Research Article: New Research | Development Distinct 3 (Opn3) expression in the developing during mammalian embryogenesis https://doi.org/10.1523/ENEURO.0141-21.2021

Cite as: eNeuro 2021; 10.1523/ENEURO.0141-21.2021 Received: 31 March 2021 Revised: 6 August 2021 Accepted: 11 August 2021

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Copyright © 2021 Davies et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

Distinct Opsin 3 (Opn3) expression in the developing nervous system

during mammalian embryogenesis

Wayne I. L. Davies1#, Soufien Sghari1#, Brian A. Upton2-4#, Christoffer Nord1, Max Hahn1,

Ulf Ahlgren1, Richard A. Lang3-6, Lena Gunhaga1*

1Umeå Centre for Molecular Medicine (UCMM), Umeå University, Umeå, 901 87, Sweden

2Medical Scientist Training Program, University of Cincinnati, Cincinnati, OH, USA

3The Visual Systems Group, Abrahamson Pediatric Eye Institute, Cincinnati Children's

Hospital Medical Center, Cincinnati, OH, USA

4Center for Chronobiology, Division of Pediatric Ophthalmology, Cincinnati Children's

Hospital Medical Center, Cincinnati, OH, USA

5Division of Developmental Biology, Cincinnati Children's Hospital Medical Center,

Cincinnati, OH, USA

6Department of Ophthalmology, University of Cincinnati, College of Medicine, Cincinnati,

OH, USA

#Equal authorship contribution

*Corresponding author: Lena Gunhaga, [email protected]

Abbreviated title

Embryonic Opn3 expression in neural development

1

Author contributions

Wayne I L Davies, Methodology, Data curation, Formal analysis, Writing – original draft,

Writing – review & editing.

Soufien Sghari, Methodology, Data curation, Formal analysis, Writing – original draft,

Writing – review & editing.

Brian A Upton, Methodology, Data curation, Formal analysis, Writing – review & editing.

Christoffer Nord, Methodology, Software, Writing – review & editing.

Max Hahn, Methodology, Software, Writing – review & editing.

Ulf Ahlgren, Funding acquisition, Resources, Software, Writing – review & editing.

Richard A Lang, Conceptualization, Funding acquisition, Resources, Writing – review & editing.

Lena Gunhaga, Conceptualization, Funding acquisition, Resources, Writing – original draft,

Writing – review & editing.

Number of figures, tables and extended data figures/tables

Main Figures: 9

Main Table: 1

Movies: 2

Suppl. Figures: 6

Number of words

Abstract: 225

Introduction: 866

Discussion: 1 811

2

Funding sources

The research conducted in the Gunhaga laboratory was supported by the Swedish Research

Council (2017-01430), Insamlingsstiftelsen, Medical Faculty at Umeå University, and

Kempestiftelserna (SMK-1763). Work in the Lang laboratory was supported by the National

Institutes of Health (R01EY027077, R01EY027711, R01EY032029 and T32GM063483), and in the Ahlgren lab by the Swedish Research Council (2017-01307) and

Insamlingsstiftelsen, Medical Faculty at Umeå.

Conflicts of interest

The authors declare that no conflict of interest exist.

Acknowledgements

We thank Maria Eriksson and Federico Morini, Umeå University, for technical advice related to 3D imaging protocols, and Paul Speeg, Cincinnati Children's Hospital, for mouse colony management. We thank the following persons for kindly providing antibodies; Anna Överby

Wernstedt (GFAP) and Helena Edlund (donkey anti-goat Alexa 488), both at Umeå

University.

3

1 Abstract

2 Opsin 3 (Opn3) is highly expressed in the adult , however, information for spatial and

3 temporal expression patterns during embryogenesis is significantly lacking. Here an Opn3-

4 eGFP reporter mouse line was utilized to monitor cell body expression and axonal projections

5 during embryonic and early postnatal to adult stages. By applying 2D and 3D fluorescence

6 imaging techniques, we have identified the onset of Opn3 expression, which predominantly

7 occurred during embryonic stages, in various structures during brain/head development. In

8 addition, this study defines over twenty Opn3-eGFP positive neural structures never reported

9 before. Opn3-eGFP was first observed at E9.5 in neural regions, including the ganglia that

10 will ultimately form the trigeminal, facial and vestibulocochlear cranial nerves. As

11 development proceeds, expanded Opn3-eGFP expression coincided with the formation and

12 maturation of critical components of the central and peripheral nervous systems, including

13 various motor-sensory tracts, such as the dorsal column- sensory tract, and

14 olfactory, acoustic and optic tracts. The widespread, yet distinct, detection of Opn3-eGFP

15 already at early embryonic stages suggests that Opn3 might play important functional roles in

16 the developing brain and to regulate multiple motor and sensory circuitry systems,

17 including proprioception, , ocular movement and olfaction, as well as memory,

18 mood and . This study presents a crucial blueprint from which to investigate

19 autonomic and cognitive opsin-dependent neural development and resultant behaviors under

20 physiological and pathophysiological conditions.

21

4

22 Significance Statement

23 The expression of the mammalian Opsin 3 (Opn3) has only recently been characterized in

24 adults, with no significant study during embryonic development. This study utilized an Opn3-

25 eGFP mouse line to identify Opn3-related development of the CNS and PNS. 2D and 3D

26 fluorescence imaging revealed cell body and axonal Opn3-eGFP expression, which indicated

27 an early onset of Opn3-eGFP in cranial and spinal nerve ganglia, and sensory organs.

28 Embryonic expression of Opn3-eGFP was also identified in the , ,

29 and , whereas Opn3-eGFP expression in the and

30 associated regions were observed postnatally. The presence of Opn3, a short-

31 wavelength-sensitive , in many brain regions during embryogenesis is of great

32 interest when investigating autonomic and cognitive photo-dependent neural development.

33

5

34 Introduction

35 Light detection (photoreception) and its related disorders are often focused on the visual

36 system, where eyes and the specialized rod and cone photoreceptors they contain are crucial

37 for forming images of the external world. However, it has become evident that there is a

38 multiplicity of non-visual light detection processes, where specialized G -coupled

39 receptors (i.e. ) mediate both visual and non-visual photoreception. Briefly, the opsin

40 protein moiety is covalently linked to a light-sensitive retinoid , which together

41 form a photopigment (Davies et al., 2012; Yokoyama, 2000). In the presence of particular

42 wavelengths of light, absorb the energy of a and, thereafter, activate

43 functionally relevant phototransduction signaling cascades (Lamb et al., 2016; Shichida and

44 Matsuyama, 2009).

45

46 For many years, it was assumed that visual opsins in the rods and cones, and a subset of

47 intrinsically photosensitive ganglion cells (RGCs; see Extended Data for a list of

48 abbreviations) were the only opsin-based photoreceptors present in , all of which

49 were restricted to the . However, around 20 years ago, so-called atypical or non-visual

50 opsins, including Opsin 3 (Opn3, also known as encephalopsin or panopsin), Opn4

51 () and Opn5 (neuropsin), were identified in photoreceptors that were not thought

52 to be involved in directly (Blackshaw and Snyder, 1999; Provencio et al.,

53 1998; Tarttelin et al., 2003b). Of these three opsins, melanopsin has been the most studied in

54 terms of function in visual and non-visual light detection processes, where it has been shown

55 to play a key role in photoentrainment and pupillary constriction in mammals

56 (Freedman et al., 1999; Lucas et al., 2003; Sghari et al., 2020; Xue et al., 2011). Other known

57 non-visual effects of Opn4 include DNA repair activation (Hirayama et al., 2009) and cell

6

58 cycle regulation (Dekens et al., 2003). Nevertheless, in mammals, relatively little is known

59 about non-visual light detection processes.

60

61 Consistent with reported light-regulated processes other than vision, the presence of

62 atypical photopigments in multiple non-retinal adult tissues has been observed. For example,

63 Opn4 has been detected in melanocytes (Provencio et al., 1998), the (Sghari et al., 2020;

64 Xue et al., 2011), cornea and associated trigeminal ganglia (Delwig et al., 2018), blood

65 vessels and heart (Regard et al., 2008; Sikka et al., 2014), and Opn5 being found in the eye,

66 brain, testes, ear and skin (Buhr et al., 2019; Buhr et al., 2015; Haltaufderhyde et al., 2015;

67 Kojima et al., 2011). The initial identification and expression of Opn3 in the adult mouse

68 brain, including regions like the cerebral cortex and cerebellum (Blackshaw and Snyder,

69 1999), have been verified and extended (Nissila et al., 2012; Olinski et al., 2020b). Recent

70 studies in the adult mammalian brain, especially that of rodents and some , indicate

71 that Opn3 exhibits a far wider expression profile (Nissila et al., 2012; Olinski et al., 2020b),

72 which suggests the potential for many unknown roles of light signaling in the brain and other

73 organs. Notably in adult , including higher mammals, Opn3 is expressed in

74 adipocytes and several tissues, such as,, heart, intestine, kidneys, liver, ovaries, ,

75 placenta, pulmonary arteries, retina and associated eye structures, skeletal muscle, skin,

76 testes, uterus, trachea and lungs (Blackshaw and Snyder, 1999; Halford et al., 2001;

77 Haltaufderhyde et al., 2015; Nayak et al., 2020; Olinski et al., 2020a; Ortiz et al., 2018;

78 Tarttelin et al., 2003a; Wu et al., 2021; Yim et al., 2020). In addition, phylogenetic analyzes

79 indicate that related orthologues of Opn3 are present in all major classes studied to

80 date (Davies et al., 2015; Shichida and Matsuyama, 2009), suggesting a conserved function

81 for this photopigment. The widespread adult expression pattern of these non-visual opsins

82 suggests that light may play a broader functional role than previously thought. Whether non-

7

83 visual opsins are also spatially and temporally expressed during embryonic stages, and if so,

84 in which structures and when (i.e. the time of onset), have not been defined.

85

86 A sufficient number of is able to penetrate the skin, skull, soft tissues, including

87 the mammalian brain itself, at both fetal and adult stages to activate photoreceptors (Dunn et

88 al., 2015; Nayak et al., 2020; Reid et al., 2017; Sun et al., 2016; Zhang et al., 2020).

89 Therefore, it is essential to determine if Opn3 might play a significant role in brain

90 development and function. As such, the primary aims of this study were to utilize an Opn3-

91 eGFP reporter mouse model to investigate the expression of Opn3 throughout embryonic (E)

92 development, just after birth, as well as juvenile and adult stages. A combination of

93 immunofluorescence techniques, including 3D imaging by optical projection tomography

94 (OPT) was used.

95

96 This study provides a detailed appreciation of the early onset of Opn3 at around E9.5 and

97 broad, yet distinct, expression of Opn3 in the developing mammalian embryo. Specifically,

98 numerous Opn3-eGFP positive cranial ganglia, neural projections and nuclei were detected in

99 the developing brain and spinal cord, which together identify key structures of major neural

100 pathways and circuits. Collectively, these results form the basis of further investigations into

101 how Opn3-dependent signaling might regulate the development of the nervous system and

102 other key structures, which might prove significant in understanding the season-of-

103 conception/birth dependent risk of developing several metabolic, neurological and psychiatric

104 diseases (Foster and Roenneberg, 2008).

105

106 Materials and Methods

107 Ethics statement

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108 All procedures were performed in accordance with the [Author University] animal

109 care committee’s regulations.

110

111 Opn3-eGFP reporter mice

112 A genetically-modified Opn3-eGFP (enhanced green fluorescent protein) reporter mouse

113 strain (Tg[Opn3-EGFP]JY3Gsat; MMRRC/GENSAT stock number 030727-UCD, Mutant

114 Mouse Resource & Research Centers, University of California, Davis, California) was

115 generated as detailed previously (Nayak et al., 2020). This reporter mouse strain contains an

116 eGFP reporter inserted at the start of 1 of the coding sequence of Opn3, but

117 downstream of the Opn3 promoter. As such, eGFP expression was driven by the Opn3

118 promoter and is stated as Opn3-eGFP herein. were housed on a 12 h:12 h light-dark

119 (LD) cycle in a temperature and humidity-controlled environment.

120

121 Genotyping and PCR

122 Genotyping to confirm the presence of the Opn3-eGFP allele was performed using an

123 oligonucleotide pair (forward primer, 5´-CAGAGCGTGAGATCCACCCTGTT-3´; reverse

124 primer, 5´-TAGCGGCTGAAGCACTGCA-3´) that generated an amplicon of 320 bp under

125 the following PCR cycling conditions: an initial step of 94°C for 5 min, followed by 30

126 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 30 s, and a final elongation step of 72°C

127 for 7 min.

128

129 Tissue preparation

130 Adult mice and pregnant females (1-5 months) were perfused with fresh 4%

131 paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) (pH 7.4) that was filtered through a

132 0.2 μm syringe filter to remove particulates. Where stated, whole embryos, heads or

9

133 from embryonic stages (E8.5, E9.5, E10.5, E13.5, E15.5 and E17.5), as well as from

134 postnatal (P) stages 0.5, P10 and adults, were collected. At least three and up to five

135 specimens of each time point were analyzed. In all cases, retrieved embryonic, postnatal or

136 adult tissues were immersion-fixed in fresh filtered PFA (4%) at 4°C for 4 h to overnight

137 (ON), before being stored in fresh 0.1 M PB at 4°C until further use.

138

139 Immunohistochemistry

140 Gradual cryo-protection of whole embryos or selected tissues using 3.25% to 25% sucrose

141 in PB at 4°C was performed, and thereafter embedded in NEG-50 (Cellab, Stockholm,

142 Sweden). Samples were stored at -80°C and cryo-sectioned with thickness at 20 μm (for

143 E8.5-E10.5), 40 μm (for E13.5 to adult stages), or 10 μm (for E17.5 Opn3-eGFP/GFAP

144 staining). For Opn3-eGFP reporter detection, immunohistochemistry was used by applying

145 standard protocols (Wittmann et al., 2014) with a rabbit anti-GFP antibody (1:500 dilution;

146 Thermo Fisher Scientific, Göteborg, Sweden; #A11122) and an anti-rabbit IgG Cy3-

147 conjugated secondary antibody (1:400 dilution; Jackson ImmunoResearch Europe, Ely, UK;

148 #111-165-003), or an anti-rabbit Alexa 488-conjugated secondary antibody (1:300, Jackson

149 ImmunoResearch, West Grove, Pennsylvania). For glial fibrillary acidic protein (GFAP) and

150 Opn3-eGFP co-labelling, rabbit anti-GFAP (1:500 dilution; gift from Anna Överby

151 Wernstedt, Umeå University, Sweden) and goat anti-GFP (1:500 dilution; Thermo Fisher

152 Scientific, Göteborg, Sweden; #A600-101-215M) primary antibodies were used. Secondary

153 antibodies used were; donkey anti-rabbit Alexa 594-conjugated (1:400 dilution; Thermo

154 Fisher Scientific, Göteborg, Sweden; #A21207) and donkey anti-goat Alexa 488-conjugated

155 antibodies (1:400 dilution; Thermo Fisher Scientific, Göteborg, Sweden; #A11055; gift from

156 Helena Edlund, Umeå University, Sweden), respectively. In all cases, 4′,6-diamidino-2-

157 phenylindole (DAPI; 1: 400 dilution; Sigma-Aldrich, Stockholm, Sweden) was included to

10

158 counterstain nuclei. Slides were mounted with fluorescent mounting medium (Agilent

159 Technologies, Stockholm, Sweden). Immunohistochemistry sections were examined by

160 fluorescence microscopy (Nikon Eclipse, E800; Nikon Instruments Europe, Amsterdam,

161 Netherlands), with images being captured using a Nikon DS-Ri1 digital camera and Nikon

162 NIS-Elements F v4.6 software, before being processed with Photoshop Creative Cloud (CC)

163 2019 (Adobe, San Jose, California).

164

165 Optical projection tomography (OPT)

166 OPT scanning was performed on whole embryos at E10.5 and brains at P0.5. Sample

167 preparation was performed essentially as previously described (Alanentalo et al., 2007;

168 Eriksson et al., 2013; Sharpe et al., 2002), except for the addition of further freeze-thaw

169 methanol steps to increase antibody permeabilization (for both samples at E10.5 and P0.5)

170 and extended washing steps for P0.5 brain samples. For Opn3-eGFP detection, filtered (0.45

171 μm) rabbit anti-GFP (1:500 dilution, Thermo Fisher Scientific, Göteborg, Sweden; #A11122)

172 and goat anti-rabbit IgG Cy3-conjugated secondary (1:400 dilution; Jackson

173 ImmunoResearch Europe, Ely, UK; #111-165-003) antibodies were used, incubating at RT

174 for 48-72 h at each step. Specimens were briefly equilibrated and placed in filtered low

175 melting point agarose (1.5%; SeaPlaque Agarose, Lonza, Basel, Switzerland) and mounted

176 into positioned, before methanol treatment and clearing in benzyl alcohol and benzyl

177 benzoate (ratio 1:2). Once adequately transparent, specimens were imaged using a Bioptonics

178 3001 OPT scanner (SkyScan, Antwerp, Belgium) using Texas Red (Ex/Em: 560±20 nm/610

179 nm LP; exposure time: 800 ms for E10.5 and 425 ms for P0.5) and FITC (Ex/Em:

180 425±20 nm/475 nm LP; exposure time: 450 ms for E10.5 and 200 ms for P0.5) filter sets to

181 maximize Opn3-eGFP and anatomical (autofluorescence) signals, respectively.

182 Computational post-scanning processing were carried out as previously described (Cheddad

11

183 et al., 2012). 3D tomographic reconstructions were performed using NRecon v1.6.9.18

184 software (SkyScan, Antwerp, Belgium), with visualization and video production conducted

185 by implementing Imaris 9.3.1 software (Bitplane, Belfast, UK).

186

187 Results

188 In this study, the spatial and temporal expression of Opsin 3 (Opn3) was analyzed by

189 examining an Opn3 promoter-driven eGFP mouse line (Opn3-eGFP) with a main focus on

190 embryonic (CNS) and peripheral (PNS) nervous system. The

191 detection of Opn3-eGFP expression was optimized, by using a sequential combination of a

192 primary GFP and a secondary Cy3 antibody, to amplify positive staining and reduce

193 unspecific background labelling. To define both temporal and spatial expression of Opn3-

194 eGFP, embryos and postnatal animals of several stages were analyzed using sections in

195 coronal, transversal and sagittal orientations. The below presented Opn3-eGFP expression

196 patterns were consistent observations comprised from analyzing a minimum of three and up

197 to five specimens of each time point.

198

199 Early onset of Opn3 expression in the developing nervous system

200 Opn3-eGFP expression was detected in the developing CNS and PNS at embryonic day

201 (E) 9.5 (Extended Data Fig. 1-1), but not at E8.5 (data not shown). Cranial nerves (CN) are

202 part of the PNS that have both sensory and/or motor components that direct multiple sensory,

203 motor and autonomic functions. The sensory part, including the ganglia, derives from cranial

204 neural crest cells and ectodermal placode cells (Patthey and Gunhaga, 2011; Schlosser, 2014;

205 Vermeiren et al., 2020). At E9.5, Opn3-eGFP expression was detected in cells of the early

206 forming trigeminal ganglia and facio-acoustic ganglion complex, which will later give rise to

207 the sensory branches of the trigeminal (CN V), facial (CN VII) and vestibulocochlear (CN

12

208 VIII) cranial nerves (Extended Data Fig. 1-1B,C,J,K). Furthermore, Opn3-eGFP positive

209 (Opn3-eGFP+) cells were observed in the basal plate of the neural tube along the antero-

210 posterior axis of the future spinal cord (Extended Data Fig. 1-1A-D,L), as well as the second,

211 third and fourth pharyngeal pouches (Extended Data Fig. 1-1A-C). Additional Opn3-eGFP+

212 non-neural regions, which are not the subject of this study, included the gastrointestinal tract,

213 the dorsal pancreatic bud and the developing heart (Extended Data Fig. 1-1A,B,D).

214

215 Scattered Opn3-eGFP expression was also observed in the olfactory placode (Extended

216 Data Fig. 1-1E,F), the most ventral and dorsal regions of the diencephalic neuroepithelium

217 (Extended Data Fig. 1-1G,H) and the dorsal outer lining of the mesencephalic

218 neuroepithelium (Extended Data Fig. 1-1I). Thus, the onset of Opn3 expression is around

219 E9.5 in both the CNS and PNS.

220

221 At E10.5, Opn3-eGFP is expressed in several motor-sensory related cells

222 At E10.5, Opn3-eGFP expression was detected in the expanding trigeminal ganglia near to

223 the brainstem (Fig. 1A,C,H,I), in the facio-acoustic ganglion complex in close connection to

224 the Opn3-eGFP negative (Opn3-eGFP-) otic vesicle (Fig. 1B,C,H,I), in the developing

225 glossopharyngeal and inferior vagal ganglia, including peripheral projections of the vagal

226 ganglia (Fig. 1B,C,H). In coronal sections, scattered Opn3-eGFP+ cells were located in the

227 neuroepithelium of the brainstem at the level of the trigeminal ganglia, in contrast to the level

228 of the facio-acoustic complex and otic vesicle, where Opn3-eGFP+ cells were primarily

229 located in the lateral lining of the brainstem neuroepithelium (Fig. 1H,I). Moreover, Opn3-

230 eGFP was detected in the dorsal root ganglia, derived from neural crest cells, throughout the

231 antero-posterior axis of the spinal cord (Fig. 1D,E,J). Several afferent processes of the dorsal

232 root ganglia were also observed in the hindbrain region (Fig. 1D,E). Consistent with findings

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233 at E9.5 (Extended Data Fig. 1-1H-K), Opn3-eGFP+ cells were observed along the basal plate

234 of the caudal neural tube, including motor exit points and related peripheral spinal nerve

235 projections (Fig. 1J,K; Extended Data Fig. 1-2A-C).

236

237 By E10.5, the thickened olfactory placode has invaginated: at this stage Opn3-eGFP

238 expression was observed in both the olfactory epithelium and the nearby mesenchyme (Fig.

239 1F), which resembled patterns of post-mitotic olfactory neurons (Miller et al., 2010;

240 Panaliappan et al., 2018). Expression of Opn3-eGFP was also detected in the developing

241 optic stalk, but at this stage no expression was observed in the optic cup (Fig. 1G).

242

243 To obtain a global appreciation of Opn3-eGFP expression, with particular reference to the

244 identified projections, 3D imaging by optical projection tomography (OPT) was conducted,

245 which verified the aforementioned Opn3-eGFP expression pattern noted in E10.5 sections.

246 This included detection of Opn3-eGFP in different components of the PNS, such as nerves,

247 ganglia, as well as sensory (otic) and somatosensory (skin, thorax, abdomen and limbs)

248 projections towards their CNS targets, the hindbrain via the rootlets of cranial ganglia and the

249 spinal cord via the dorsal root ganglia, respectively (Fig. 2; Movie 1). The uniform

250 upregulation of Opn3-eGFP in the dorsal root ganglia along the antero-posterior axis of the

251 embryo, as well as in projections emanating from dorsal root and cranial ganglion, and the

252 olfactory placode were clearly visible (Fig. 2; Movie 1). Consistent with expression observed

253 in E9.5 sections (Fig. 1), Opn3-eGFP was also observed in abdominal structures and the

254 developing heart (Fig. 2; Movie 1).

255

256 Opn3-eGFP detection in distinct ganglia and nerve fibers at E13.5

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257 At E13.5, Opn3-eGFP was detected in many regions of the CNS, including cells of both

258 the mantle and marginal layers of the spinal cord (Fig. 3A). The visualization of Opn3-eGFP

259 labelled spinal motor neurons at E10.5, prior to sensory neurons located in the mantle layer,

260 are consistent with the chronological differentiation of spinal motor neurons that precedes

261 sensory neurons (Rodier, 1980). At the level of the medulla, Opn3-eGFP was detected in the

262 spinal trigeminal nucleus, which receives information about deep/crude touch, and

263 temperature from the face via all three branches of CN V (trigeminal), in addition to branches

264 of CN VII (facial), CN IX (glossopharyngeal) and CN X (vagus) (Fig. 3B,C). Consistently,

265 Opn3-eGFP detection was also noted in trigeminal ganglia rootlets projecting to the principal

266 sensory trigeminal nucleus (Fig. 3H), which receive information about discriminative

267 sensation and light touch of the face, as well as conscious proprioception of the jaw via first

268 order neurons of CN V.

269

270 Opn3-eGFP expression was also observed in the mesencephalic trigeminal nuclei (Fig.

271 3G), which are involved in both proprioception and motor functions of the jaw. Moreover,

272 Opn3-eGFP was detected in the facial nerve (CN VII), including the genu of the facial nerve,

273 which carries both sensory and autonomic fibers (Fig. 3G; Extended Data Fig. 1-2D-F). By

274 this stage, Opn3-eGFP+ vagal and glossopharyngeal ganglionic projections have reached the

275 posterolateral region of the medulla to form the solitary tract (Fig. 3C), which conveys

276 afferent information from stretch receptors and chemoreceptors in the walls of the

277 cardiovascular and respiratory systems, as well as the gastrointestinal tract. Opn3-eGFP was

278 also detected in the (Fig. 3F; Fig. 4C), which relay information from the

279 solitary tract to the thalamus.

280

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281 At E13.5, Opn3-eGFP was detected in both the hypoglossal nucleus and nucleus

282 prepositus (also called nucleus prepositus hypoglossi) (Fig. 3C,F), the latter of which plays

283 an integral role in both vestibular and visual systems by integrating velocity and positional

284 information for horizontal eye movements. Opn3-eGFP+ vestibular fibers of the

285 vestibulocochlear nerve (CN VIII) reach the medulla where they synapse with medullary

286 , also detected as Opn3-eGFP+ (Fig. 3C,E,F). Furthermore, Opn3-eGFP was

287 detected in the medial longitudinal fasciculus (Fig. 3C), which form the ascending tract from

288 the superior and medial vestibular nuclei. The medial longitudinal fasciculus terminates in the

289 oculomotor-associated sub-nuclei that are located along the dorso-ventral and caudo-rostral

290 regions of the tectum of the (Fig. 4A-C), as well as the abducens nucleus in the

291 caudal portion of the (Fig. 3E). The cochlear nuclei, located in the junction of the pons

292 and medulla where the auditory part of CN VIII synapses, were also identified as Opn3-

293 eGFP+ (Fig. 3D-F). In turn, the medullary cochlear nuclei send information to the superior

294 olivary complex (Fig. 3D,E), the latter of which are a collection of nuclei important for

295 ascending and descending auditory pathways.

296

297 In the cerebellum, Opn3-eGFP expression was detected in the external granular layer and

298 (Fig. 3D,G), as well as the superior cerebellar peduncle (Fig. 3F; Fig.

299 4A). This latter structure mainly consists of cerebellar efferent fibers that via the cerebello-

300 rubral tract project to, among others, the contralateral , which also was observed

301 to be Opn3-eGFP+ (Fig. 4B,C). Similarly, the tegmental decussations of ascending and

302 descending fibers were Opn3-eGFP+ (Fig. 4A,B).

303

304 At the level of the , Opn3-eGFP was detected in the subthalamic area and the

305 dorsal thalamus (Fig. 4D-F), as were the projections of the to the dorsal

16

306 thalamus and the medial forebrain bundles in the (Fig. 4D,E). In

307 addition, the dorso- and ventro-medial hypothalamic nuclei (Fig. 4D,E), which are related to

308 the , and the stria medullaris (Fig. 4G), which is part of the

309 , were shown to express Opn3-eGFP. At this stage, the developing cerebral

310 cortex did not express Opn3-eGFP (Fig. 4F). In both visual and olfactory systems, Opn3-

311 eGFP was observed inRGCs, the optic nerve and tract (Fig. 4G,H), as well as the olfactory

312 epithelium, nerve and bulb, and the vomeronasal organ and nerve (Fig. 4I).

313

314 Expanded Opn3-eGFP expression in sensory organs, tracts and nuclei important for

315 motor-sensory functions at E15.5

316 At E15.5, additional and expanded Opn3-eGFP+ regions were observed (Figs. 5-6). In the

317 medulla, scattered cells expressing Opn3-eGFP were located in the medullary reticular nuclei

318 (Fig. 5A,B), which are related to sleep behavior, and the magnocellular reticular nucleus (Fig.

319 5A,B), which project to the branchiomotor nuclei. Moreover, Opn3-eGFP expression was

320 observed in the raphe obscurus, which are localized in the midline of the brainstem and

321 involved with autonomic and motor activities (Fig. 5A,B). Close to the solitary tract, Opn3-

322 eGFP was detected in the medullary ascending cuneate and gracile fasciculus (Fig. 5D).

323 These fasciculi are part of the dorsal column-medial lemniscus (DCML) pathway, which

324 transfer information about proprioception, exteroception and vibratory sensations from the

325 trunk and extremities. This is consistent with findings at E10.5, where the posterior dorsal

326 root ganglia, which are first order neurons in the DCML pathway, were confirmed to be

327 Opn3-eGFP+ (Fig. 1D,E,J). Furthermore, Opn3-eGFP expression was observed in the medial

328 longitudinal fasciculus located in the lateral wall of the (Fig. 5D,E), as well as

329 the genu of the facial nerve (Fig. 5D,E) and the nucleus prepositus (Fig. 5F). The lateral

330 vestibular nucleus in the upper medulla and the superior vestibular nucleus in the pons, which

17

331 are both related to the vestibulo-ocular reflex when changing head and eye positions, were

332 shown to express Opn3-eGFP (Fig. 5F,G).

333

334 In the pons, marked Opn3-eGFP expression was detected in the (also called

335 pontine gray) (Fig. 5A,C) and the medial cerebellar peduncle (Fig. 5A,C), the latter of which

336 conveys motor information regarding intended movements from the pontine nuclei to the

337 contralateral cerebellum. At E15.5, a clear segregation of the Opn3-eGFP+ spinal, motor and

338 sensory trigeminal nuclei were observed (Fig. 5D,F,G), as well as noted Opn3-eGFP

339 expression in auditory structures such as the nucleus of the (Fig. 5D,G) and

340 the brachium of the (Fig. 6A). The pontine reticular nuclei (Fig. 5E-G) and

341 the parabrachial nuclei (Fig. 6A), which relay solitary tract information to the forebrain, were

342 detected as Opn3-eGFP+. In the cerebellum, Opn3-eGFP expression was observed in the

343 dentate, fastigial and interposed nuclei (Fig. 5H).

344

345 In the midbrain, Opn3-eGFP expression was observed in the reticular nuclei (Fig. 6A), the

346 oculomotor-associated sub-nuclei (Fig. 6A; Extended Data Fig. 6-1) and the subthalamic area

347 (Fig. 6F). Opn3-eGFP was also detected in the stria medullaris, containing afferent fibers

348 from, among others, the Opn3-eGFP+ septal nuclei, projecting to the in the

349 medial (Fig. 6A,E,F). At this stage, Opn3-eGFP expression was identified in the

350 decussation of the superior cerebellar peduncle and the ventral tegmental decussation (Fig.

351 6B,C). Consistently, the superior cerebellar peduncle consists of fibers carrying both

352 cerebello-rubral and cerebello-thalamic tracts, here detected as Opn3-eGFP+ (Fig. 6B-D), in

353 which project from deep cerebellar nuclei (Fig. 3G) to the Opn3-eGFP+ contralateral

354 red nucleus (Fig. 6D) or the ventral lateral nucleus of the thalamus, respectively. Some

355 thalamic nuclei, known to act as relay stations between the midbrain and cortical areas, were

18

356 also Opn3-eGFP+; these include the periventricular thalamus nucleus (Fig. 6B,E) that are

357 associated with the limbic system, as well as the dorsal lateral geniculate nucleus (Fig. 6F),

358 which is related to the visual pathway. Other central projections of the were

359 clearly labelled as Opn3-eGFP+, including the optic nerve, chiasm and tract (Fig. 6G,H),

360 where visual information is transferred to the dorsal lateral geniculate nucleus. Opn3-eGFP

361 expression was also observed in the olfactory sensory epithelium (Fig. 6H) and in parts of the

362 auditory system, namely the pinna of the ear, vestibular nerve and geniculate ganglion (Fig.

363 6I). In addition, scattered Opn3-eGFP expression was also noted in the septal nuclei (Fig. 6F)

364 and the paraventricular hypothalamic nucleus (Fig. 6G).

365

366 Several Opn3-eGFP positive forebrain nuclei are observed at E17.5

367 Additional Opn3-eGFP+ structures were more clearly detected at E17.5 compared to

368 earlier stages. Specifically, Opn3-eGFP expression was observed in the inferior colliculus

369 (Fig. 7A), which receives auditory input from the superior olivary complex (Fig. 5A) via the

370 lateral lemniscus (Fig. 7C,H), which was also observed to be Opn3-eGFP+. Moreover, the

371 (Fig. 7B), which plays a critical role in the autonomic system, motivated

372 behavior and pain modulation, was Opn3-eGFP+. In the cerebellum, the was

373 Opn3-eGFP+, as was the uncinate fasciculus of the cerebellum (also known as the hooked

374 bundle of Russell) (Fig. 7B), which consists of fibers that descend to the superior cerebellar

375 peduncle. In the midbrain, the cuneiform nucleus and midbrain reticular nuclei (Fig. 7C,E),

376 both included in the midbrain , were identified as Opn3-eGFP+. Other

377 Opn3-eGFP+ midbrain-located nuclei comprised the deep nuclei of the

378 (Fig. 7B,E) and the parafascicular nucleus (Fig. 7G,J), the latter of which is considered to be

379 an essential thalamic structure in the feedback systems of -thalamo-cortical

380 circuits involved in cognitive processes. At this stage, the rubro-spinal tract, which originates

19

381 from the red nucleus and crosses the midline via the ventral tegmental decussation, were all

382 detected by Opn3-eGFP (Fig. 7E,F), as was the dorsal tegmental decussation (Fig. 7E,F).

383 Furthermore, Opn3-eGFP expression was observed in the (Fig. 7G), a basal

384 ganglia structure, and in the three areas of the , including the

385 , thalamic fasciculus and field H (Fig. 7I-K), in addition to the

386 (Fig. 7I).

387

388 At E17.5, Opn3-eGFP was detected in the (Fig. 7J), an important structure

389 well-known to be involved in photoreception by producing during the dark phase

390 of the sleep-wake cycle, as well as the ventro-medial thalamic nucleus (Fig. 7J,K), which

391 projects pain-temperature information to the insular cortex. In the forebrain, Opn3-eGFP

392 expression was observed in several hormone-releasing nuclei; namely the paraventricular and

393 periventricular hypothalamic nuclei (Fig. 7L-N), and the medial preoptic nucleus (Fig. 7P),

394 which all remained Opn3-eGFP+ at postnatal/adult stages (Extended Data Fig. 7-1B,F,H).

395 Other Opn3-eGFP+ regions included the pyriform (olfactory) cortex (Fig. 7P); the subfornical

396 organ (Fig. 7O), which is one of the circumventricular organs of the brain; and the nucleus

397 accumbens (Fig. 7P), which is part of the reward and reinforcement systems. Moreover,

398 scattered Opn3-eGFP expression was observed throughout the septum of the brain (Fig.

399 7O,P).

400

401 Studies of adult Opn3::mCherry reporter mice have reported that Opn3 is expressed also in

402 non-neuronal astrocytes (Olinski et al., 2020b). In mouse, the differentiation of astrocytes

403 begins around E17-18 (Matsue et al., 2018; Reemst et al., 2016). To examine whether Opn3

404 is expressed in astrocytes at embryonic stages, double-immunohistochemistry of Opn3-eGFP

405 and glial fibrillary acidic protein (GFAP), a known astrocyte marker (Reemst et al., 2016),

20

406 was performed at E17.5. At this stage, GFAP+ astrocytes were identified in the ,

407 in particular the and the fimbria, and around the third ventricle at the level of

408 the paraventricular hypothalamic nucleus, but GFAP was not co-expressed with Opn3-eGFP

409 (Extended Data Fig. 7-1A-D). Moreover, GFAP+ astrocytes were observed along the optic

410 tract, in the glia limitans, the granular layer of , septohippocampal nucleus,

411 habenula, some regions of the stria medullaris and pyramidal tract, but again not co-

412 expressed with Opn3-eGFP (Extended Data Fig. 7-1A,C,D and data not shown). Thus, these

413 results indicate that Opn3-eGFP is not expressed in GFAP+ astrocytes in the embryonic brain.

414

415 Expanded Opn3-eGFP expression in the brain at postnatal and adult stages

416 Just after birth, at P0.5, an expansion of Opn3-eGFP expression was observed in the

417 forebrain, including the cerebral cortex, with defined expression in the anterior cingulate

418 cortex, involved in higher-level social functions (Fig. 8A,D), which was even more

419 pronounced at P10 (Extended Data Fig. 8-1D). At P0.5, Opn3-eGFP expression was also

420 noted in the and in parts of the hippocampus, including the dentate gyrus (Fig.

421 8A,C). Moreover, Opn3-eGFP+-thalamic regions were identified, including the posterior

422 thalamus, lateral dorsal nucleus,as well as ventral posteromedial and lateralnuclei (Fig. 8A,B).

423 Notably, the ventral posterolateral nucleus of the thalamus (Fig. 8A) receives information

424 from, among others, the DCML pathway, which incorporated several Opn3-eGFP+ structures

425 detected at E15.5 (Fig. 5D). Also, Opn3-eGFP was detected in the dorsal lateral geniculate

426 nucleus (Fig. 8B), which is a relay center for visual information from the optic nerve. At this

427 stage, OPT imaging, to appreciate the 3D perspective, clearly confirmed the expression of

428 Opn3-eGFP in the medulla and pons, optic chiasm and tracts, dorsal lateral geniculate nuclei

429 and areas of the primary visual cortex, as well as the olfactory bulb and several hypothalamic

430 nuclei (Fig. 9; Movie 2).

21

431

432 Additional Opn3-eGFP+ regions were identified at P10, namely the mammillary bodies

433 and medial hypothalamic nuclei (Extended Data Fig. 8-1A), which are important for memory.

434 Other parts of the limbic system that express Opn3-eGFP included the anterior and posterior

435 fibers of the (Extended Data Fig. 8-1B,D,E), which connect the hippocampus with the

436 forebrain. Furthermore, part of the cortico-striatal circuitry, between the anterior cingulate

437 cortex and the striatum, which includes the reward system, were Opn3-eGFP+ (Extended

438 Data Fig. 8-1D). The posterior hypothalamus was also noted to express Opn3-eGFP

439 (Extended Data Fig. 8-1B). The entorhinal cortex, important for spatial memory in relation to

440 the hippocampus, was Opn3-eGFP+, as were the dentate gyrus and of the

441 (Extended Data Fig. 8-1C,D). In the thalamus, Opn3-eGFP

442 expression was observed in the medial geniculate nucleus and the dorsal lateral geniculate

443 nucleus (Extended Data Fig. 8-1C), which are related to auditory and visual pathways,

444 respectively. The outer membrane, surrounding the brain also expressed Opn3-eGFP

445 (Extended Data Fig. 8-1C).

446

447 During adulthood, Opn3-eGFP expression was identified in the perifornical lateral

448 hypothalamic nuclei, which are related to respiratory control, and in α-tanycytes that line the

449 lateral walls of the third ventricle (Extended Data Fig. 8-1E). Furthermore, Opn3-eGFP was

450 identified in the hormone-secreting of the adult hypothalamus, including

451 both the supraoptic nucleus proper and retro-chiasmatic supraoptic nucleus, and in the bed

452 nucleus of the , which is a structure related to stress responses (Extended Data

453 Fig. 8-1G,H). A few examples of areas that expressed Opn3-eGFP already at embryonic

454 stages that also were identified at early postnatal stages and in the adult brain, were the

455 substantia nigra, the paraventricular and periventricular hypothalamic nuclei, the dorsal

22

456 lateral geniculate nucleus, the septal nuclei, the dorsomedial hypothalamic nucleus and the

457 (Extended Data Fig. 8-1B-F).

458

459 Discussion

460 Whereas most visual and non-visual opsins appear to be expressed in more or less

461 restricted patterns, Opn3 has been shown to be expressed in a broad range of multiple adult

462 tissues (Blackshaw and Snyder, 1999; Nissila et al., 2012; Olinski et al., 2020b). Opn3

463 mRNA was first discovered in the cerebral cortex and cerebellum of the adult mouse brain

464 (Blackshaw and Snyder, 1999), results that have been subsequently verified and expanded,

465 most recently in studies using an Opn3::mCherry mouse model (Olinski et al., 2020b).

466 Despite more detailed knowledge about the spatial expression of Opn3 in the adult mouse

467 brain, there is a significant lack of information concerning when and where in the nervous

468 system Opn3 is expressed during embryogenesis.

469

470 To address this issue, an Opn3 promoter-driven eGFP mouse line was used to investigate

471 Opn3 expression in the developing nervous system with a focus on the brain/head region. A

472 summary of Opn3-eGFP+ key structures in relation to temporal onset of expression is outlined

473 in Table 1. Notably, our study identified 25 new Opn3-eGFP+ structures in the CNS and PNS,

474 including sensory ganglions, the olfactory epithelium, vestibular nuclei and parafascicular

475 nucleus, that have not been previously reported to express Opn3 (Table 1). The analyzed

476 Opn3-eGFP knock-in mouse line possesses two intact Opn3 wild-type alleles that are

477 consistent with an observed normal phenotype, which is in contrast to the recently developed

478 Opn3::mCherry fusion protein mouse line (Olinski et al., 2020b). Being under the influence

479 of the native Opn3 promoter, both detected eGFP and Opn3::mCherry expression patterns

480 appear to mirror the cellular expression profile of endogenous Opn3 from earlier reports.

23

481 Moreover, Opn3-eGFP cellular expression in the adult brain, as characterized by this current

482 study, mirrors that shown for Opn3::mCherry (Olinski et al., 2020), which implicates that

483 both Opn3 reporter mice are accurately detecting Opn3-positive cells. As eGFP is a

484 cytoplasmic protein, the reporter molecule is free to diffuse throughout the cell. As such, not

485 only cell bodies, but also axonal projections are readily visible in the Opn3-eGFP reporter

486 mouse line used here, which is greatly informative when analyzing anatomical-functional

487 relationships.

488

489 In this study, evidence is provided that Opn3 is expressed during most of embryonic

490 development, and Opn3-eGFP was detected already at E9.5, much earlier than previously

491 reported, in structures within the CNS and PNS. This timeline of expression indicates that

492 Opn3 may be the earliest of the opsin family of to be expressed in mammalian

493 development. AtE9.5-E10.5, Opn3-eGFP expression was detected in most, if not all, spinal

494 nerves, their associated dorsal root ganglia, as well as several sensory ganglia, all of which

495 are part of the PNS. The sensory ganglia form first order sensory neurons projecting to

496 somatic tissues via ascending sensory tracts to brainstem and forebrain regions, while the

497 spinal cord motor neurons form first order motor neurons connected directly to muscles

498 (Chen et al., 2017). Results presented here show Opn3-eGFP expression in motor neurons and

499 spinal nerves at E10.5, prior to observed Opn3-eGFP expression in sensory neurons at E13.5,

500 which are in agreement with the temporal differentiation of first order spinal motor neurons

501 before sensory neurons (Rodier, 1980).

502

503 Following embryonic growth, the temporal onset of Opn3-eGFP expression correlated

504 with the development of individual neural structures in specific pathways and circuits (Chen

24

505 et al., 2017), which indicates a potential role for Opn3 in early neural development and

506 subsequent maturation. Among the different sensory systems, the olfactory-associated

507 pathway is the first to express Opn3-eGFP, with expression observed in the olfactory placode

508 at E9.5, followed by post-mitotic migratory neurons at E10.5, the nerve and bulb at E13.5 and

509 the olfactory cortex at E17.5. Opn3 is also expressed in the visual system, where Opn3-eGFP

510 was evident in the optic stalk at E10.5, as well as RGCs at E13.5, including their Opn3-

511 eGFP+ axons in the optic nerve. Consistently, a previous study demonstrated by RT-PCR that

512 Opn3 is expressed at E10.5 during ocular development in mouse (Tarttelin et al., 2003a). The

513 optic nerves send central projections to the dorsal lateral geniculate nucleus of the thalamus,

514 in this study detected by Opn3-eGFP from E15.5, and to the primary visual cortex, shown to

515 express Opn3 at adulthood (Olinski et al., 2020b). From E13.5 to adult stages, Opn3-eGFP

516 expression expands throughout the spinal cord, brainstem and forebrain. Ventral,

517 intermediate and dorsal gray matters in the spinal cord were identified as Opn3+ regions that

518 form motor, autonomic and sensory spinal cord components. Moreover, the onset of Opn3-

519 eGFP in the hypothalamic paraventricular nucleus around E15.5, was followed by the

520 detection of Opn3-eGFP in the periventricular nucleus, dorsomedial hypothalamic nucleus

521 and the medial preoptic nucleus, all of which were obvious at E17.5. Moreover, Opn3-eGFP

522 expression was noted in the basal ganglia at E15.5, which connects to multiple thalamic

523 nuclei with clear expression at E17.5, including the parafascicular nucleus, a part of the

524 medial and intralaminar nuclei that is hypothesized to play a critical role in the sensorimotor

525 process of attentional orienting and behavioral flexibility (Brown et al., 2010; Yamanaka et

526 al., 2018). Other Opn3-eGFP+ subthalamic structures that are related to the basal ganglia, and

527 involved in modulated motor control, include the white and gray matters of the fields of

528 Forel, a structure implicated in epilepsy, movement and behavioral disorders (Neudorfer and

529 Maarouf, 2018), as well as the subthalamic nucleus. All of the above are examples of that

25

530 Opn3-eGFP onset of expression follows the individual developmental maturation of these

531 neural structures.

532

533

534

535 In brief, thalamic nuclei connect various CNS structures with the cerebral cortex.

536 Interestingly, no Opn3-eGFP expression was detected in association with the thalamic nuclei

537 until after birth at P0.5. Consistently, the cerebral cortex was also Opn3-negative before birth,

538 with cortical Opn3-eGFP expression coinciding with the onset of expression in the thalamic

539 lateral dorsal, ventral posteromedial and ventral posterolateral nuclei at P0.5. In the cerebal

540 cortex, observed Opn3-eGFP+ areas included the pyriform cortex (around E17.5), cingulate

541 cortex and (the hippocampus and associated structures) (around P0.5), and the

542 entorhinal cortex (around P10). In subcortical areas, Opn3-eGFP was detected in the septal

543 nuclei and at around E17.5. Other extra-cortical structures that form part

544 of limbic system were also shown to express Opn3-eGFP, namely the hypothalamic

545 mammillary bodies (around P10) and the ventral medial thalamic nucleus (around E17.5).

546 Consistently, previous reports have shown that Opn3 is detectable in the cingulate, insula and

547 frontal cortical regions of the adult brain, mainly in layer V pyramidal neurons that are

548 characterized mainly by their cortico-striatal projections (Blackshaw and Snyder, 1999;

549 Olinski et al., 2020b). These latter structures are required for appropriate goal-directed

550 behaviors, including motivation and cognitive processes that mediate appropriate actions to

551 obtain a specific outcome (Gerfen et al., 2013; Haber, 2016). As such, the presence of Opn3-

552 eGFP in limbic-associated regions suggests potential (photoreceptive) roles in motivation,

553 emotion, learning and memory.

26

554

555 The absence of Opn3-eGFP expression is also of functional significance and importance.

556 In particular, Opn3-eGFP was not detected in the cerebral crus that carry cortico-spinal,

557 cortico-bulbar (brainstem) and cortico-pontine tracts. As such, these data suggest that cortical

558 nuclei expressing Opn3-eGFP in the frontal cortex are part of the inter-telencephalic cortico-

559 striatal circuitry, with the exclusion of pyramidal neural network. Thus, Opn3 is most likely

560 restricted to either the premotor and/or supplementary motor areas in the frontal cortex,

561 where all cortico-striatal connections are required before facilitating movement.

562

563 By utilizing Opn3-eGFP as a cytoplasmic reporter, it was possible to track and trace

564 neuronal cell bodies and their axonal projections for all Opn3+ cell types. As a result, Opn3-

565 eGFP was detected in the dorsal, lateral and ventral marginal layers that form the nerves of

566 the second order DCML sensory tract, including both gracile and cuneate fasciculi, as well as

567 spinothalamic, spinocerebellar, spino-tectal, spino-reticular and spino-vestibular tracts. These

568 data suggest a possible involvement of Opn3 in modulating sensory information pertaining to

569 proprioception and fine touch. Also, from E13.5, the presence of Opn3-eGFP in higher CNS

570 structures located in the brainstem identified second order ascending (brainstem to forebrain)

571 and descending (brainstem to spinal cord) tracts. These include:i) the

572 (carrying sensory information from sensory and spinal trigeminal nuclei to the ventral

573 posteromedial nucleus of the thalamus); ii) the vestibulo-ocular tract (from medullary

574 vestibular nuclei to the oculomotor associated sub-nuclei via the medial longitudinal

575 fasciculus); and iii) tracts of the auditory system (from the cochlear nucleus to the superior

576 olivary complex, followed by the lateral lemniscus, then the inferior colliculus, and finally

577 the medial geniculate nucleus). Further tracts identified by Opn3-eGFP were: iv) the solitary-

27

578 parabrachial tract (carrying sensory information from CN VII, CN IX and CN X to the

579 solitary nucleus and parabrachial nuclei); v) the cerebellar peduncles [including the superior

580 (deep cerebellar nuclei projections), medial (pontine nuclei projections) and inferior (spino-

581 cerebellar tract)]; and vi) the cerebral peduncle that mainly carries sensory input to the

582 forebrain. Together these observations emphasize that studies of Opn3-eGFP expression can

583 be used to follow projections for various neural systems and that Opn3 appears to play a role

584 in the development of multiple sensory pathways.

585

586 Given that Opn3 has been shown to functionally form a short-wavelength-sensitive

587 photopigment, with a spectral peak of absorbance in the blue range (Sugihara et al., 2016),

588 the widespread expression pattern of Opn3 during embryogenesis and well into adulthood

589 suggests that mammalian photoreception could be far more complex than previously

590 understood. Furthermore, the mechanisms by which they transduce photic signals to control

591 physiology, or are affected in pathophysiological conditions, are either poorly understood or

592 unknown. Thus, it remains to be shown that physiological levels of light can activate Opn3 in

593 the mammalian brain. Mental health disorders, such as depression, seasonal affective disorder

594 (SAD) (via a Pro10Leu mutation in a related opsin gene, OPN4, (Roecklein et al., 2009)),

595 anxiety, schizophrenia, bipolar disorder, Alzheimer’s disease and other forms of dementia are

596 associated with photosensory disruption (Foster et al., 2013). Moreover, there is a season-of-

597 conception/birth dependent risk to develop these disorders (Foster and Roenneberg, 2008),

598 which further indicates that light may play a crucial role for establishing optimal neural

599 connections. Recently, studies have begun to uncover the neural basis linking light detection

600 and depression/elation. One such study identified a -independent

601 pathway, where photosensitive RGCs project to the perihabenular region of the thalamus, a

602 retina-brain circuit that is strongly implicated in modulating mood (Fernandez et al., 2018).

28

603 Given the detection of Opn3-eGFP in both the retina and thalamus, it is possible that

604 dysfunction of Opn3-dependent photosensory systems may also be involved in mood

605 regulation. What functional disturbances a modulation of Opn3 might cause in regions of the

606 developing embryo, as well as adults, remains to be determined. Nonetheless, the temporal

607 and spatial control of widespread, yet distinct, Opn3 expression raises the possibility that this

608 photopigment and/or other components of the Opn3-dependent phototransduction cascade

609 play important roles in critical neuronal circuits. In conclusion, this study presents a crucial

610 blueprint from which to investigate autonomic and cognitive opsin-dependent neural

611 development and resultant behaviors under physiological and pathophysiological conditions.

612

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786

36

787 Figures and Figure Legends

788

789 Figure 1. Opn3-eGFP immunodetection (red) at E10.5 in sagittal (A-E) and coronal (F-K)

790 serial sections counterstained with DAPI (blue). On the left, schematics of E10.5 embryos

791 including planes of sections shown in A-K. A, Di: diencephalic vesicle, Mes: mesencephalic

792 vesicle, opv: optic vesicle, Rh: rhombencephalic vesicle, v.g: trigeminal ganglion. B, ix.g:

793 glossopharyngeal ganglion, Mes: mesencephalic vesicle, o: otic vesicle, oe: olfactory

794 epithelium, Rh: rhombencephalic vesicle, vii-viii.g: facio-acoustic ganglia, x.g: vagus

795 ganglion. A, B, Parts of the amnion surrounding the embryo were digitally removed (raw data

796 in Extended data Fig. 1-3). C, ix.g: glossopharyngeal ganglion, o: otic vesicle, Rh:

797 rhombencephalic vesicle, v.g: trigeminal ganglion, vii-viii.g: facio-acoustic ganglia, x.g:

798 vagus ganglion, x.n: vagal nerve. D, drg: dorsal root ganglia, drg.x: dorsal root afferent

799 processes. E, ap: alar plate, bp: basal plate, drg: dorsal root ganglia, drg.x: dorsal root afferent

800 processes, Rh: rhombencephalic vesicle. F, oe: olfactory epithelium. G, Di: diencephalic

801 vesicle, opv: optic vesicle, os: optic stalk. H, ix.n: glossopharyngeal nerve, o: otic vesicle,

802 v.g: trigeminal ganglion, vii.g geniculate ganglion, viii.g: vestibulocochlear ganglion, x.n:

803 vagal nerve. I, rs: rootlets of the v/vii/viii.g, v.g: trigeminal ganglion, vii.g geniculate

804 ganglion, viii.g: vestibulocochlear ganglion. J, bp: basal plate, drg: , SC:

805 spinal cord. K, mnl: mantle layer, s.n: spinal nerves. Scale bars: 100 μm. Figure 1-1 and

806 Figure 1-2 are supporting Figure 1.

37

807 Figure 2. 3D optical projection tomography (OPT) imaging of whole E10.5 embryo showing

808 Opn3-eGFP immunodetection. A, Sagittal view showing detection of Opn3 (red) against a

809 background of auto-fluorescing anatomical structures (blue). Developing ganglia that form

810 the sensory branches of cranial nerves (CN) V, CN VII and CN VIII cranial nerves are shown

811 in B, and spinal nerves highlighted in C and D. D, Dorsal view of embryo shown in A.

812 Abbreviations: bp: basal plate, dpb: dorsal pancreatic bud, drg: dorsal root ganglia, git:

813 gastrointestinal tract, hrt: heart; ix.g: glossopharyngeal ganglion, Mes: mesencephalic vesicle,

814 oe: olfactory epithelium, Pro: prosencephalic vesicle, Rh: rhombencephalic vesicle, s.n:

815 spinal nerves, v.g: trigeminal ganglion, vii-viii.g: facio-acoustic ganglia (comprising the vii.g

816 geniculate ganglion and viii.g: vestibulocochlear ganglion), x.g: vagus ganglion. Scale bars:

817 250 μm.

818

819

38

820 Figure 3. Opn3-eGFP immunodetection (red) at E13.5 in horizontal (A, C, D, F-H) and

821 coronal (B, E) serial sections in caudo-rostral direction from the spinal cord to hindbrain

822 areas counterstained with DAPI (blue). On the left, schematics of E13.5 embryonic heads

823 including planes of sections shown in A-H. A, iz: intermediate zone, mnl: mantle layers, mrl:

824 marginal layers, SC: spinal cord. B, ME: medulla, SC: spinal cord, v.sp n: spinal trigeminal

825 nerve, v.sp: spinal trigeminal nucleus. C, ix.g: glossopharyngeal ganglion, ME: medulla, mlf:

826 medial longitudinal fasciculus, sol: solitary tract, v.sp: spinal trigeminal nucleus, vb:

827 vestibular nuclei, x.g: vagus ganglion, xii: hypoglossal nucleus. D, CB: cerebellum, co:

828 cochlear nucleus, egl: external granular layer of cerebellum, ME: medulla; P: pons, so:

829 superior olivary complex, Tec: tectum of midbrain, vii.g: geniculate (facial) ganglion. E, co:

830 cochlear nucleus, so: superior olivary complex, vb: vestibular nuclei, vi: abducens nucleus. F,

831 pb: parabrachial nuclei, prp: nucleus prepositus, scp: superior cerebellar peduncle, Tec:

832 tectum of midbrain, v.sp.n: spinal trigeminal nerve, viii: vestibular nerve. G, dpcb: deep

833 cerebellar nuclei, Tec: tectum of midbrain, v.mes: mesencephalic trigeminal nucleus, v.sp.n:

834 spinal trigeminal nerve, vii.gn: genu of facial nerve, vii: sensory/parasympathetic facial

835 nerve. H, rs: rootlets of the v.g, v.g: trigeminal ganglion, v.sen: sensory trigeminal nucleus.

836 Scale bars: 100 μm.

39

837 Figure 4. Opn3-eGFP immunodetection at E13.5 (red) in coronal serial sections caudo-

838 rostral direction from midbrain to forebrain areas counterstained with DAPI (blue). On the

839 left, a schematic of an E13.5 embryonic head including planes of sections shown in A-I. A,

840 mlf: medial longitudinal fasciculus, oas: oculomotor associated sub-nuclei, scp: superior

841 cerebellar peduncle, td: tegmental decussations, Tec: tectum of midbrain, Teg: midbrain and

842 pontine , vtg: ventral tegmentum. B, M: midbrain, oas: oculomotor associated sub-

843 nuclei, P: pons, r: red nucleus, td: tegmental decussations. C, cpd: cerebral peduncle, oas:

844 oculomotor associated sub-nuclei, pb: parabrachial nuclei, r: red nucleus, sn: substantia nigra.

845 D, cpd: cerebral peduncle, dmh: dorsomedial hypothalamic nucleus, dt: dorsal thalamus, H:

846 hypothalamus, ll.n: nucleus of lateral lemniscus, sta: subthalamic area, T: thalamus, v.g:

847 trigeminal ganglion. E, dt: dorsal thalamus, H: hypothalamus, mfb: medial forebrain bundles,

848 str: striatum, T: thalamus, dmh: dorsomedial hypothalamic nucleus. F, CC: cerebral cortex,

849 cpd: cerebral peduncle, dt: dorsal thalamus, str: striatum. G, stm: stria medullaris, ot: optic

850 tract. H, op.n: optic nerve, rgc: retinal ganglion cells. I, OB: olfactory bulb, oe: olfactory

851 epithelia, on: olfactory nerve, vo: vomeronasal organ, von: vomeronasal nerve. Scale bars:

852 100 μm.

853

40

854 Figure 5. Opn3-eGFP immunodetection (red) at E15.5 in horizontal serial sections in

855 caudorostral direction from the medulla to pons areas counterstained with DAPI (blue). On

856 the left, a schematic of an E15.5 embryonic head including planes of sections shown in A-H.

857 A, ME, medulla, P: pons, so: superior olivary complex, sol.n: solitary nucleus, xii:

858 hypoglossal nucleus. B, marn: magnocellular reticular nucleus, mern: medullary reticular

859 nuclei, ro: nucleus raphe obscurus. C, mcp: medial cerebellar peduncle, pn: pontine nuclei. D,

860 cf: cuneate fasciculus, gf: gracile fasciculus, ll.n: nucleus of lateral lemniscus, ME: medulla,

861 P: pons, sol: solitary tract, v.sen: sensory trigeminal nucleus, v.sp: spinal trigeminal nucleus.

862 E, mlf: medial longitudinal fasciculus, prn: pontine reticular nuclei, vii.gn: genu of facial

863 nerve. F, ME: medulla, P: pons, prp: nucleus prepositus, sol.n: solitary nucleus. G, lav: lateral

864 vestibular nucleus, ll.n: nucleus of lateral lemniscus, prn: pontine reticular nuclei, suv:

865 superior vestibular nucleus, v.m: motor trigeminal nucleus, v.sen: sensory trigeminal nucleus,

866 v.sp: spinal trigeminal nucleus. H, CB: cerebellum, dn: , fn: fastigial nucleus,

867 IntA: , scp: superior cerebellar peduncle. Scale bars: 100 μm.

868

41

869 Figure 6. Opn3-eGFP immunodetection (red) at E15.5 in horizontal serial sections in caudo-

870 rostral direction from the midbrain to forebrain areas counterstained with DAPI (blue). On

871 the left, a schematic of an E15.5 embryonic head including planes of sections shown in A-I.

872 A, bic: brachium of inferior colliculus, CB: cerebellum, CC: cerebral cortex, fr: fasciculus

873 retroflexus, M: midbrain, mrn: midbrain reticular nuclei, P: pons, pb: parabrachial nuclei,

874 stm: stria medullaris, T: thalamus. The inserted box indicates area shown in Extended Fig. 6-

875 1. B, C: , dlg: dorsal lateral geniculate nucleus, M: midbrain, P: pons, pvt: pvt:

876 periventricular thalamic nucleus, T: thalamus. C, scp: superior cerebellar peduncle, vtd:

877 ventral tegmental decussation, xscp: decussation of superior cerebellar peduncle. D, crt:

878 cerebellorubral tract, ctt: cerebellothalamic tract, fr: fasciculus retroflexus, M: midbrain, r:

879 red nucleus, T: thalamus. E, fr: fasciculus retroflexus, ham: medial habenula, pvt:

880 periventricular thalamic nucleus, stm: stria medullaris. F, sp: septal nuclei, sta: subthalamic

881 area. G, H: hypothalamus, ot: optic tract, pavh: paraventricular hypothalamus nucleus. H, oc:

882 optic chiasm, oe: olfactory epithelium, op.n: optic nerve, ot: optic tract. I, gg: geniculate

883 ganglion, pin: pinna of the ear, viii: vestibulocochlear nerve. Scale bars: 100 μm.

884

42

885 Figure 7. Opn3-eGFP immunodetection (red) at E17.5 in horizontal (B) and coronal (A, C-P)

886 serial sections from the hindbrain to forebrain areas counterstained with DAPI (blue). On the

887 left, a schematic of E17.5 embryonic heads including planes of sections shown in A-P. A, ic:

888 inferior colliculus. B, CA: , CB: cerebellum, dnsc: deep nuclei of superior

889 colliculus, fn: fastigial nucleus, hbr: hooked bundle of Russel, M: midbrain, pag:

890 periaqueductal gray. C, CB: cerebellum, cun: cuneiform nucleus, dncb: deep nuclei of

891 cerebellum, ll.d dorsal component of lateral lemniscus, P: pons, Tec: tectum of midbrain,

892 Teg: tegmentum of midbrain. D, CB: cerebellum, ME: medulla, mern: medullary reticular

893 nuclei, P: pons, pb: parabrachial nuclei, prn: pontine reticular nuclei. E. dnsc: deep nuclei of

894 superior colliculus dtd: dorsal tegmental decussation, mlf: medial longitudinal fasciculus,

895 mrn: midbrain reticular nuclei, oas: oculomotor associated sub-nuclei. F, M: midbrain, oas:

896 oculomotor associated sub-nuclei, r: red nucleus, rst: , Tec: tectum of

897 midbrain, Teg: tegmentum of midbrain, vtd: ventral tegmental decussation. G, sn: substantia

898 nigra, pf: parafascicular nucleus. H, ll: lateral lemniscus, MB: mamillary body, mcp: medial

899 cerebellar peduncle, pn: pontine nuclei (gray), prf: pontine reticular formation. I, fh: field H,

900 pvt: paraventricular thalamic nucleus, sta: subthalamic area, stm: stria medullaris, sut:

901 subthalamic nucleus. J, CC: cerebral cortex, cpd: cerebral peduncle, fh: field H, H:

902 hypothalamus, lf: lenticular fasciculus, pf: parafascicular nucleus, pi: pineal gland, sta:

903 subthalamic area, T: thalamus, vmt: ventromedial thalamic nucleus. K, fh: field H, H:

904 hypothalamus, hal lateral habenula, ot: optic tract, stm: stria medullaris, T: thalamus, thf:

905 thalamic fasciculus, dmh: dorsomedial hypothalamic nucleus, vmt: ventromedial thalamic

906 nucleus. L, hip: hippocampus, H: hypothalamus, pavh: paraventricular hypothalamic nucleus,

907 T: thalamus. M, H: hypothalamus, ot: optic tract, pavh: paraventricular hypothalamic nucleus,

908 pvh periventricular hypothalamic nucleus, T: thalamus. N, H: hypothalamus, ot: optic tract,

909 pvh periventricular hypothalamic nucleus, T: thalamus. O, C: cerebrum, sfo: subfornical

43

910 organ, sp: septum of brain, T: thalamus. P, nac: nucleus accumbens, cpf: pyriform (olfactory)

911 cortex, mpn: medial preoptic nucleus, sp: septum of brain, str: striatum. Scale bar: 100 μm.

912 Figure 7-1 is supporting Figure 7.

913

914 Figure 8. Opn3-eGFP immunodetection (red) at P0.5 in horizontal sections of forebrain areas

915 counterstained with DAPI (blue). On the left, a schematic of a P0.5 brain including planes of

916 sections shown in A-D. A, CC: cerebral cortex, Hip: hippocampus, po: posterior thalamus, rt:

917 thalamic reticular nucleus, str: striatum, vpl: ventral posterolateral nucleus, vpm: ventral

918 posteromedial nucleus. B, C: cerebrum, dlg: dorsal lateral geniculate nucleus, ld: lateral

919 dorsal nucleus, sfo: subfornical organ, sp: septal nuclei, T: thalamus, Tec: tectum of

920 midbrain. C, dg: dentate gyrus, str: striatum. D, acc: anterior , str: striatum.

921 Scale bars: 100 μm. Figure 8-1 is supporting Figure 8.

922

923 Figure 9. 3D optical projection tomographic (OPT) imaging of Opn3-eGFP

924 immunodetection in the brain at P0.5. A, Sagittal view showing the presence of Opn3-eGFP

925 (red) against a background of auto-fluorescing anatomical structures (blue). B, Repeat of (A)

926 showing unmasked Opn3-eGFP (red) staining only. C, D, Ventral views of the brain shown

927 in A and B, respectively. Abbreviations: dlg: dorsal lateral geniculate nucleus, dpcb: deep

928 nuclei of cerebellum, ic: inferior colliculus, mcp: medial cerebellar peduncle, mern:

929 medullary reticular nuclei, mpn: medial preoptic nucleus, nac: nucleus accumbens, oas:

930 oculomotor associated sub-nuclei, OB: olfactory bulb, oc: optic chiasm, op.n: optic nerve, ot:

931 optic tract, pn: pontine nuclei (also pontine nuclei gray), pvh: periventricular hypothalamic

932 nucleus, r: red nucleus, sc: superior colliculus, so: superior olivary complex, v.m: motor

933 trigeminal nucleus, v.sp: spinal trigeminal nucleus. Scale bars: 1 mm.

934

44

935 Table legend.

936 Table 1. A summary outlining the onset of Opn3-eGFP expression in various structures of

937 the CNS and PNS. • indicates novel Opn3-eGFP+ structures identified in this study.

938

939 Movies.

940 Movie 1. Video of 3D imaging of a whole E10.5 embryo (along the y axis) showing Opn3-

941 eGFP immunodetection (red) against a background of auto-fluorescing anatomical structures

942 (blue).

943

944 Movie 2. Video of 3D imaging of a P0.5 brain (along the x axis) showing Opn3-eGFP

945 immunodetection (red) against a background of auto-fluorescing anatomical structures (blue).

946

947 Extended Data Figures and Figure Legends

948 Extended Data Fig. 1-1. Opn3-eGFP immunodetection (red) at E9.5 in sagittal (A-D) and

949 coronal (E-L) serial sections counterstained with DAPI (blue). On the left, schematics of E9.5

950 embryos including planes of sections shown in A-L. A, bp: basal plate, dpb: dorsal pancreatic

951 bud, nt: neural tube, php: pharyngeal pouches. B, dpb: dorsal pancreatic bud, nt: neural tube,

952 php: pharyngeal pouches, v.g: trigeminal ganglion. C, bp: basal plate, nt: neural tube, o: otic

953 vesicle, php: pharyngeal pouches, vii-viii.g: facio-acoustic ganglia. D, git: gastrointestinal

954 tract, Mes: mesencephalic vesicle, Pro: prosencephalic vesicle, Rh: rhombencephalic vesicle.

955 E, Te: telencephalic vesicle. F, op: olfactory placode, Te: telencephalic vesicle. G, Di:

956 diencephalic vesicle, inf: infundibulum. H, Di: diencephalic vesicle. I, Mes: mesencephalic

957 vesicle. J, Mes: mesencephalic vesicle. K, o: otic vesicle, Rh: rhombencephalic vesicle, v.g:

958 trigeminal ganglion, vii-viii.g: facio-acoustic ganglia. L, bp: basal plate, nt: neural tube. Scale

959 bars: 100 μm.

45

960

961 Extended Data Fig. 1-2. No DAPI labelled nuclei were detected in Opn3-eGFP positive

962 projection areas. A-F, Opn3-eGFP immunodetection (red) at E10.5 in (A-C) and at E13.5 (D-

963 F) horizontal sections counterstained with DAPI (blue). A, bp: basal plate, drg: dorsal root

964 ganglion, SC: spinal cord. B, s.n: spinal nerves, mnl: mantle layer. C, s.n: spinal nerves. D,

965 dpcb: deep nuclei of cerebellum, Tec: tectum of midbrain, v.mes: mesencephalic trigeminal

966 nucleus. E, v.spn: spinal trigeminal nerve, vii: facial nerve, vii.gn: genu of facial nerve. F,

967 v.spn: spinal trigeminal nerve, vii: facial nerve, vii.gn: genu of facial nerve. Scale bars: 100

968 μm.

969

970 Extended Data Fig. 1-3. Raw data of the main images in figure 1A and 1B. Opn3-eGFP

971 immunodetection (red) at E10.5 in horizontal serial sections counterstained with DAPI (blue)

972 showing the (in Fig. 1A,B) digitally removed Opn3-eGFP positive amnion surrounding the

973 embryo (arrowheads). Scale bar: 100 μm.

974

975 Extended Data Fig. 6-1. Confirmation of DAPI labelled nuclei in the region of the

976 oculomotor associated sub-nuclei. A-C, Opn3-eGFP immunodetection (red) at E15.5 in

977 horizontal (A-C) sections counterstained with DAPI (blue). A, bic: brachium of inferior

978 colliculus, CB: cerebellum, CC: cerebral cortex, fr: fasciculus retroflexus, M: midbrain, mrn:

979 midbrain reticular nuclei, P: pons, pb: parabrachial nuclei, stm: stria medullaris, T: thalamus.

980 B, oas: oculomotor associated sub-nuclei. C, 3dv: third ventricle. Scale bars: 100 μm.

981

982 Extended Data Fig. 7-1. Opn3-eGFP expression was not observed in GFAP+ astrocytes. On

983 the left, a schematic of an E17.5 head including planes of sections shown in A-D. A-D, Opn3-

984 eGFP (red) and glial fibrillary acidic protein (GFAP, green) immunodetection at E17.5 in

46

985 coronal sections counterstained with DAPI (blue). Opn3-eGFP and GFAP was not co-

986 expressed in GFAP+ astrocytes in the hippocampal, thalamic or hypothalamic regions. C,D,

987 Minimal co-localization, but not co-expression, of Opn3-eGFP and GFAP was observed in

988 parts of the optic tract. A, gl: glia limitans, Hip: hippocampus, stm: stria medullaris. B, dg:

989 dentate gyrus, fim: fimbria, stm: stria medullaris. C, 3dv: third ventricle, dg: dentate gyrus,

990 fim: fimbria, ot: optic tract, pvt: paraventricular thalamic nucleus, stm: stria medullaris, vmt:

991 ventromedial thalamic nucleus. D, 3dv: third ventricle, mpn: medial preoptic nucleus, ot:

992 optic tract, pavh: paraventricular hypothalamic nucleus, pvh periventricular hypothalamic

993 nucleus. Scale bars: 100 μm.

994

995 Extended Data Fig. 8-1. Opn3-eGFP immunodetection (red) at P10 in horizontal (A-D) and

996 at adult stage in coronal (E-H) serial sections of forebrain areas counterstained with DAPI

997 (blue). On the left, schematics of P10 and adult brains including planes of sections shown in

998 A-H. A, MB: mammillary bodies, mh: medial hypothalamic nuclei. B, fx: posterior fibers of

999 the fornix, pavh: paraventricular hypothalamic nucleus, ph: posterior hypothalamus, sn:

1000 substantia nigra. C, dg: dentate gyrus, dlg: dorsal lateral geniculate nucleus, EC: entorhinal

1001 cortex, mg: medial geniculate nucleus, om: outer membrane, sub: subiculum. D, acc: anterior

1002 cingulate cortex, CC: cerebral cortex, fx: fibers of fornix, hc: hippocampal commissure, ld:

1003 lateral dorsal thalamic nucleus, sfo: subfornical organ, str: striatum, sp: septal nuclei. E-H,

1004 Opn3-eGFP immunodetection using an anti-rabbit 488 antibody, here pseudo-colored (from

1005 green to red). E, dmh: dorsomedial hypothalamic nucleus, fx: fibers of the fornix, pfn:

1006 perifornical lateral hypothalamic nuclei, tn: tanycytes. F, pavh: paraventricular hypothalamic

1007 nucleus, pvh: periventricular hypothalamic nucleus. G, son.rx: retrochiasmatic supraoptic

1008 nucleus. H, bnst: bed nucleus of stria terminalis, mepn: median preoptic nucleus, mpn:

1009 medial preoptic nucleus, son.p: supraoptic nucleus proper. Scale bars: 100 μm.

47

PNS and Age CNS Raphe obscurus • sensory organs (day) Cuneate and gracile Trigeminal Neural tube Basal plate E9.5 nuclei • ganglion • Pontine nuclei Facio-acoustic Brain vesicles Basal plate of Medial cerebellar complex peduncle • ganglion • Motor trigeminal Olfactory Dorsal outer cells of nucleus placode mesencephalon Nucleus of lateral Dorsal root Spinal cord Motor neurons E10.5 lemniscus ganglion Brachium of inferior Inferior vagus colliculus ganglion • Midbrain Midbrain reticular Glossopharynge Brain vesicles Optic stalk nuclei al ganglion • Inferior colliculus Migratory Brachium of inferior olfactory colliculus neurons Forebrain Periventricular Retinal Spinal cord Dorsal, lateral and E13.5 thalamic nucleus ganglion cells ventral mantle and Dorsal lateral Optic nerve marginal layers geniculate nucleus Olfactory Hindbrain Spinal trigeminal Septal nuclei • epithelium • nucleus • Paraventricular Olfactory Principal sensory hypothalamic nucleus trigeminal Cerebellum Uncinate fasciculus of E17.5 nerve • cerebellum nucleus • Midbrain Vomeronasal Mesencephalic Periaqueductal gray • Cuneiform nucleus organ • trigeminal Rubrospinal tract nucleus • Substantia nigra Vomeronasal Genu of facial nerve • Epithalamus Pineal gland nerve • Solitary tract Thalamus Parafascicular Parabrachial nuclei • nucleus • Hypoglossal nucleus • Ventromedial thalamic nucleus Nucleus prepositus • Subthalamus Subthalamic nucleus Vestibular Hypothalamus Periventricular nuclei • hypothalamic nucleus Medial longitudinal Medial hypothalamic fasciculus nucleus Abducens Cerebrum Subfornical organ Nucleus accumbens nucleus • Pyriform cortex Cochlear nuclei • Thalamus Posterior thalamus P0.5 Superior olivary Lateral dorsal nucleus complex Ventral posteromedial External granular nucleus layers of cerebellum Ventral posterolateral Deep cerebellar nuclei nucleus Superior cerebellar Subpallium Dentate gyrus of peduncle hippocampus Midbrain Red nucleus Anterior cingulate Oculomotor associated cortex sub nuclei • Frontal cortex Tegmental fibres Hypothalamus Mammillary nuclei P10 Forebrain Subthalamic area Posterior nucleus Dorsal thalamus Subpallium Fornix Dorsal medial Subiculum hypothalamic nucleus Corticostrial circuitry Stria medullaris Meninges Outer membrane Optic tract Hypothalamus Perifornical nuclei Adult Olfactory bulb Tanycytes Pinna of the ear Hindbrain Medullary reticular E15.5 Supraoptic nucleus nuclei Bed nucleus of stria Magnocellular reticular terminalis nucleus

PNS and Age CNS sensory organs (day)

1