RESEARCH REPORT

Rorβ regulates selective axon-target innervation in themammalianmidbrainHaewon Byun1, Hae-Lim Lee2, Hong Liu3, Douglas Forrest3, Andrii Rudenko4 and In-Jung Kim1,5,*

ABSTRACTDevelopmental control of long-range neuronal connections in themammalian midbrain remains unclear. We explored the mechanismsregulating target selection of the developing superior colliculus (SC).The SC is a midbrain center that directs orienting behaviors anddefense responses. We discovered that a transcription factor, Rorβ,controls establishment of axonal projections from the SC to twothalamic nuclei: the dorsal lateral geniculate nucleus (dLGN) and thelateral posterior nucleus (LP). A genetic strategy used to visualizeSC circuits revealed that in control animals Rorβ+ neurons abundantlyinnervate the dLGN but barely innervate the LP. The oppositephenotype was observed in global and conditional Rorb mutants:projections to the dLGN were strongly decreased, and projections tothe LP were increased. Furthermore, overexpression of Rorb in thewild type showed increased projections to the dLGN and decreasedprojections to the LP. In summary, we identified Rorβ as a keydevelopmental mediator of colliculo-thalamic innervation. Suchregulation could represent a general mechanism orchestratinglong-range neuronal connections in the mammalian brain.

KEY WORDS: Rorβ, Axon targeting, Midbrain, Superior colliculus,Thalamus, FLP-DOG

INTRODUCTIONThe development of long-range neuronal connections employsmultiple mechanisms. Although axon pathfinding, topographicmapping and laminar-specific connections have been extensivelystudied, some related phenomena, including target selection, remainpoorly understood (Dickson, 2002; Sanes and Yamagata, 2009).Axon-target selection is the process of axons choosing their targets andavoiding the adjacent ones. Multiple studies using invertebrates andperiphery-to-brain connections have uncovered the basic mechanismsof axon-target selection (Dickson, 2002). However, how suchselection is executed within the mammalian brain remains unclear.The superior colliculus (SC) is a midbrain center that regulates

orienting behaviors and defense responses to threat (Basso andMay,2017; Cang et al., 2018). Previous studies revealed that the SCprojects axons to several subcortical areas (Ahmadlou et al., 2018;Bickford et al., 2015; May, 2006; Shang et al., 2018; Wei et al.,

2015). Projections to the dorsal lateral geniculate nucleus (dLGN)are uniquely localized within the nucleus and possibly regulatedirection selectivity. Projections to the lateral posterior nucleus (LP)and the parabigeminal nucleus (PBGN) control visual cue-triggeredbehaviors, such as freezing and escaping.

Traditionally, developmental studies have focused on thearrangement of sensory inputs to the SC, including topographicmap formation (Constantine-Paton et al., 1990; Feldheim andO’Leary, 2010). However, little is known about the mechanismsregulating the development and output pathways of SC neurons.

To examine such mechanisms, we previously sought moleculeslabeling specific types of SC neurons (Byun et al., 2016), focusing onthe superficial layer of the SC (sSC), which receives visual inputsfrom the retina and cortex and projects axons to subcortical areas.Wediscovered that a transcriptional factor, retinoid-related orphanreceptor β (Rorβ), is highly expressed within the sSC. As sSCneurons from specific sublayers selectively innervate differentthalamic nuclei (Harting et al., 1991; May, 2006), we hypothesizedthat sSC Rorβ+ neurons may project axons to distinct midbrain areas.

Several studies have demonstrated the importance of Rorβ in thenervous system (Abraira et al., 2017; Jabaudon et al., 2012). GlobalRorb mutants show impaired circadian behavior and neuronaldifferentiation (André et al., 1998; Liu et al., 2013; Oishi et al.,2016), and disruption of the human RORB locus has been reportedin neurodevelopmental disorders (Baglietto et al., 2014; Rudolfet al., 2016). However, little is known about the role of Rorβ in theestablishment of the long-range axonal projections (Moreno-Juanet al., 2017).

We assessed the role of Rorβ in sSC neuronal development usingglobal and midbrain-specific Rorb mutants. A novel genetictechnique that allows targeted GFP expression was used tovisualize Rorβ+ neurons. We found that they project axons todistinct subcortical areas: dorsal LGN (dLGN), ventral LGN(vLGN), pretectum (PT), PBGN and LP. Rorb loss led to a severedecrease in dLGN projections and an increase in LP projections; noobvious changes were detected in other areas. Rorb overexpressionincreased projections to the dLGN and decreased projections to theLP. Altogether, our study identifies a molecular mechanismregulating sSC target selection and sSC-thalamic connections.

RESULTS AND DISCUSSIONBasic characterization of sSC Rorβ+ neuronsTo assess the role of Rorβ in sSC development, we performed basiccharacterization of Rorβ+ neurons. We previously reported that Rorβ+

somas are localized at the top layerof the sSC (Byun et al., 2016).Here,we confirmed this by analyzing axonal projections of retinal ganglioncells (RGCs) using cholera toxin β subunit (CTB)-conjugatedfluorescent dyes (Fig. 1A,C). Strong CTB labeling marks the axonalarborization layer of RGCs (stratum griseum superficiale, SGS), andweak labeling marks the axonal bundle layer (stratum opticum, SO).To identify Rorβ+ neurons, we used Rorb1g/+ mice, in which theReceived 17 September 2018; Accepted 23 June 2019

1Department of Ophthalmology and Visual Science, Yale University School ofMedicine, New Haven, CT 06511, USA. 2Department of Cellular and MolecularPhysiology, Yale University School of Medicine, New Haven, CT 06511, USA.3Laboratory of Endocrinology and Receptor Biology, National Institutes of Health,NIDDK, Bethesda, MD 20892, USA. 4Department of Biology and GraduateProgram, The City College and City University of New York, New York, NY 10031,USA. 5Department of Neuroscience, Yale University School of Medicine, NewHaven, CT 06511, USA.

*Author for correspondence (in-jung.kim@yale.edu)

I.-J.K., 0000-0001-9469-5357

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© 2019. Published by The Company of Biologists Ltd | Development (2019) 146, dev171926. doi:10.1242/dev.171926

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reading frame of GFP replaces the Rorb1-specific exon leading tocomplete loss of Rorb1 mRNA (Liu et al., 2013; Fig. 1B). Here, werefer toRorb1g/+ asRorb+/−mice, asRorb1 is a predominant isoform inthe sSC (Byun et al., 2016). CTB labeling showed that Rorβ+ neuronsoccupy the upper layer of the sSC (SGS; Fig. 1C).We then examined expression of Rorb in the sSC during

development (Fig. S1A,B). At embryonic day (E)13, Rorb isexpressed by a few cells in the sSC, an intermediate zone (IZ)(Edwards et al., 1986). We used a Ki67 antibody, which labelsproliferating cells, to visualize the ventricular and subventricularzones (VZ/SVZ) of the SC. Rorb signal was barely detectable in theVZ/SVZ, suggesting that expression occurs mostly post-mitotically.At E16, Rorb expression was increased in the top layer, close to thepia, and remained there at postnatal day (P)1-P6.Layer-restricted expression of Rorb suggested that sSC Rorβ+

neurons may innervate distinct subcortical targets. As GFP-based

tracing was challenging because of strong widespread GFP signalin Rorb+/− brains, we utilized a novel strategy exploiting aGFP-dependent recombinase, FLP-DOG (Tang et al., 2017) andFLP-dependent mCherry expression (fDIO-mCherry). FLP-DOGcontains destabilized GFP-binding proteins (dGBP1) and codon-optimized FLP (Flpo). Binding to GFP protects dGBP1 fromdegradation resulting in active Flpo expression (Fig. 1D). fDIO-mCherry contains two pairs of incompatible FLP recognition (FRT)sequences flanking an inverted mCherry. Interaction of stabilizedFlpowith FRT places mCherry into the forward orientation, allowingexpression. We generated adeno-associated viruses (AAVs) thatexpress FLP-DOG and fDIO-mCherry and co-injected them into thesSC of Rorb+/− mice at P0-P1. Immunostaining with anti-GFP andanti-mCherry at ∼P20 revealed that ∼98% of mCherry+ cellsexpressed GFP (n=745 cells, 3 mice), suggesting high specificity ofthe FLP-DOG strategy. We found that Rorβ+ neurons innervate

Fig. 1. Basic characterization of sSC Rorβ+ neurons. (A) Schematic of the SC after CTB injection into the contralateral eye. (B) Replacement of Rorbexon with GFP sequence inRorb+/–mouse, leading to complete loss ofRorbmRNA in mutants (Liu et al., 2013; Byun et al., 2016). (C)Rorb expression (green) ismostly confined to the upper layer of the sSC in the Rorb+/− mouse at P13. Dense labeling of CTB (magenta) delineates a boundary between stratum griseumsuperficiale (SGS) and stratum opticum (SO). (D) Schematic of the FLP-DOG strategy using fDIO-mCherry. The presence of GFP stabilizes unstable GFP-binding proteins fused to FLP and allows mCherry expression in fDIO-mCherry. (Flpo, codon optimized FLP; dGBP1, destabilized GFP-binding protein; yellow,ubiquitin molecules; CAG, promoter). (E) Left: mCherry expression after injecting AAV-FLP-DOG and AAV-fDIO-mCherry into the sSC of Rorb+/− mouse(Rorb, green; mCherry, magenta). Right: Magnified views of the boxed area. (F-I) Axonal projections from the sSC to the PBGN (F), dLGN, vLGN and LP (G) andPT (H) (GFP, green; mCherry, red). The PBGN was identified by choline acetyltransferase (ChAT) staining (cyan in F; Mufson et al., 1986). The dLGN,vLGN and PT were identified by their anatomical localization and high GFP level (DAPI, blue in H). The LP was identified by calretinin (CR) staining (cyan in G;Zhou et al., 2017) or anatomical localization and low GFP level. (I) Schematic of Rorb+ neuronal projections. Thicker arrow indicates the most abundantprojections. Dashed arrow indicates the least abundant projections. Scale bars: 300 µm in C,E (left),G,H; 100 µm in E (right),F.

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several subcortical areas (Fig. 1E-I): dLGN, vLGN, PT and PBGN.Axonal projections to the vLGN and PT were less abundant.Projections to the LP were detectable as long as medial sSC neuronswere mCherry labeled.To assess the percentage of Rorβ+ neurons among the total sSC

population projecting to each target, we employed retrogradelabeling. We chose to focus on the dLGN and LP – the target areasshowing the most and least abundant projections, respectively.Neurons were labeled by injecting CTB-555 into the dLGN or LP ofadult mice (Fig. S1C-H). We found that most dLGN-projecting sSCneurons were located at the SGS, and∼62% of the labeled cells wereGFP+ (Rorb expressing). Most LP-projecting sSC neurons werelocated at the SO and only ∼13% of them were GFP+. Our resultsare consistent with previous findings that neurons in the upper layersof the sSC project mainly to the dLGN but barely to the LP (Hartinget al., 1991; May, 2006).

Rorβ regulates sSC neuronal developmentBased on temporal and spatial expression of Rorb, we hypothesizedthat Rorβ might control neuronal positioning or postmitoticdifferentiation, including axonal projections. To test thesehypotheses, we conducted loss-of-function experiments.No obvious differences were observed in the cytoarchitecture of

the SC in control andRorbmutants at P1 (Fig. S2A-D). Similar to thecontrol, Rorβ+ neurons were enriched in the upper layer of the sSC ofthe mutants. However, the Rorβ+ neuronal layer was visibly narrowerin the mutants at P12 (Fig. S2E-G). Examination of neurofilament(SMI312) staining demarcating the layer below the SGS (SO) andquantification of Rorβ+ neurons confirmed a decrease of neuronaldistribution and number. These results suggest that Rorb loss mightaffect late developmental aspects, such as axonal projections.To examine whether Rorb is required for sSC axon development,

we injected AAV-FLP-DOG and AAV-fDIO-mCherry into the SCof E15.5 embryos and examined the brains at P2. Because of noprior reports on sSC output pathways, time points were chosenbased on the Rorb expression and cortical projection studies (Arlottaet al., 2005; Galazo et al., 2016). Very few, if any, labeled cells werefound in the sSC (not shown). As this could be due to weakembryonic GFP expression insufficient to produce enoughstabilized FLP-DOG (Fig. 1D), we injected AAVs at P0-P1 andexamined brain tissues at P10-P11. Owing to obvious changes inRorβ+ neuronal number and distribution at P12, analysis was notconducted beyond this stage. We discovered a strong decrease inprojections to the dLGN in the mutants (Fig. 2A-D,I), which wasmost pronounced in the rostral dLGN. In contrast, projections to theLP were increased. Quantification of mCherry fluorescence(Materials and Methods) revealed ∼87% decrease in projectionsto the dLGN and ∼5-fold increase in projections to the LP in themutants. Axonal innervation in other areas, including the PBGN,showed no obvious changes (Fig. 2E-I). Projections to the vLGNand PT were not quantified because the boundaries between axonbundles and terminals were less clear. Examination of the otherareas failed to detect any mistargeting or random stalling of axons.Altogether, our data indicate that Rorb deletion strongly affectssSC-thalamic projections.

Conditional deletion of Rorβ in the SC phenocopies alteredsSC axonal development in global Rorb mutantsChanges in the sSC Rorβ+ neuronal layer and alterations of axonalprojections in the Rorb mutants might be directly related to eachother. However, such changes could be caused by general retinaldisorganization (André et al., 1998; Sakurai and Okada, 1992;

Smith and Bedi, 1997). To rule this out, we employed a conditionalknockout (cKO) strategy.

We utilized a Rorb mutant, in which an exon encoding a partof the DNA-binding domain is flanked by loxP sites (Rorbflox/flox;Fig. S3A). Engrailed1-Cre (En1-Cre) mice were used as colliculus-specific Cre line (Dhande et al., 2012; Kimmel et al., 2000). Tovalidate the En1-Cre line, we crossed it to the Ai14 line expressingCre-dependent td-Tomato (Madisen et al., 2010) and confirmed therestricted Cre expression (Fig. S3B). To demonstrate ablationefficiency, we crossed Rorbflox/flox to Rorb+/−; En1-Cre (Rorb cKO)and confirmed Rorb deletion by RT-PCR (Fig. S3C).

We examined whether conditional deletion of Rorb affects thedevelopment of sSC neurons. Given that Rorb expression isdetected at E13, we examined early developmental aspects,including cell survival and laminar positioning. Quantification ofcleaved-caspase 3+ cells at E16 and P1, and NeuN (Rbfox3)+ cells atP12 showed no difference between genotypes, suggesting that Rorbis not required for neuronal survival (Fig. S3D-F). Quantification ofRorβ+ neurons throughout development (Fig. S3G,H) demonstratedthat in both controls and mutants, the number of Rorβ+ neuronsdecreased from embryonic to postnatal stages. We found nodifferences in layer distributions between genotypes (Fig. S3I).Together, these results demonstrate that conditional deletion ofRorb does not disrupt SC development and that Rorb is not requiredfor cell survival and migration in the sSC.

Next, we examined alterations of sSC neuronal projections inconditional mutants. Using the FLP-DOG strategy, we discoveredthat, similar to the global knockout, Rorb loss led to a decrease inaxonal innervation in the dLGN and an increase in axonalinnervation in the LP of cKO mice at ∼P20 (Fig. 3A-C).Quantification of mCherry fluorescence revealed ∼66% decreaseof projections to the dLGN and ∼2.4-fold increase to the LP. Noobvious changes were detected in the PBGN, vLGN and PT(Fig. 3D-H). These findings suggest that alterations of axonalprojections in the Rorb mutants are caused by Rorβ ablation in SC.

Finally, we injected AAV-FLP-DOG and AAV-fDIO-mCherryinto the sSC at P0-P1 and investigated axonal innervation in the dLGNand LP at P4/P5, P7/P8 and P10/P11 (Fig. 3I-O). In controls, axonalprojections to the dLGNwere detectable at P4, became abundant at P7and were complete by P10. The cKO mutants showed no obviousprojections to the dLGN at P4 and relatively few at P7 and P10.Remarkably, analysis of projections to the LP revealed the oppositephenotype. In controls, axonal targeting (not just passing) wasundetectable at P4 but became ratherobvious at P7 and P10.However,the mutants showed clear axonal branching at P7. Quantificationdemonstrated decreased projections to the dLGN (∼85% at P4,∼89%at P7 and∼57%at P10) and increased projections to theLP (∼4.2-foldat P4, ∼3.9-fold at P7 and ∼3.5-fold at P10).

Overexpression of Rorβ in SC redirects sSC-thalamicprojectionsTo investigate whether mis-expression of Rorb is sufficient toredirect SC neuronal projections to thalamus, we delivered Rorb byelectroporation into wild-type mice at E13 and AAV injections atE13 and E15.5. Delivery at E13 produced no interpretable resultsbecause of very little labeling in the sSC (electroporation) orexcessive labeling in thalamus (AAV). Therefore, we analyzed theresults obtained by delivering AAV-Rorβ and AAV-YFP into theSC at E15.5 and characterizing axonal projections at ∼P18(Fig. 4A). The fidelity of co-expression was validated by injectingAAV-YFP and AAV-mCherry (∼90% of YFP+ cells expressedmCherry; n=325 cells; Fig. 4B). Rorb overexpression was

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confirmed by RT-qPCR (Fig. 4C). Quantification of YFPfluorescence revealed that Rorb overexpression increasedprojections to the dLGN by ∼1.6 fold and decreased projectionsto the LP by ∼69% (Fig. 4D-K). This result strongly suggests thatRorb is sufficient to bias axon-target selection of sSC neurons.Here, we examined how long-range neuronal connections are

established during development of the mouse midbrain. Using anovel FLP-DOG genetic strategy, we identified Rorβ as a keymolecule regulating sSC-thalamic projections. We cannotcompletely rule out Rorβ involvement in specifying neuronalidentity because of the lack of the markers for other sSC neuronaltypes. However, the normal layer distribution of Rorβ+ neurons inthe mutants suggests that Rorβ has no obvious effects on the identityacquisition of neurons projecting to the dLGN/LP.Based on axonal phenotypes at different time points, we

speculate that Rorβ might regulate the developmental timing ofsSC projections: Rorb loss seems to delay projections to the dLGN

but accelerate them to the LP. Projections to the dLGN, althoughnever reaching the control level by P20, increase over time (∼15%of control at P4 and ∼34% at P20), suggesting a delay in targeting.Reduced projections to the dLGN could be also explained by failedaxonal arborization. Although we saw no obvious projections to thedLGN in P4 mutants, supporting the former possibility, single cellanalysis or advanced imaging may provide more definitive answers.

Specific recognition molecules crucial for establishing neuronalconnections (Maness and Schachner, 2007; Riccomagno andKolodkin, 2015) could acts as downstream effectors of Rorb. Wepreviously reported that several adhesionmolecules are differentiallyexpressed in sSC Rorβ+ neurons (Byun et al., 2016). It will beinteresting to examine whether their altered expression couldphenocopy the projection changes observed in Rorb mutants. AssSC projections to the dLGN have been implicated in directionselectivity (Bickford et al., 2015), it may be important to assesswhether Rorb deletion alters this function in sSC or dLGN neurons.

Fig. 2. Rorb deletion alters sSC neuronalprojections. Rorβ+ projections visualizedusing FLP-DOG strategy (GFP, green;mCherry, magenta/red). (A-D) Rorb lossdecreases axonal projections to the dLGNand increases axonal projections to the LP atP10. Arrows (C,D) indicate axonal targeting tothe caudal vLGN in control and globalknockout (KO) mice. LP was identified by itsunique location and low GFP level (no clearcalretinin expression). (A′-D′) Magnified viewsof the dLGN and LP. (E-H) No obviouschanges of axonal innervation in the vLGN,PT and PBGN (DAPI, blue). Barely detectableChAT staining at P10 (cyan in G,H). (I)Quantification of axonal projections to thedLGN, LP and PBGN. Data are presented asrelative changes in fluorescence intensitynormalized to control (n=4 mice/group); mean±s.e.m.; *P<0.05; ***P<0.001. Scale bars:250 µm in A-F; 100 µm in G,H.

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MATERIALS AND METHODSAnimalsAll animal procedures were approved by the Institutional Animal Care andUse Committee at Yale University and were in compliance with federalguidelines. We used both female and male animals in the study. The age ofthe animals was specified in each experiment. For timed pregnancies, thedate of the vaginal plug detection was considered as E0.5.

Generation of the Rorb1g/+ mouse, in which a reporter construct wasknocked into the retinoid-related orphan nuclear receptor β (Rorb) gene, hasbeen described previously (Liu et al., 2013). Briefly, the coding sequence ofgreen fluorescent protein (GFP) replaced the Rorb1-specific exon, leading tocomplete loss of Rorb1mRNA in the Rorb1g/1g mouse. Given that Rorb1 is apredominantly expressed isoform in the sSC (Byun et al., 2016), we referredto Rorb1g/+ mouse as Rorb+/− mouse throughout the text. A conditionalRorb mutant, in which an exon encoding the first zinc finger of the DNA-binding domain is flanked by loxP sites (Rorbflox/flox), was derived by

targeted homologous recombination in embryonic stem cells. The Neoselection cassette was deleted by FLP recombinase, then FLP was removedby out-crossing (Ozgene, Australia). For genotyping wild-type and floxalleles, the following primers were used: Rorb-F2, 5′-TTTAGCGGAAG-CCCAGGAAGG-3′; Rorb-R5, 5′-TCAAATGGAGTCAGTGTTGC-3′,which yielded a 520 bp band for the wild type, a 720 bp band for thefloxed allele and a 210 bp band for deletion of the exon between the loxPsites. For deletion of Rorb in the colliculus, we used engrailed1-Cre(En1-Cre) mice. Cre expression in En1-Cre mice is limited to the midbrainand hindbrain and turns on around E9.5 (Kimmel et al., 2000; JacksonLaboratory, #007916). To validate the properties of the En1-Cre line, wecrossed it to the Ai14 line, which expresses Cre-dependent tdTomato(Madisen et al., 2010; Jackson Laboratory, #007914) and confirmedrestricted Cre expression in the midbrain and hindbrain. Conditionaldeletion of Rorb in the Rorbflox/−; En1-Cre mouse was confirmed by thestandard reverse transcription PCR. The primers used for amplification

Fig. 3. Conditional deletion of Rorb in the SC phenocopies altered sSC neuronal projections in global Rorb mutants. FLP-DOG-based visualization(GFP, green; mCherry, magenta/red). (A,B) Decreased projections to the dLGN and increased projections to the LP of cKO mice at P20 (CR, cyan).(C) Quantification of axonal innervation in the dLGN and LP. Data are presented as relative changes in fluorescence intensity normalized to control (n=5-7mice/group). (D-H) No obvious changes of axonal innervation in the vLGN, PTandPBGNat P20 (ChAT, cyan; DAPI, blue). (F) Quantification of axonal projectionsto the PBGN. (I-O) Developmental analysis of Rorβ+ neuronal projections to the dLGN and LP. Axonal branches in the dLGN were detectable at P4/P5 in control,but not until P7 in cKO. Axonal branches in the LP were barely detectable at P4/P5, but noticeable at P4/P5 in cKO. (O) Quantification of axonal branches inthe dLGN and LP (n=5 mice/group/stage); mean±s.e.m.; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Scale bars: 300 µm in A,B,G-N; 100 µm in D,E.

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were: forward 5′-TCATCACGTGTGAAGGCTGCAAG-3′ and reverse 5′-ATCCTCCCGAACTTTACAGCATC-3.

ImmunohistochemistryMice were anesthetized by intraperitoneal injection of a combination of100 mg ketamine plus 10 mg xylazine/kg of bodyweight and perfusedtranscardially with 4% paraformaldehyde (PFA)/PBS. For embryonicstudies, the heads of the embryos were separated from the bodies, post-fixed for 3-4 h at 4°C, then incubated with 15% sucrose/PBS for∼6 h at 4°Cfollowed by 30% sucrose/PBS overnight at 4°C and sectioned using acryostat (16-20 µm sections). For postnatal studies, whole brains weredissected, post-fixed overnight at 4°C and prepared for sectioning. Forcryosectioning, tissue was incubated with 15% sucrose/PBS followed by30% sucrose/PBS overnight at 4°C and sectioned at 20-35 µm. For free-floating sectioning, tissue was post-fixed with 4% PFA/PBS overnight at4°C, washed with PBS and sectioned at 35-50 µm using a vibratome. Forquantification of axonal projections to the subcortical areas, 35 µm-thicksections were chosen. For immunostaining, the sections were washed twicefor 5 min with PBS, blocked with 3-5% donkey serum/0.1% Triton X-100/PBS for 30 min at room temperature, and incubated with the primaryantibodies for 2-3 days at 4°C. Then, the sections were incubated withappropriate secondary antibodies for 2 h at room temperature.

Primary antibodies used were: rabbit anti-GFP (1:1000, Millipore,AB3080P), chicken anti-GFP (1:1000, Aves Laboratories, GFP-1020),rabbit anti-DsRed (1:1000, Clontech, 632496), goat anti-ChAT (1:500,

Millipore, AB1440), mouse anti-NeuN (1:1000, Millipore, MAB377),mouse anti-calretinin (1:2000, Milipore, MAB1568), mouse anti-SMI312(1:1000, BioLegend, 837904), rabbit anti-cleaved caspase 3 (1:1000, CellSignaling Technology, 9661), rabbit anti-Ki67 (1:500, Thermo Scientific,RM9106). Secondary antibodies were conjugated to Alexa Fluor-488, Cy3or Cy5 (all from Jackson ImmunoResearch Laboratories; 703-545-155,711-545-152, 711-165-152, 715-165-151, 715-175-151, 705-175-147) anddiluted 1:500.

Construction and production of recombinant AAVTo generate the plasmid that carries FLP-dependent mCherry, we amplified themCherry sequence by PCR, created AscI and NheI sites at each end andreplaced PCR products with the DNA fragment in the AAV vector (Addgene#74291). The final product contained mCherry between two nested pairs ofincompatible FRT sequences (pAAV-CAG-fDIO-mCherry-WPRE-SV40pA).The AAV plasmid carrying FLP-DOG was obtained from Addgene (#75469).

For overexpression experiments, full-length Rorbwas excised with EcoRIfrom the previously generated plasmid Rorb-pCR8/GW/TOPO TA vector(Invitrogen; Byun et al., 2016), treated with Klenow fragment and sub-cloned into the AAV vector (Addgene #18917) blunted after BamHI andEcoRI digestion. Generation of AAV-YFP construct or AAV-mCherry hasbeen previously described (Fink et al., 2017).

Virus production was based on a triple-transfection, helper-free method,and virus was purified as described previously (Park et al., 2015). Briefly,HEK293 cells in exponential growth phase were transfected with the DNAs

Fig. 4. Overexpression of Rorβ in the SCredirects sSC-thalamic projections. (A)Schematic of in utero injection at E15.5 andanalysis in the sSC and thalamus at P18. (B)Colocalization of YFP (green) and mCherry(magenta) in the sSC revealing significantlyoverlapping expression (n=2 mice).(C) Increase of Rorb mRNA level afteroverexpression (RT-qPCR; n=2 mice). (D-K)Rorb overexpression increases projectionsto the dLGN and decreases projections tothe LP at P18 (YFP, green; CR, magenta).Dashed lines (F,H,J) demarcate sSC areaswhere labeled neurons were counted. ThedLGN and LP were identified by CR staining.Arrows (I,K) indicate possible projectionsfrom the labeled neurons in the deep layer ofthe SC. (D,E) Quantification of axonalinnervation in the dLGN and LP. Data arepresented as relative changes influorescence intensity normalized to control(n=5-7 mice/group); mean±s.e.m.; **P<0.01;****P<0.0001. Scale bars: 100 µm in B;300 µm in F-K.

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using polyethylenimine. DNA mixtures contained a plasmid carrying AAVcapsid 2/1 genes (UPennVector Core), delta F6 plasmid (UPennVector Core)and a plasmid carrying the gene of interest. Cells were harvested 72 h aftertransfection. Viral vectors were purified using a step gradient of iodixanolby ultracentrifugation, buffer-exchanged to PBS, and concentrated usingUltracel (Millipore). The titer was determined by quantitative PCR usingprimers that recognize WPRE or human growth hormone poly A sequences;the concentrated titers were >1013 viral genome particles/ml in allpreparations. Viral stocks were stored at −80°C.

Intraocular injectionTo visualize axonal distribution of retinal ganglion cells in the sSC, Rorb+/−

mice were anesthetized by intraperitoneal injection of a combination of100 mg ketamine plus 10 mg xylazine/kg of bodyweight. A small hole wasmade in the eyewith an insect pin to release intraocular pressure. Cholera toxinB subunit conjugated to Alexa Fluor-555 (CTB-555; 1-2 µl of 1 mg/ml,Invitrogen) was injected through the same hole using a Hamilton syringe.Intraperitoneal injections of the analgesic buprenorphine (0.05-0.1 mg/kg ofbodyweight) were given before and after the surgery.

Anterograde and retrograde labelingTo analyze axonal projections of sSCRorβ+ neurons, we injected AAV-FLP-DOG andAAV-fDIO-mCherry (ratio of 1:5 or 5:1) into the sSC.We kept the5:1 ratio between FLP-DOG and fDIO-mCherry in most experiments exceptwhen labeling the brains that were examined at P4 and at P7.We changed theratio of FLP-DOG to fDIO-mCherry to 1:5 to increase the level of mCherryexpression. Quantification revealed that ∼98% of mCherry+ cells expressGFP (n=745 cells, 3 mice) at P20 and ∼95% of mCherry+ cells express GFP(n=364 mCherry+ cells, 3 mice) at P7. These results indicate that the FLP-DOG to fDIO-mCherry ratio did not affect the fidelity of the FLP-DOGstrategy. For injections, P0-P1 pups were placed on ice to inducehypothermia, and a small incision was made on the skin over the SC. Theskull and brain tissue were simultaneously penetrated with disposable glasspipettes containing AAVs. Then ∼200 nl of AAVs were administrated by apressure injector. After injection, pipettes were gently withdrawn to preventbackflow and the skin was sealed using cyanoacrylate glue. Oral injections ofnonsteroidal anti-inflammatory analgesics (meloxicam, 0.3 mg/kg ofbodyweight) were given before and after the surgery for 2 days. Animalswere sacrificed at P4/P5, P7/P8, P10/P11 and P20/P21. The brains weredissected, sectioned and immunostained with antibodies to GFP (to amplifyRorβ+ signal) and DsRed (to amplify mCherry signal). Axonal projections ofthe labeled neurons were examined in sections of the entire brain extendingfrom the suprachiasmatic nucleus to the cerebellum.

To retrogradely label sSC neurons that project to the dLGN or LP, we useda fluorescent dye (CTB-555). For stereotaxic injections, the mice wereanesthetized by intraperitoneal injection of a combination of 100 mgketamine plus 10 mg xylazine/kg of bodyweight and a small craniotomywas made over the dLGN or LP. Coordinates used for dLGN injection werebregma −2.3 mm, lateral ±2.5 mm, dura −2.5 mm. Coordinates used forLP injection were bregma −2.0 mm, lateral ±1.8 mm, dura −2.4 mm.Fluorescent dye (∼200 nl) was injected with a glass pipette at a rate of∼15 nl/min and the pipette was left in place for 5 min after injection.Intraperitoneal administration of the analgesic buprenorphine (0.05-0.1 mg/kgof bodyweight) was given before and after the surgery. Two days later, wedissected the brain and performed immunostaining with an antibody to GFP todetect Rorβ localization. Labeled sSC neurons (CTB+ or CTB+ andGFP+)werequantified in each animal using three coronal sections, 200 µm apart. Neuronswere counted in areas of equal size within the colliculus.

In utero injectionIn utero injections were conducted following an in utero electroporationprocedure previously described (Saito, 2006) without electric shock. Foroverexpression experiments, pregnant CD-1 females (E15.5) wereanesthetized by intraperitoneal injection of a combination of 100 mgketamine plus 10 mg xylazine/kg of bodyweight. The embryos wereexposed after laparotomy and a mixture of AAV-Rorβ and AAV-YFP (1:1ratio) or AAV-YFP alone (1:1 with PBS) were administered into the SC ofeach embryo by a pressure injector. Intraperitoneal injections of the

analgesic buprenorphine (0.05-0.1 mg/kg of bodyweight) were given beforeand after the surgery. Embryos were allowed to develop and pups weresacrificed at P18. The brains were dissected, sectioned and immunostainedwith antibodies to GFP (to amplify YFP signal) and calretinin (to delineatedLGN and LP). For quantification, the number of labeled neurons in the sSCand fluorescence intensity of axonal terminals in the thalamic nuclei wereanalyzed.

To examine co-transfection efficiency, AAV-YFP and AAV-mCherry(1:1 ratio) were injected. For quantification, sectioned brains wereimmunostained with antibodies to GFP and DsRed.

Quantitative RT-PCR (RT-qPCR)To assess overexpression of Rorb after in utero injections, the brains weredissected at P2 and the SCs were isolated. Total RNAwas prepared using theRNeasy Mini Kit (Qiagen) and cDNA was synthesized using either theSuperscript III First-Stand system (Thermo Fisher) or the iScript gDNAclear cDNA synthesis kit (Bio-Rad). RT-qPCR was performed in duplicateusing iQ SYBR Green Supermix (Bio-Rad) on the CFX96 real-time system(Bio-Rad). The Ct values of samples were normalized to that of Gapdh andoverexpression levels of Rorb in relation to controls were calculated by theΔΔCt method. Primers used for qPCR were as follows: Gapdh, 5′-GTGG-AGTCATACTGGAACATGTAG-3′ and 5′-AATGGTGAAGGTCGGTG-TG-3′; Rorb, 5′-TCATCACGTGTGAAGGCTGCAAG-3′ and 5′-ATCCTCCCGAACTTTACAGCATC-3′.

Image acquisition and analysisImages were acquired using a Zeiss Imager M2 fluorescence microscopeand a Zeiss LSM 800 confocal microscope. z-stacks were obtained with1 µm steps using a 20× objective (NA 0.8) and 0.5 µm steps using a 40×objective (NA 1.4). ImageJ software (National Institutes of Health) was usedfor data analysis.

To quantify axonal projections to subcortical areas, images were takenusing 35 µm-thick coronal sections, ∼200 µm apart, extending from rostralto caudal areas of the brain. Sections containing both the dLGN and LP wereselected, and the regions of the dLGN and LP covered by axon terminalswere demarcated. Fluorescence intensity of each demarcated area was me-asured using ImageJ as the sum of pixel values after subtracting backgroundsignal; the total intensity from two or three sections (depending on theanimal’s age) was calculated for each nucleus. To account for injectionvariability, the summed fluorescence intensities from the dLGN and LPsections were divided by the total number of labeled sSC cells that werecounted in the sSC sections, ∼200 µm apart, covering the entire infectedsSC area. Cell counting for overexpression experiments was conducted inthe sSC areas spanning 350 µm from the pia. Data obtained for dLGN andLP in mutants were normalized to controls and presented as percentages.

To quantify the total number and distribution of Rorβ+ neurons,anatomically matched sections were selected. For embryonic analysis,images were taken using sections that spanned ∼120 µm from the medialedge to the lateral edge of the ventricular zone. For postnatal analysis, imageswere taken using sections that spanned ∼200 µm from the medial edge to∼200 µm from the lateral edge. For cell counting, the images were cropped to350 µm×450 µm (width×height). Such cropping covers an entire area from thepia to the ventricular zone in embryonic tissues and visual input areas from thepia in postnatal tissues. To analyze the distribution of neurons, images werehorizontally divided from the pia and cells were grouped into 25 µm bins.

To quantify retrogradely labeled sSC neurons in adult brains, we adaptedcounting strategies employed in the analyses of cortical projection neurons(Arlotta et al., 2005; Galazo et al., 2016). Briefly, images were taken usingthree coronal sections, 200 µm apart. For cell counting, images werecropped at ∼200 µm from the midline to avoid the most medial part wherelabeling of axons and neurons was mixed. The cropped sizes were either450 µm×450 µm or 450 µm×600 µm (width×height).

Experimental design and statistical analysisAxonal projections were quantified using two or three different sections formeasurement of fluorescent intensity in thalamic nuclei, three to sevendifferent sections for counting the labeled sSC cell number per animal andsummed for each of five to seven animals per condition. Total number or

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layer distribution of Rorβ+ or NeuN+ neurons were analyzed using three tosix different sections per animal and averaged for each of three or fouranimals per condition. Animals of either sex were collected from two to fivedifferent litters for each time point.

All the data are reported asmean±s.e.m. and analyzedusingGraphPadPrismsoftware (GraphPad Software). Means between two groups (control and eitherglobal or conditional mutants or control and overexpression) were comparedusing an unpaired two-tailed Student’s t-test. The value of n represents thenumber of animals used per condition. The following significance levels wereused: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

AcknowledgementsWe thank Joshua Sanes (Harvard University) for helpful discussions. This work wassupported by a NIH grant (EY026878: a Core Grant for Vision Research for YaleUniversity) and by an unrestricted grant from Research to Prevent Blindness tothe Department of Ophthalmology and Visual Science at Yale University.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: I.-J.K.; Methodology: H.L., D.F., I.-J.K.; Validation: H.B., H.-L.L.,A.R., I.-J.K.; Formal analysis: H.B., H.-L.L., A.R., I.-J.K.; Investigation: H.B., H.-L.L.,A.R., I.-J.K.; Resources: H.-L.L., H.L., D.F.; Writing - original draft: A.R., I.-J.K.;Writing - review & editing: A.R., I.-J.K.; Visualization: A.R., I.-J.K.; Supervision:I.-J.K.; Project administration: I.-J.K.; Funding acquisition: D.F., A.R., I.-J.K.

FundingThis research was supported byWhitehall Foundation grants (2014-05-103 to I.-J.K;2017-08-39 to A.R.), the E. Matilda Ziegler Foundation for the Blind (10-001306 toI.-J.K.) and partially by the intramural research program at the National Institutesof Health (H. Liu and D.F.). Deposited in PMC for release after 12 months.

Supplementary informationSupplementary information available online athttp://dev.biologists.org/lookup/doi/10.1242/dev.171926.supplemental

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