, there are also miRNAs that are specific toretina either in adults (e.g., miR-183) or during development (miR-130b). (b) Some of the key miRNAs in developing retina include themiR-106b–93–25 and miR-17–18a–19a/b–20a–92 clusters that regulate mitotic proliferation in many tissues. Violet indicates highexpression, while green indicates low levels of expression. Figure adapted from Hackler et al. (2010).

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Reh 2016). A further approach to localize miRNAs has relied on analysis of miRNA expressionafter changes in retinal cellular composition: Georgi & Reh (2010) used a Notch inhibitor toforce the differentiation of progenitor cells into neurons to identify progenitor-specific miRNAs,while Hackler et al. (2010) analyzed miRNAs in Nrl mutant mice to identify rod-specific miRNAs.The various approaches to localization of miRNAs have all provided information for some of themore abundant miRNAs, but for most of the miRNAs in the developing retina, we still do not knowexactly which cells express them. However, recent advances in single-cell transcriptomics shouldprovide a more complete picture of the miRNAs expressed in specific retinal cell populationsduring development.

3. DETERMINING THE FUNCTIONAL ROLE FOR miRNAsIN DEVELOPING RETINA: DICER DELETION

One approach to determine the role of miRNAs in retinal development has been to target theenzymes required for miRNA processing (Figure 1). The most popular target to date has beenDicer, since knockout or knockdown of this enzyme will prevent mature miRNAs from beingproduced. Several studies in mice, zebrafish, and Xenopus frogs have examined the effects of Dicerdeletion or knockdown in the developing retina (Akhtar et al. 2015, Damiani et al. 2008, Daviset al. 2011, Decembrini et al. 2009, Georgi & Reh 2010, Iida et al. 2011, La Torre et al. 2013,Maiorano & Hindges 2013, Pinter & Hindges 2010) (Table 2). Given the large numbers ofmiRNAs in the developing retina, diversity of cell types, and potentially very large number oftargets for each miRNA, it is surprising that the retina develops as well as it does without functionalDicer. However, several clear phenotypes have emerged from these studies, highlighting thosedevelopmental processes that are most sensitive to a reduction in miRNA levels.

Reduction in eye size and retinal cell number has been observed in nearly every study(Table 2). This is likely a consequence of increased cell death, as most studies have observeda higher rate of apopotosis. Several studies have specifically shown loss in retinal ganglion cells(RGCs), but other cell types also appear to be affected. A reduction in mitotic proliferation hasalso been noted in some studies, though this is variable and dependent on the specific line ofCre recombinase–expressing mice or knockdown strategy in other species. Other, more specificphenotypes have also been documented, particularly alterations in cell fate determination andaxon guidance of RGC axons, and these are discussed below. Deletion of Dicer in mature cellshas less dramatic immediate consequences; however, apoptosis is observed over a longer timecourse. Inhibition of miRNA production, specifically in photoreceptors with a rod-specific Dicerconditional deletion, leads to rod cell death beginning around 8 weeks postnatal and causing anear complete loss by 14 weeks (Sundermeier et al. 2014).

Although the increase in apoptosis observed after Dicer deletion may be due to relativelynonspecific dysregulation of gene expression, which can trigger cell death pathways, there isalso evidence for miRNAs being important regulators of apoptosis genes in the retina. Sanukiet al. (2011) generated a loss-of-function mouse mutant of miR-124a by targeted disruptionof Rncr3, the transcript that produces miR-124a. Rncr3−/− mice had smaller brains and otherabnormalities. In the retina, the cone photoreceptors were specifically affected and underwentcell death over several weeks. One target of miR-124a is Lhx2, and the increase in this gene inthe Rncr3 knockout mouse cones was implicated in the observed cell death. However, deletionof specific miRNAs can also directly elicit apoptosis, suggesting that loss of Dicer may lead toa loss in miRNAs that specifically repress cell death pathways. Walker & Harland (2009) found,for example, that miR-24a represses apoptosis in the developing Xenopus retina; knockdown ofmiR-24a results in an increase in apoptosis, while proliferation and other developmental processes


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Table 2 Summary of Dicer deletion studiesa

Study DeletionAberrant retinal

phenotype detected Proliferation changes Apoptosis changes

Damiani et al. 2008 Chx10-Cre; Dicerfl/fl Rosettes/Retinaldegeneration

No change Pychnotic nucleipresent

Iida et al. 2011 Dkk3-Cre; Dicerfl/fl Brn3-positive RGCsincrease

PH3-positive cellsunchanged

Caspase-3 increase notquantified

Decembrini et al.2008

Dicer KO usingmorpholino approach(Xenopus)

Otx2 and Otx5b bipolarcells increase

BrdU-positive cellsincrease

TUNEL increase

Georgi & Reh 2010 Alpha-Pax6-Cre;Dicerfl/fl

Brn3-positive RGCsincrease; bipolar cells,rods, and Muller gliadecrease

Slight decline inPH3-positive cells

Caspase-3 increase

Pinter & Hindges2010

Rx-Cre; Dicerfl/fl Eye size decreases; RGCaxon guidanceproblems

Proliferation not assayed Caspase-3 increase

Davis et al. 2011 Alpha-Pax6-Cre andtryp2-Cre; Dicerfl/fl

Brn3-positive RGCsincrease; amacrine,rods, and Muller gliadecrease

Ki67 not quantified Caspase-3 increase

Maiorano &Hindges 2013

Alpha-Pax6-Cre;Dicerfl/fl

Loss of all Dicer−/−

RGCs by P8Ki67 relative to Dicer+

retinal area assessedCaspase-3 relative toDicer+ retinal areaassessed

Akhtar et al. 2015 Dicer mutation(zebrafish)

Small eye, lethal Proliferation notassessed

Cell death not assessed

aDeletion of Dicer leads to a variety of developmental defects depending on the method of knockout/knockdown and the species used in the study. Nearlyall studies have reported some cell death and changes in the relative numbers of retinal cell types.Abbreviations: KO, knockout; RGC, retinal ganglion cell; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

were not affected. In addition, miR-24a directly represses translation of the proapoptotic factorsApaf1 and Caspase-9 via binding sites in their 3′ UTRs. A role for miRNAs in specificallyinhibiting cell death pathways was also elegantly demonstrated by Zhu et al. (2011). By creatinga mouse model where a sponge for the photoreceptor-enriched miRNA cluster composed ofmiR-96, miR-182, and miR-183 is expressed under the opsin promoter, they found that thephotoreceptors were more susceptible to light-damage-induced stress. In this case, Caspase-2was normally targeted by these miRNAs, and this inhibition was blocked by the introducedsponge. The fact that particular miRNAs can affect apoptotic pathways may provide a way toreduce or delay degeneration of specific retinal cells in progressive degenerative diseases, such asglaucoma or retinitis pigmentosa, and the possibility of developing miRNA-based therapies forthese diseases has been proposed (Sundermeier & Palczewski 2016).

4. ROLE OF miRNAs IN EARLY EYE DEVELOPMENT AND EYE-FIELDSPECIFICATION

Although miRNAs have been shown to be important in a number of early developmental events invertebrate embryos, most studies of miRNAs in the eye have been conducted after the period of eye-field specification. Nevertheless, there is evidence that miRNAs also play a role in the early eventsof eye morphogenesis. Knockdown of miR-204 in medaka fish causes microphthalmia, abnormal

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lens development, and altered patterning of the retina along the dorsal-ventral axis (Conte et al.2010). These phenotypes are mediated primarily by an increase in the expression of the Meis2transcription factor. In addition, overexpression of a mutant form of miR-204 (associated withocular coloboma) also results in microphthalmia in the fish (Conte et al. 2015). Pax6, an eye-fieldtranscription factor, is also important in the regulation of expression of miR-204, which suppressesthe neuronal gene Sox11 in the lens (Shaham et al. 2013). The network between miR-204, Pax6,and Meis2 is thus a critical link in the diversification and patterning of the ocular tissues early indevelopment.

5. miRNAs AND DEVELOPMENTAL TIMING

The various types of retinal neurons, and the Muller glia, are generated by mitotically active cells,known as retinal progenitors (RPs). These cells are capable of many rounds of cell division andexpress many of the same transcription factors present in the eye-field cells, along with proneuraland neural differentiation genes, such as Ascl1 and Neurog2. In all vertebrates, the RPs generatethe various types of neurons in a relatively conserved sequence (Figure 3a). Ganglion cells, conephotoreceptors, and horizontal cells are “born” from RP cell divisions early in retinal development,whereas most amacrine cells, rod photoreceptors, bipolar cells, and Muller glia are produced bythe RPs in the second half of histogenesis (Livesey & Cepko 2001a, Reh & Cagan 1994). Inmammals and birds, the RP cells can be further classified by differences in their expression ofparticular genes; for example, mouse progenitor cells present in early embryonic stages of retinalneurogenesis express Prtg, while RP cells from later embryonic stages and postnatal retina expressSox9 and Ascl1 (Brzezinski et al. 2011, Georgi & Reh 2010, La Torre et al. 2013, Livesey & Cepko2001b). The so-called early retinal progenitors (ERPs) and late retinal progenitors (LRPs) canbe further characterized by their response to mitogenic factors: ERPs do not respond to EGF(epidermal growth factor), while this is a potent mitogen for LRPs (Anchan et al. 1991, Lillien &Cepko 1992). The competence for production of different types of neurons also changes as theretina develops: Most of the rods, bipolar cells, and Muller glia are produced by Ascl1-positiveLRPs, but ERPs produce the RGCs (Brzezinski et al. 2011). A great deal has been learned over thepast 25 years about the transcription factors that specify the various types of retinal neurons andthe Muller glia; however, the factors that define the temporal sequence of neuronal productionare only beginning to be elucidated.

The timing of events in retinal development is an example of a strict developmental sequence.Often, these sequences are controlled by a timing mechanism, and changes in that timing canproduce very different phenotypic outcomes. The very different number of vertebrae in snakesand birds is an example of where relatively small heterochronic changes in development (like thetime it takes to make each somite) can dramatically affect the morphology of an animal (Keyte &Smith 2014). miRNAs have been associated with developmental timing since their discovery inCaenorhabditis elegans. In a series of remarkable publications, Ambros & Horvitz (1984) definedthe heterochronic pathway in these simple organisms by screening for mutants that affected thepattern of postembryonic cell divisions in the seam cell lineages. Ambros and colleagues went onto identify the first miRNA, lin-4, as one of these key regulators of timing (Lee et al. 1993), andsubsequently let-7 and lin-28 were discovered as part of this pathway (Moss et al. 1997, Wightmanet al. 1993). These genes make up the core heterochronic pathway in C. elegans, and since thesefirst components were identified, several additional proteins and miRNAs have been added to thepathway (Moss & Romer-Seibert 2014).

In mice, normal developmental timing also requires miRNAs. In Dicer conditional knock-out (CKO) mice, one of the most dramatic phenotypes is an inhibition of the normal changein competence of the RPs, from producing so-called early generated cell types, like RGCs, to


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Figure 3 (Figure appears on preceding page)

(a) Simplified model of retinal neurogenesis. An early retinal progenitor (ERP) that does not express Ascl1generates retinal ganglion cells (RGCs) at early stages of embryonic development (E12), but as developmentproceeds, the ERP gives rise to late retinal progenitors (LRPs), which express Ascl1 and have the potential togenerate the other types of retinal cells. Abbreviations: CON, cone photoreceptors; HOR, horizontal cells;AMA, amacrine cells; ROD, rod photoreceptors; BIP, bipolar cells; MUL, Muller glia. (b) Conditionaldeletion of Dicer in mouse retina using the alpha-Pax6-Cre line prevents the transition from ERP to LRPcells, and as a result, Ascl1 is not expressed in the progenitors and they generate excess Brn3-positiveganglion cells [seen in the ganglion cell layer (GCL) and migrating in the neuroblastic layer (Nbl)].Abbreviation: CKO, conditional knockout. (c) Heatmap of microRNA (miRNA) levels in mouse retina attwo stages of development (red = high; green = low). Several miRNAs increase substantially in LRPs (P1),compared with ERPs (E14), including members of the let-7 family, miR-9, and miR-125b. (d) TheRNA-binding protein lin-28 prevents the transition from ERP to LRP. Overexpression of the let-7inhibitor, lin-28, in developing mouse retina phenocopies the Dicer conditional deletion, showing anincrease in Brn3-positive ganglion cell production at an age when these cells are no longer produced in thewild-type mouse; mCherry is used to show the region of transfection in the control and lin-28-transfectedretinas. Abbreviations: OE, overexpression; RGCL, retinal ganglion cell layer. (e) Overexpression of let-7,miR-125, and miR-9, collectively named LP-miRNAs, in early embryonic retina accelerates the transition ofERPs to LRPs, as shown by the early onset of Nrl-GFP-positive rod photoreceptors. mCherry is used toshow the region of transfection. ( f ) Model of heterochronic gene regulation by miRNAs during retinaldevelopment. Many of these same molecules were discovered for their role in the regulation ofdevelopmental timing in Caenorhabditis elegans. Panels a–e modified from La Torre et al. (2013).

producing late fates, like glia and rod photoreceptors (Davis et al. 2011, Georgi & Reh 2010, LaTorre et al. 2013). ERP markers (Prtg, Fgf15, lin-28) continue to be expressed into late embry-onic and postnatal stages of retinal development, while LRP markers, like Ascl1 and Sox9, arenever expressed by the RPs after Dicer deletion (Ascl1 in Figure 3b). Although Otx2-positivecells continue to be produced in the Dicer CKO mouse retina, they do not express markers ofrods (rhodopsin) or bipolar cells. Instead, there is an overproduction of the ganglion cells, andthis persists long past the normal developmental competence window for ganglion cell production(Brn3 in Figure 3b).

La Torre et al. (2013) FACS purified the cells with Dicer deletion, since the Cre recombinasecarried a reporter. They then carried out a study of the mRNAs most significantly affected bythe loss of Dicer in developing retinal cells. They found that many of the genes normally in earlyborn cell types and their progenitors, like Lgr5 and Onecut1, 2, and 3, increased in the Dicer-negative cells, while genes associated with LRPs, like Fabp7, Ascl1, and Sox9, were reduced inthe cells, consistent with the immunolabeling results. One gene that was particularly increasedwas Prtg, a gene previously identified in early progenitors of the spinal cord. Further westernblot and immunolabeling studies demonstrated that this gene is expressed in ERPs but absent inthese cells in retina from later stages. Another gene of interest was lin-28, a core component of theC. elegans heterochronic pathway. The RNA-binding protein lin-28 interferes with the productionof functional let-7 by binding the pre-miR form. In the developing retina, lin-28 is expressed witha similar time course and distribution as Prtg.

To better understand how these genes were regulated by the loss in specific miRNAs causedby the Dicer deletion, the authors also profiled miRNAs in the developing retinas. They founda specific set of miRNAs that was more prominently expressed in P0 retina than in E14 retina(Figure 3c) and were able to determine which of these were expressed in progenitors by theirresponse to forced differentiation by Notch inhibition. This allowed them to define a set of early-progenitor miRNAs and a set of LRP miRNAs (LRP-miRNAs). They found, using RT-PCR andmiRNA sensors, that LRP-miRNAs increase in RPs over the transition from early to late stagesof neurogenesis. They focused on members of the let-7 family, miR-9, and miR-125.


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Using an antagomir knockdown approach to test the function of LRP-miRNAs, the authorsshowed that knocking down just three miRNAs—let-7, miR-9, and miR-125—phenocopies theDicer CKO. Further, overexpression of these three LRP-miRNAs as mature miRNA mimics inDicer CKO retinas rescued the phenotype and allowed progenitors to adopt a late-progenitorpattern of gene expression (La Torre et al. 2013). Even more interesting, overexpression of LRP-miRNA mimics in wild-type retina can actually accelerate developmental timing in this system(Figure 3e). The authors also were able to show that both Prtg and lin-28 are targets of the LRP-miRNAs, accounting for their downregulation, as progenitors transition from the early to late state.Moreover, these two genes, when overexpressed, can also retard normal developmental timing inthe retina (Figure 3d,f ). There is evidence that the components of the heterochronic pathwaycontinue to regulate the timing of key events in maturation and even lifespan. For example, lin-4mutants age at an increased rate, while lin-14 mutants age more slowly than wild-type animals(Boehm & Slack 2005, 2006). Both let-7b and let-7e are more highly expressed in old humanskeletal muscle than in young muscle (Drummond et al. 2011). Genome-wide association studieshave found that polymorphisms near the LIN28B locus are associated with precocious pubertyin girls (He et al. 2009, Ong et al. 2009, Sulem et al. 2009). Thus, this fundamental biologicaltiming pathway may be involved in other aspects of development, maturation, and aging (Moss &Romer-Seibert 2014).

Studies in nonmammalian vertebrates provide further insight into the potential mechanisms bywhich miRNAs regulate developmental timing of cell differentiation. In Xenopus, knockdown ofDicer, using morpholios, produces defects in retinal lamination and cell cycle exit. The translationof Xotx5b and Xotx2, two transcription factors important for bipolar cell fate, is increased, thoughmRNA for these genes was not affected (Decembrini et al. 2009). By screening highly expressed,developmentally regulated miRNAs, researchers found that miR-129, miR-155, miR-214, andmiR-222 target Xotx2 as well as Vsx1, another gene required for bipolar cells. Inactivation of thesemiRNAs with antisense oligonucleotides results in an increase in bipolar cells (Decembrini et al.2009) similar to that observed in the Dicer knockdown. These researchers concluded that thesespecific miRNAs couple cell cycle exit with bipolar cell fate, as previous data showed a relationbetween cell cycle length and miRNA expression. It appears that a different set of miRNAs controldevelopmental timing in mice and frogs, since the miRNAs in this study were different from thosestudied in developmental timing in mouse retina. Nevertheless, let-7c and miR-125b were alsoreported to be among the top miRNAs expressed in developing frog retina, so it is possible thatsimilar mechanisms as those described in mice also regulate frog retinal development.

6. miRNA REGULATION OF NOTCH SIGNALING

Notch is a key developmental regulator of retinal neurogenesis. In mice, both Notch1 and Notch3are expressed by RPs. Several lines of evidence, from genetic to small molecule inhibitors, haveshown that loss in Notch signaling leads to an increase in neurons at the expense of glia in theretina and elsewhere in the CNS. In addition, in the retina, both ERPs and LRPs require Notch;inhibition of Notch signaling at any stage of development produces characteristic phenotypes(Nelson et al. 2007, 2009).

Loss of miRNAs in the Dicer CKO mouse retina leads to a reduction in expression in severalcomponents of the Notch signaling pathway by P0, including Hes5, the Notch ligand, Dll3, andNotch1 itself, leading to a generalized decrease in Notch signaling (Georgi & Reh 2011). The lossin Notch signaling does not appear to be responsible for the effects of Dicer deletion on progenitorcompetence, however, since this was not rescued by crossing the Dicer CKO mice with Notch–intracellular domain (ICD)-expressing mice. Although the Notch signaling defect was rescued in

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these mice, the defect in gliogenesis was still present, and the normally late-generated cell types,like rods and bipolar cells, were still absent, while ganglion cells were still overproduced. Theseresults suggest that Notch signaling is regulated by a different set of miRNAs than those thatcontrol progenitor competence.

One potential mechanism by which miRNAs regulate Notch signaling comes from a studyin zebrafish. Olena et al. (2015) surveyed developing zebrafish retina and found 12 miRNAsexpressed at 2 days postfertilization (dpf ) and 23 miRNAs at 5 dpf. From in situ localizationexperiments, 3 miRNAs—miR-9, miR-124, and miR-216a—were expressed specifically in thedeveloping eye. Overexpression of miR-216a led to retinal phenotypes consistent with reducedNotch signaling: a decrease in Muller glia and an increase in photoreceptors (Olena et al. 2015).Although several components of the Notch pathway have miRNA recognition elements (MREs)for miR-216a, they focused on SNX5, a gene required for Delta endocytosis and Notch ICDinternalization. Knockdown of miR-216a causes an increase in Muller glial differentiation, withfewer photoreceptors, and this effect can be partly rescued by overexpression of SNX5.

It is not known whether miR-216 plays a similar role in mice, and it is not highly expressed indeveloping mouse retina (Georgi & Reh 2010); however, another miRNA, miR-7a, may regulateNotch signaling in developing mouse RPs. Transfection of an miR-7a decoy causes a loss in Mullerglia, while overexpression of miR-7a causes an increase in these cells. This appears to be via a directtargeting of Notch 3 (Baba et al. 2015). It is unlikely that the reduction in Notch 3 accounts for themore general loss in Notch signaling observed in the Dicer CKO mouse, and thus it is likely thatother miRNAs are normally important in regulating this very important developmental signal.

7. miRNAs AND ESTABLISHING VISUAL CONNECTIVITY

Recent findings have shown that miRNAs play essential roles in the establishment of connectivityin the visual system. Although most of the data is relevant to axon guidance and synapse formationoutside the retina per se, we discuss here some core findings concentrating on the vertebrate visualsystem.

RGCs are the only neurons projecting out of the retina to form long-range connections withtargets in the brain proper. On their journey, they encounter several choice points where theyneed to make pathfinding decisions in order to innervate the appropriate targets, including theoptic chiasm at the midline (Erskine & Herrera 2014).

The first indication that miRNAs impact long-range axon guidance decisions came from stud-ies by Pinter & Hindges (2010), who used a conditional Dicer deletion approach in mice. To deleteDicer from the developing retina, the authors used an Rx-Cre mouse line, where the Cre recombi-nase is driven by promoter elements from the eye-field transcription factor retinal-homeobox geneRx (Swindell et al. 2006). In mice, Rx is normally expressed from E7.5 onward in the anterior neuralplate and becomes subsequently restricted to the retina and ventral forebrain by E10.5 (Matherset al. 1997). The degree of visual overlap between the two eyes directly determines the magnitudeof stereovision and is reflected in the relative proportions of ipsi- and contralaterally projectingaxons at the optic chiasm (Herrera et al. 2003). In mice, with only a limited visual overlap, about95% of all RGC axons cross the midline, whereas the remaining 5% stay ipsilaterally (Drager &Olsen 1980). Pinter & Hindges (2010) showed that upon deletion of Dicer, RGC axons exhibita strong pathfinding phenotype at the optic chiasm, with many axons failing to cross the midline(Figure 4a). Instead, they either stayed ipsilaterally or extended aberrantly into the ventral di-encephalon (Pinter & Hindges 2010). Furthermore, a significant portion of axons was found toturn into the contralateral optic stalk and extend to the contralateral eye. The authors found that,in parallel with these projection errors, axons were generally defasciculated, not only within the


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Figure 4Roles of microRNAs during axon outgrowth. (a) Summary of phenotypes occurring in conditional Dicermutant mice in combination with the Rx-Cre lines. In wild-type or heterozygous Dicer mutant mice (left),the neural retina encapsulates the lens, and retinal ganglion cell axons (red) extend toward the midline,forming the optic nerve. At the optic chiasm, most of the axons cross to the contralateral side (contralateraloptic tract), whereas only 3–5% stay on the ipsilateral side (ipsilateral optic tract). The axons form tightfasciculated axon bundles at the point of crossing and when extending into the optic tracts. In homozygousDicer mutants (right), four major defects are observed: (�) a small eye, with the lens being only about 50%encapsulated by the retina; (�) an increased ipsilateral projection; (�) fibers extend further than the opticchiasm, and after crossing the midline, they form a parallel optic tract; and (�) a significant proportion ofaxons extend aberrantly into the contralateral eye (adapted from Pinter & Hindges 2010). (b) Examples ofindividual miRNAs affecting the correct timing of axon guidance receptor expression at the growth cone,fasciculation, axon branching, and the local translation of messenger RNAs involved in axon guidanceresponses.

retina failing to form a tight optic fiber layer but also at the optic chiasm, leading to the forma-tion of a secondary optic tract on the contralateral side. Although this defasciculation phenotypepoints toward a cell-autonomous effect, the study could not clearly determine whether the axonpathfinding defects at the midline are based on altered sensitivity of the growing RGC axons or achange of the chiasm cells (or a combination of both) as the Rx promoter leads to Cre expressionboth in the retina and at the chiasm (Pinter and Hindges 2010). Rx-Cre+/Dicer mutant mice dieat birth, which made it impossible to study the role of Dicer, and thus miRNAs, in the process oflater axon targeting—for example, during the process of topographic mapping.

In addition to studies eliminating most of the miRNAs through Dicer deletions, more re-cent studies have identified the roles of individual miRNAs to control the process of retinalaxon outgrowth and targeting. In Xenopus, miR-124 was shown to control the precise timingof sensitivity of RGC growth cones to Sema3A during the formation of the visual projections(Baudet et al. 2011) (Figure 4b). The authors showed that a knockdown of miR-124 leads to the

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downregulation of the Sema3A receptor neuropilin1. This is achieved indirectly through coR-EST, which was identified as a novel miR-204 target that controls Nrp1 expression. A differentstudy identified possible targets of miR-204, which has important function during eye develop-ment, using an RNA sequencing approach upon overexpression and knockdown of this miRNA(Conte et al. 2014). The resulting list contains a substantial number of molecules associated withaxon guidance, including Eph receptors and their ephrin ligands (Reber et al. 2007). Knockdownof miR-204 in medaka resulted in defasciculation of RGC axons as well as aberrant growth intodifferent retinal layers and mistargeting along the optic pathways (Conte et al. 2014) (Figure 4b).These defects were rescued by the knockdown of either EphB2 or ephrin-B3, suggesting thatthe regulation of these molecules through miR-204 is critical for the correct axon pathfinding ofRGC axons. These studies demonstrate nicely the intricate network of factors that are controlledby individual microRNAs to ensure the correct spatiotemporal presence of axon guidance cuesneeded to form a fully functioning visual system.

Once RGC axons have reached the target area, a multistep process, including axon branch-ing, remodeling, and synapse formation, takes place to finally form the appropriate connections(Hindges et al. 2002). An important regulator of RGC axon branching is brain-derived neu-rotrophic factor (BDNF)/TrkB signaling (Feldheim & O’Leary 2010). Consistent with a role ofmicroRNAs in this process, Marler et al. (2008) showed that BDNF-induced branching is de-pendent on the presence of miR-132 (Figure 4b). Here, the authors demonstrate that in boththe mouse and the chick, miR-132 is highly expressed in RGCs during the developmental win-dow when RGC axons grow into their targeting area and refine their termination zones throughbranching and synaptogenesis (Hindges et al. 2002, Yates et al. 2001). Overexpression of miR-134 leads to increase of branching, while a knockdown decreases BDNF-mediated branching.This process is achieved through the interaction of miR-134 with its target Rho family GTPase-activating protein p250GAP, an inhibitor of Rac, which in turn is a branch-promoting protein.During the refinement phase of RGC axon mapping, BDNF in the target induces the expression ofmiR-132, which then leads to a reduction of p250GAP levels and thus increase of branching activ-ity through activation of Rac (Marler et al. 2008). This interaction of miR-132 with p250GAP wasalso shown in different contexts—for example, in dorsal root ganglion axons, where it promotesaxon extension (Hancock et al. 2014).

An important mechanism of axon guidance, branching, and target selection is the local trans-lation of mRNAs (Jung et al. 2014, Shigeoka et al. 2016). Interestingly, several screens usingdifferent neuronal populations have shown that miRNAs are not restricted to the cell soma andthat they are also found in axons (Natera-Naranjo et al. 2010, Sasaki et al. 2014), in combinationwith the presence of RISC (Hengst et al. 2006). This suggested a possible role of miRNAs incontrolling the levels of locally translated proteins.

Indeed, a recent profiling study in growing Xenopus RGC axons identified a number of miRNAspresent in axons and even the growth cone, with miR-182 being the most abundant axonal miRNA(Bellon et al. 2017). Using a loss of function approach, the authors could show that miR-182 isimportant for the appropriate targeting of RGC axons in the optic tectum. Further experimentsrevealed that this change in targeting precision was dependent on the action of Slit2. The authorsfound that miR-182 regulates the responsiveness of growth cones to Slit2 through controllingthe local translation of cofilin 1 (Cfl1), an important regulator of the cytoskeleton. In normalconditions, miR-182 binds to the 3′ UTR of Cfl1 and represses its local translation. However, uponSlit2 stimulation (through its receptors Robo2 and Robo3), the miR-182 repression is releasedand allows the production of Cfl1 and thus the necessary cytoskeletal changes for a growth-cone repulsive response. Interestingly, this mechanism of modulating growth-cone responsivenessseems to be very specific, as the sensitivity to a different repulsive cue, Sema3A, was unchanged


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(Bellon et al. 2017). This suggests an even more intricate system to activate and modulate theresponses of growth cones to external signals through miRNAs.

In addition to the findings about miRNAs during axon guidance/targeting discussed here, thereare a number of studies from outside the visual system showing the involvement of miRNAs in thisprocess (Iyer et al. 2014). Similarly, miRNAs have also been found to control further steps duringthe establishment of neural circuits, including dendritogenesis and synapse formation (Rajman &Schratt 2017), and it is very likely that this also includes these processes within the retina or alongthe visual pathway.

8. CONCLUSIONS

Posttranscriptional regulatory mechanisms play an important role during the development andfunctional assembly of the retina. This includes the action of miRNAs found to be expressedat different stages of eye development. Although much progress has been made to unravel theirfunctions, one particular difficulty has been that most of these noncoding molecules are morelikely acting to fine-tune the protein output of a cell as compared to leading to a complete switch(with some exceptions). In addition, the fact that many miRNAs act in combination with eachother to bind individual target mRNAs demonstrates the necessity to understand their role in acombinatory fashion. Nevertheless, recent experimental advances—for example, multiplex genetargeting or simple base editing using CRISPR/Cas (clustered regularly interspaced short palin-dromic repeats/CRISPR-associated proteins) approaches, together with the progress in single-cellsequencing and proteomics—will certainly be helpful to the field and efforts to identify the functionof miRNAs in the eye in much more detail.

SUMMARY POINTS

1. MicroRNAs (miRNAs) are critical for controlling key developmental timing events inthe developing retina, such as the sequential generation of specific cell fates.

2. Some of the components of the well-characterized heterochronic pathway of miRNAsand RNA-binding proteins are also important regulators of developmental timing inmouse retinal neurogenesis, showing the deep conservation of this early role.

3. miRNAs play important roles at various steps during the establishment of visual systemconnectivity.

FUTURE ISSUES

1. Cell-type-specific deletions of Dicer or other genes necessary for microRNA (miRNA)processing have the potential to reveal other developmental requirements for these reg-ulators in neurons and glia.

2. Single-cell analyses of miRNA expression in the eye have the potential to reveal possibleroles in the generation of retinal cell subtypes.

3. Little is known about the role of miRNAs in regulating synaptogenesis in the retina.Given the evidence for miRNA regulation in synaptogenesis in other systems, it wouldbe very interesting to assess this, particularly for the formation of specific visual circuits.

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DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

Work in the authors’ laboratories is supported by a National Eye Institute grant (NEIR01EY021482 to T.A.R.), the Foundation Fighting Blindness (TA-RM-0614–0650-UWA toT.A.R.), the Paul G. Allen Family Foundation (Frontiers Program to T.A.R.), the Medical Re-search Council (G0601182 to R.H.), the Wellcome Trust (087883/Z/08/Z to R.H.), and theBiotechnology and Biological Sciences Research Council (BB/M000664/1 to R.H.).

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VS04_TOC ARI 21 August 2018 11:58

Annual Review ofVision Science

Volume 4, 2018Contents

A Life in VisionJohn E. Dowling � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

MicroRNAs in Retinal DevelopmentThomas A. Reh and Robert Hindges � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �25

Microglia in the Retina: Roles in Development, Maturity, and DiseaseSean M. Silverman and Wai T. Wong � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �45

Plasticity of Retinal Gap Junctions: Roles in Synaptic Physiologyand DiseaseJohn O’Brien and Stewart A. Bloomfield � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �79

Retinal Vasculature in Development and DiseasesYe Sun and Lois E.H. Smith � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 101

Parallel Processing of Rod and Cone Signals: Retinal Functionand Human PerceptionWilliam N. Grimes, Adree Songco-Aguas, and Fred Rieke � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 123

Elementary Motion Detection in Drosophila: Algorithms andMechanismsHelen H. Yang and Thomas R. Clandinin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 143

Neural Mechanisms of Motion Processing in the Mammalian RetinaWei Wei � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 165

Vision During Saccadic Eye MovementsPaola Binda and Maria Concetta Morrone � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 193

Corollary Discharge Contributions to Perceptual ContinuityAcross SaccadesRobert H. Wurtz � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 215

Visual Function, Organization, and Development of the MouseSuperior ColliculusJianhua Cang, Elise Savier, Jad Barchini, and Xiaorong Liu � � � � � � � � � � � � � � � � � � � � � � � � � � � � 239

Thalamocortical Circuits and Functional ArchitectureJens Kremkow and Jose-Manuel Alonso � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 263

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VS04_TOC ARI 21 August 2018 11:58

Linking V1 Activity to BehaviorEyal Seidemann and Wilson S. Geisler � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 287

A Tale of Two Visual Systems: Invariant and Adaptive VisualInformation Representations in the Primate BrainYaoda Xu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 311

Blindness and Human Brain PlasticityIone Fine and Ji-Min Park � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 337

How Visual Cortical Organization Is Altered by Ophthalmologicand Neurologic DisordersSerge O. Dumoulin and Tomas Knapen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 357

The Organization and Operation of Inferior Temporal CortexBevil R. Conway � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 381

Invariant Recognition Shapes Neural Representations of Visual InputAndrea Tacchetti, Leyla Isik, and Tomaso A. Poggio � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 403

Shape from Contour: Computation and RepresentationJames Elder � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 423

Geometry of Pictorial ReliefJan J. Koenderink, Andrea J. van Doorn, and Johan Wagemans � � � � � � � � � � � � � � � � � � � � � � � 451

Color Perception: Objects, Constancy, and CategoriesChristoph Witzel and Karl R. Gegenfurtner � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 475

Motion Perception: From Detection to InterpretationShin’ya Nishida, Takahiro Kawabe, Masataka Sawayama, and Taiki Fukiage � � � � � � � � 501

Errata

An online log of corrections to Annual Review of Vision Science articles may be found athttp://www.annualreviews.org/errata/vision

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