Research Article: New Research | Development Distinct Opsin 3 (Opn3) expression in the developing nervous system 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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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 brain, 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-medial lemniscus 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 spinal cord to regulate multiple motor and sensory circuitry systems,
17 including proprioception, nociception, ocular movement and olfaction, as well as memory,
18 mood and emotion. 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 brainstem, cerebellum,
29 hypothalamus and thalamus, whereas Opn3-eGFP expression in the cerebral cortex and
30 associated limbic system regions were observed postnatally. The presence of Opn3, a short-
31 wavelength-sensitive photopigment, 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 protein-coupled
39 receptors (i.e. opsins) mediate both visual and non-visual photoreception. Briefly, the opsin
40 protein moiety is covalently linked to a light-sensitive retinoid chromophore, which together
41 form a photopigment (Davies et al., 2012; Yokoyama, 2000). In the presence of particular
42 wavelengths of light, photopigments absorb the energy of a photon 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 retinal ganglion cells (RGCs; see Extended Data for a list of
48 abbreviations) were the only opsin-based photoreceptors present in mammals, all of which
49 were restricted to the retina. 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 (melanopsin) and Opn5 (neuropsin), were identified in photoreceptors that were not thought
52 to be involved in visual perception 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 circadian clock 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 iris (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 primates, 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 vertebrates, including higher mammals, Opn3 is expressed in
74 adipocytes and several tissues, such as,, heart, intestine, kidneys, liver, ovaries, pancreas,
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 vertebrate 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 photons 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 animal 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 gene inserted at the start of exon 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. Animals 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 brains
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 central nervous system (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
13
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
14
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, pain 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 parabrachial nuclei (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 vestibular nuclei, 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 midbrain (Fig. 4A-C), as well as the abducens nucleus in the
291 caudal portion of the pons (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 deep cerebellar nuclei (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 red nucleus, 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 forebrain, Opn3-eGFP was detected in the subthalamic area and the
305 dorsal thalamus (Fig. 4D-F), as were the projections of the cerebral peduncle to the dorsal
16
306 thalamus and the medial forebrain bundles in the lateral hypothalamus (Fig. 4D,E). In
307 addition, the dorso- and ventro-medial hypothalamic nuclei (Fig. 4D,E), which are related to
308 the autonomic nervous system, and the stria medullaris (Fig. 4G), which is part of the
309 epithalamus, 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 third ventricle (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 pontine nuclei (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 lateral lemniscus (Fig. 5D,G) and
340 the brachium of the inferior colliculus (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 habenular nuclei in the
349 medial habenula (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 axons 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 visual system 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 periaqueductal gray (Fig. 7B), which plays a critical role in the autonomic system, motivated
372 behavior and pain modulation, was Opn3-eGFP+. In the cerebellum, the fastigial nucleus 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 reticular formation, were identified as Opn3-eGFP+. Other
377 Opn3-eGFP+ midbrain-located nuclei comprised the deep nuclei of the superior colliculus
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 basal ganglia-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 substantia nigra (Fig. 7G), a basal
384 ganglia structure, and in the three white matter areas of the subthalamus, including the
385 lenticular fasciculus, thalamic fasciculus and field H (Fig. 7I-K), in addition to the
386 subthalamic nucleus (Fig. 7I).
387
388 At E17.5, Opn3-eGFP was detected in the pineal gland (Fig. 7J), an important structure
389 well-known to be involved in photoreception by producing melatonin 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 hippocampus,
407 in particular the dentate gyrus 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 olfactory bulb, 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 striatum 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 fornix (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 subiculum of the
441 hippocampal formation (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 supraoptic nucleus of the adult hypothalamus, including
451 both the supraoptic nucleus proper and retro-chiasmatic supraoptic nucleus, and in the bed
452 nucleus of the stria terminalis, 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 preoptic area (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 genes 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 archicortex (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 nucleus accumbens 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 trigeminal lemniscus
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 suprachiasmatic nucleus-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: dorsal root ganglion, 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 tegmentum, 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: dentate nucleus, fn: fastigial nucleus,
867 IntA: interposed nucleus, 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: cerebrum, 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: cerebral aqueduct, 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: rubrospinal tract, 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 cingulate cortex, 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 diencephalon 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 Pallium 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