bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Identifying Isl1 genetic lineage in the developing olfactory system and in GnRH-1 neurons 2 3 Ed Zandro M. Taroc1, Raghu Ram Katreddi1 and Paolo E. Forni1*. 4 5 1Department of Biological Sciences; The RNA Institute, and the Center for Neuroscience 6 Research; University at Albany, State University of New York, Albany, NY 12222, USA. 7 8 * Corresponding Author 9 [email protected] 10 11 12 Keywords: Olfactory neurons, vomeronasal sensory neurons, GnRH neurons, Islet-1/Isl1, Isl1 13 Conditional knock-out; genetic lineage tracing, olfactory placode, . 14 15 Abstract (299, at 245 after editing) 16 During , symmetric ectodermal thickenings (olfactory placodes) give rise 17 to several cell types that comprise the olfactory system, such as those that form the terminal nerve 18 ganglion (TN), gonadotropin releasing hormone-1 (GnRH-1) neurons and other migratory 19 neurons in rodents. Even though the genetic heterogeneity among these cell types are 20 documented, unidentified cell populations arising from the olfactory placode remain. One 21 candidate to identify placodal derived neurons in the developing nasal area is the transcription 22 factor Isl1, which was recently identified in GnRH-3 neurons of the terminal nerve in fish, as well 23 as expression in neurons of the nasal migratory mass. Here, we analyzed the Isl1 genetic lineage 24 in chemosensory neuronal populations in the nasal area and migratory GnRH-1 neurons in mice 25 using in-situ hybridization, immunolabeling a Tamoxifen inducible Isl1CreERT and a constitutive 26 Isl1Cre knock-in mouse lines. In addition, we also performed conditional Isl1 ablation in developing 27 GnRH neurons. We found Isl1 lineage across non sensory cells of the respiratory epithelium and 28 sustentacular cells of OE and VNO. We identified a population of transient embryonic Isl1+ 29 neurons in the olfactory epithelium and sparse Isl1+ neurons in postnatal VNO. Isl1 is expressed 30 in almost all GnRH neurons and in approximately half of the other neuron populations in the 31 Migratory Mass. However, Isl1 conditional ablation alone does not significantly compromise 32 GnRH-1 neuronal migration or GnRH-1 expression, suggesting compensatory mechanisms. 33 Further studies will elucidate the functional and mechanistic role of Isl1 in development of 34 migratory endocrine neurons. 35 36 1) Introduction (1044) bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

37 Cranial placodes are specialized regions of ectoderm, that give rise to the pituitary gland, 38 sensory organs and ganglia of the vertebrate head [1; 2]. They form as a result of specific 39 expression patterns of transcription factors in pre-placodal ectoderm surrounding the anterior 40 neural plate [2; 3]. In mice, the olfactory placodes (OPs) are morphologically identifiable at 41 embryonic day 9.5 (E9.5), as a bilateral ectoderm thickening in the antero-lateral region of the 42 head (fig1). The transcription factors Oct1, , Pou2f1, Pax6, Eya1, Six1, Six4, Ngnr1,Hes1, 43 Hes5, MASH1/Ascl1, Ngn1 and Gli3 have been identified to control various steps of olfactory 44 placode induction/formation, neurogenesis, neuronal development, and expression of olfactory 45 specific [4; 5; 6; 7; 8; 9; 10]. 46 The olfactory pit of vertebrates generates neurogenic and non-neurogenic progenitors. The non- 47 neurogenic portions of the olfactory pit give rise to the respiratory epithelium, which is located in 48 the rostral olfactory pit, while the neurogenic portions give rise to specific neuronal and 49 glial/support cells (Fig.1,2). The respiratory epithelium, which is the main source of Fgf8, controls 50 the development of the neural crest derived nasal mesenchyme and bones [11]. Neurogenic 51 waves in the olfactory pit of mice first gives rise to mostly migratory neurons [11; 12] such as early 52 pioneer olfactory neurons, neurons of the terminal nerve, including Gonadotropin releasing 53 hormone neurons (GnRHns), NPY positive migratory neurons, and other neurons with unknown 54 identity and function or neurons of the migratory mass (MM) [13; 14; 15; 16]. The nasal area of 55 mice contains several other neuronal cell types that include specialized olfactory sensory neurons 56 such as the guanylyl cyclase-D (GC-D) neurons of the necklace olfactory system [17; 18; 19], 57 microvillar cells (MVCs)[20], sensory neurons of the septal organ (SO) [21], the Grueneberg 58 ganglion (GG) [22; 23; 24; 25; 26], and cells forming the terminal nerve ganglion (TN) [27; 28; 29; 59 30; 31; 32; 33; 34] including the GnRH-1 ns [14; 15]. The mechanisms and molecules that drive 60 progenitors of the developing olfactory pit into early migratory cells types remains largely 61 unknown. 62 Between E 9.5 and E10.5, the OPs begin to ingress to form the olfactory pits that give rise to 63 various cellular components of the nose during development [6; 35]. The olfactory pit is largely 64 populated by proliferative progenitors expressing the transcription factors Hes1+/Hes5. These 65 early progenitors undergo mitosis in the apical portion of the epithelium and give rise over time 66 to neurogenic progenitors positive for the bHLH Ascl-1 [35; 36; 37; 38]. Gli3 67 influences proliferative Hes1+ positive progenitors to give rise to Ascl-1+ neurogenic progenitors 68 that eventually generate olfactory sensory neurons (OSNs) and vomeronasal sensory neurons 69 (VSNs) [36; 37]. Notably, Gli3 loss of function does not prevent neurogenesis of GnRH-1 70 neurons (GnRH-1ns), suggesting that these have a distinct lineage from the olfactory neurons. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

71 GnRH-1ns are the initial neuronal population to form in the developing olfactory pit [12; 14]. 72 During embryonic development, GnRH1ns migrate along the axons of the terminal nerve [28] 73 from the nasal area to the basal forebrain. Once in brain the GnRH-1ns control the release of 74 gonadotropins from the pituitary gland [39]. Aberrant development or function of GnRH-1ns 75 causes hypogonadotropic hypogonadism (HH) [40], that can lead to a spectrum of reproductive 76 disorders [41]. GnRH-1 neuronal migration strictly depends on correct development and 77 maturation of the neural crest derived olfactory ensheathing cells [42; 43; 44; 45]. Cre genetic 78 lineage tracing and bac transgenic reporters have revealed extensive genetic heterogeneity 79 among neurons that originate in the olfactory area of mice including the GnRHns, suggesting a 80 potential integration of neural crest cells in the developing OP [28; 46; 47; 48; 49]. However, the 81 mechanisms that give rise to the GnRH neurons and the required transcription factors for these 82 regulatory processes remain unresolved. 83 The LIM-homeodomain transcription factor Isl1 has been recently identified in subsets of neuronal 84 derivatives forming from the olfactory placode [49; 50; 51]. Isl1/2 expression occurs in GnRH 85 expressing neurons and other putative neurons of the terminal nerve [49; 50; 51; 52] in chick, 86 zebrafish, mouse, and humans. In zebrafish [50], Isl1/2 is expressed in terminal nerve GnRH-3 87 neurons associated with the olfactory epithelium. These neuro-modulatory cells, which are non- 88 migratory in fish, exert the same physiological function as migratory GnRH-1 neurons in mice [39]. 89 The terminal nerve and migratory mass in birds contains heterogeneous populations of cells that 90 1) express GnRH-1 but are negative for Isl1/2 and Lhx2, 2) cells only expressing Lhx2, 3) cells 91 only positive for Isl1, and 4) cells that co-express Lhx2, Isl1/2, GnRH-1 [51]. In birds, cells from 92 the terminal nerve ganglion (TN) are also positive for Isl1 and Lhx2 that differ from the GnRH- 93 1ns. A third study in mouse [49] also confirmed Isl1/2 immuno-reactivity GnRH-1ns at early stages 94 of GnRH neuronal development (E11.5). A subset of GnRH-1ns, positive for Wnt1Cre genetic 95 lineage [46], were negative for Isl1 immunoreactivity. 96 One prediction is that Wnt1Cre tracing, a controversial neural crest marker, and Isl1 97 immunoreactivity define two distinct embryonic lineages for GnRH-1ns. The rival hypothesis is 98 that the Wnt1Cre positive subpopulation of GnRH-1ns differ in its timing and/or expression levels 99 of Isl1 from the majority of GnRH-1ns [46; 49]. Understanding the molecular cascade that 100 establishes the genetic lineage of different cell types is a fundamental step to identify potential 101 cellular differences between cell types and to unravel the molecular mechanisms underlying 102 neurodevelopmental pathologies. Here, we examined which regions in the OP express Isl1/2 and 103 and analyzed Isl1 genetic lineage in different neuronal types in the nasal area of mice. We further 104 generated Isl1 conditional KO mice to study the role of Isl1 in GnRH development . bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

105 106 MATERIAL AND METHODS 107 108 Animals: Isl1flox(Isl1tm2Sev/J) [53]; Isl1CreERT(Isl1tm1(cre/Esr1*)Krc/SevJ) [54], Isl1Cre 109 (Isl1tm1(cre)Sev/J) [55] Rosa26tdTomato (B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J) 110 [56] and GnRH-Cre (Gnrh1-cre 1Dlc/J) [57] mouse lines were all purchased from JAX. Mice of 111 either sex were used for ISH and IHC experiments. All experiments involving mice were approved 112 by the University at Albany Institutional Animal Care and Use Committee (IACUC). 113 114 Tamoxifen treatment : Tamoxifen (Sigma‐Aldrich), CAS # 10540‐29‐1 was dissolved in Corn Oil. 115 Tamoxifen was administered once via intraperitoneal injection at indicated developmental stages. 116 Tamoxifen was administered at 180mg/Kg based on the weight of the pregnant dam on the day 117 of injection. 118 119 Tissue preparation: Embryos were collected from time-mated dams where the emergence of 120 the copulation plug was taken as E0.5. Collected embryos were immersion-fixed in 3.7% 121 Formaldehyde/PBS at 4°C for 3-5 hours. Postnatal animals were perfused with 3.7% 122 Formaldehyde/PBS. All samples were then cryoprotected in 30% sucrose overnight, then frozen 123 in O.C.T (Tissue-TeK) and stored at -80ºC. Samples were cryosectioned using CM3050S Leica 124 cryostat and collected on Superfrost plus slides (VWR) at 14m thickness for embryo’s and 16m 125 for post-natal tissue. 126 127 Immunohistochemistry: Primary antibodies and dilutions used in this study were: rabbit- α- 128 Isl1(1:1000, Abcam), mouse-α-Isl1/2 (1:100, DSHB), SW rabbit-α-GnRH-1 (1:6000, Susan Wray, 129 NIH), goat-α olfactory marker (1:4000, WAKO), rabbit-α-phospho-Histone-H3 (1:400, Cell 130 Signaling), goat-α-Sox2 (1:400, R & D systems), rat-α-phospho-Histone-H3 (1:500, Abcam), 131 mouse-α-Ki67 (1:500, Cell Signaling), rabbit-α-DsRed (1:1000, Clontech), goat-DsRed (1:1000, 132 Rockland), HuC/D 8ug/ml (Molecular Probes). Antigen retrieval was performed in a citrate buffer 133 prior to incubation with rabbit-α-Isl1, mouse-α-Isl1/2, rabbit-α-phospho-Histone-H3, rat-α- 134 phospho-Histone-H3, mouse-α-Ki67. For immunoperoxidase staining procedures, slides were 135 processed using standard protocols [11] and staining was visualized (Vectastain ABC Kit, Vector) 136 using diaminobenzidine (DAB) in a glucose solution containing glucose oxidase to generate 137 hydrogen peroxide; sections were counterstained with methyl green. For immunofluorescence, 138 species-appropriate secondary antibodies were conjugated with Alexa-488, Alexa-594, or Alexa- bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

139 568 (Molecular Probes and Jackson Laboratories) as specified in the legends. Sections were 140 counterstained with 4′,6′-diamidino-2-phenylindole (1:3000; Sigma-Aldrich) and coverslips were 141 mounted with Fluoro Gel (Electron Microscopy Services). Confocal microscopy pictures were 142 taken on a Zeiss LSM 710 microscope. Epifluorescence pictures were taken on a Leica DM4000 143 B LED fluorescence microscope equipped with a Leica DFC310 FX camera. Images were further 144 analyzed using FIJ/ImageJ software. Each staining was replicated on at least three different 145 animals for each genotype.

146 In situ hybridization: Digoxigenin-labeled cRNA probes were prepared by in vitro transcription 147 (DIG RNA labeling kit; Roche Diagnostics) from the following templates: Isl1 from Allen Brain 148 Atlas (Probe RP_080807_04_G12). In situ hybridization was performed as described [58] and 149 visualized by immunostaining with an alkaline phosphatase conjugated anti-DIG (1:500), and 150 NBT/BCIP developer solution (Roche Diagnostics).

151 Cell quantifications: GnRH-1/Isl1 double IHC quantifications were performed at E11.5 and 152 E15.5 parasagittal non-serial sections on a single slide using the Rabbit-α-GnRH-1 primary and 153 Mouse-α-Isl1/2 primary antibodies. Embryonic co-localized cell counts on Isl1Cre or Isl1CreERT 154 lineage traced animals were done on E14.5 and E15.5 respectively parasagittal non-serial whole 155 head sections within the nasal region on a single slide where we see co-localization of both 156 markers indicated (DsRed/tdTomato GnRH-1, or HuCD). Post-natal OMP cell counts were done 157 on P8 coronal nose sections. To get the density of mature OSNs positive for constitutive Isl1 158 lineage tracing at P8, number of OMP positive neurons colocalized for tdTom were counted in the 159 caudal olfactory epithelium and calculated the area of the OMP positive cells within the traced 160 epithelium. In VNO, to get the percentage of mature VSNs positive for constitutive Isl1 lineage 161 tracing, firstly OMP positive cells colocalized for tdTom were counted. To get the total OMP 162 positive cells in VNO section, density of OMP cells in smaller areas of VNO was determined and 163 extended to the total area of OMP positive cells in the VNO. 164 GnRH-1 cell counts were performed on GnRH-1Cre/Islet1flox/flox, Isl1CreERT/flox, and control at E15.5 165 on two immunostained non-serial slides. 166 167 Validation of conditional Isl1 knockout: The Isl1flox animals have the lox-p sites flanking exon 168 4 of the Isl1 which codes for the homeodomain and consists of the amino acids in positions 169 181-240. To detect conditional loss of Isl1flox in GnRHCre/Isl1flox/flox mutants we performed 170 immunolabeling using the mouse anti-Isl1/2 (DSHB) primary antibody. This antibody recognizes 171 the amino acids 178-349 of the Isl1 protein and was previously shown to be unable to detect bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

172 Isl1flox , (Isl1tm2Sev/J) mice, after Cre mediated recombination [53] . However, in Isl1CreERT 173 (Isl1tm1(cre/Esr1*)Krc/SevJ) which is null mouse model, the amino acids in positions 181-240 are 174 maintained, therefore this null protein is still detectable in Isl1 null cells. Since Isl1CreERT [54] and 175 Isl1flox alleles [53] have been targeted in different gene regions we could not validate 176 recombination efficency of Isl1CreERT/flox cKOs. 177 178 Statistical Analyses: All statistical analyses were carried out using GraphPad Prism7 software. 179 All cell counts were averaged ± standard error (SE) among animals of the same age and 180 genotype. Means ± SEs were calculated on at least three animals per genotype. The statistical 181 difference between genotypes and groups were determined using an unpaired student’s t-test. 182 183 RESULTS 184 185 Isl1 expression in proliferative progenitors and neurons during early development of the 186 olfactory pit 187 Using in-situ hybridization at E11.5, we found Isl1 mRNA expression in the more caudal region of 188 the putative developing olfactory epithelium and proximal to the developing VNO (Fig. 3 A). We 189 previously described the latter as the region where GnRH-1ns likely form and become 190 immunoreactive (Fig 3 B) [11]. By pairing immunohistochemistry against Isl1/2 and the mitotic 191 marker phospho-histone-3 (PHH3) at E11.5, we found (Fig. 3 C,C’) some proliferative progenitors 192 in the apical regions of the developing pit that were also positive for Isl1. Isl1/2 protein expression 193 was consistent with the mRNA expression pattern (Fig 1A, C). However, strong Isl1/2 194 immunoreactivity appeared localized in sparse nuclei ventral to the developing VNO and the 195 putative respiratory epithelium (Fig 1B-D). Immunolabeling with anti Isl1/2 and GnRH-1 at E11.5, 196 E13.5 and E15.5 (Fig 3D-F’’’) indicated a dynamic Isl1/2 expression across GnRH-1ns [49]. In 197 fact, we could identify at E11.5 approximately 89% (SE +/-3.33%) of GnRH-1+ cells positive for 198 Isl1/2 immunoreactivity (Fig 3D-D”). However, GnRH-1ns were negative for Isl1/2 199 immunoreactivity in both nasal area and brain by E15.5. 200 201 202 203 Differential lineage among olfactory, vomeronasal and GnRH-1 neurons 204 As we observed sparse and a heterogenous distribution of Isl1 mRNA and protein expression in 205 the developing olfactory pit at E11.5 (Fig. 3) and found Isl1/2 protein expression with the bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

206 proliferative cell marker PHH3, we decided to investigate which cells formed from the Isl1/2+ 207 proliferative cells. We utilized a tamoxifen inducible Isl1CreERT knock-in mouse line [54] mated 208 with a sensitive Rosa-reporter [56]. Isl1CreERT allows temporal control and restricts Cre mediated 209 recombination to the time of Tamoxifen injection. Pregnant dams were treated with a single 210 injection of Tamoxifen (180mg/Kg) at E11.5 and embryos were analyzed 4 days later (Fig. 4A). 211 At E15.5, we observed Isl1 recombination in trigeminal nerve, inner ear, Rathke’s pouch and oral 212 mucosa (Fig.4B,C) [59; 60; 61; 62]. We also found Isl1 tracing in the developing nasal area (Fig. 213 4D-E”) in sparse OMP+ sensory neurons of both the main olfactory and vomeronasal epithelium. 214 Notably, Isl1Cre+ neurons positive for the pan neuronal marker HuC/D were found at the border 215 between the respiratory epithelium and sensory epithelium (Fig. 4E-E’). We found extensive Cre 216 recombination penetrance throughout the nasal respiratory epithelium (Fig. 4,D,D’). 217 We detected putative nasopalatine and ethmoidal trigeminal axons positive for Isl1 through-out 218 the nasal area and tangential to the olfactory bulb (Fig 4B,C,D,D’). By analyzing the olfactory 219 projections via olfactory marker protein (OMP) and Peripherin immunostaining in combination with 220 anti tdTomato staining, we confirmed that Isl1Cre recombination occurred in neurons that 221 appeared to bundle with the olfactory neurons. Notably, most Isl1CreERT traced axons did not 222 appear to project to the main or accessory olfactory bulb (Fig. 4D,D’). Analysis of the olfactory 223 epithelium showed a very sparse colocalization between OMP and Isl1Cre mediated 224 recombination. However, immunostaining against the pan neuronal marker HuC/D showed the 225 presence of Isl1+ neurons within the developing olfactory epithelia (Fig. E, E’). Triple 226 immunostaining against GnRH-1, tdTomato and the pan neuronal protein HuC/D showed that 227 temporally controlled Isl1CreERT recombination at E11.5 yielded 85% (SE +/-1.88%) of GnRH-1ns 228 were positive for tracing, while only 15%(SE +/-0.91) of the migratory (HuC/D+/GnRH-) cells were 229 Isl1 positive. These data suggest that Isl1 expression at E11.5 (Fig. 3 A-C) is mostly limited to 230 progenitors or precursors of GnRH-1ns and the subsets of other neurons of the migratory mass. 231 232 Analysis of Constitutive Isl1 Cre genetic lineage tracing at E14.5. 233 Tamoxifen controlled Cre recombination at E11.5 indicated that few neuronal progenitors and 234 sparse neurons in the OE and VNO were positive for Isl1 as the olfactory pit forms, while the 235 respiratory epithelium, oral mucosa and the majority of the GnRH-1ns resulted expressing Isl1. 236 To further perform a temporally unbiassed lineage tracing for Isl1, we exploited a traditional 237 Isl1Cre knock-in mouse line. In these mice recombination is expected to occur whenever Isl1 is 238 expressed [55]. We performed a Cre/Rosa reporter lineage analysis Isl1Cre+/-/R26tdTomato+/- at E14.5 239 and P8. After temporally controlled recombination (Fig.4), constitutively active Isl1 Cre at E14.5 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

240 (Fig.5) showed recombination in the trigeminal nerve, cells of the inner ear [63], the Rathke’s 241 pouch forming the anterior pituitary gland [64], the oral mucosa and the nasal respiratory 242 epithelium. 243 During gross visual observation, we noticed that more cells positive for Isl1 lineage occurred in 244 both olfactory and vomeronasal epithelia compared to that after temporally controlled Cre 245 activation (Compared Fig.5 and Fig.4). 246 247 Isl1 Cre recombination in GnRH and terminal nerve neurons 248 At E14.5 we performed OMP, Peripherin and tdTomato immunostaining. We observed labeled 249 olfactory and terminal axonal projections (Fig. 5 D-E’’’). In the olfactory system, we noticed that 250 Isl1 Cre tracing could be seen in fibers forming bundles with the olfactory neurons after temporally 251 controlled Isl1 Cre recombination (Fig. 5 D’-D’’’). However, most axons positive for recombination 252 at this stage appeared to project only to the ventral portions of the olfactory bulb with some verging 253 on invading the basal forebrain brain (Fig.5 E-E’’’’). The terminal nerve enters the brain ventral to 254 the olfactory bulb [28]. Fibers of the putative terminal nerve associated with Isl1+ migratory GnRH- 255 1ns. Using immunostaining against peripherin and tdTomato, we visualized Isl1+ positive cell 256 somas and fibers of GnRH-1ns migrating on peripherin positive axons of the TN. Some putative 257 TN’s axons showed sparse positivity for tracing (Fig.5 E-E’’’’). To confirm Isl1 Cre tracing in cells 258 of the terminal nerve, we immunostained against the antigen Robo3, which is selectively 259 expressed by these cells [28]. Robo3/tdTomato staining confirmed the presence of cell bodies 260 from putative TN cells, proximal and within the developing vomeronasal organ, some of these 261 appeared to be positive for Isl1 lineage. We also observed TN fibers, positive for Robo3, in the 262 brain (data not shown). GnRH-1, tdTomato and HuC/D (Fig. 6 D-D’’’) immunostaining confirmed 263 Isl1 lineage in virtually all GnRH-1ns (95%(SE +/-0.68), while 55% (SE +/-4.63) of the HuC/D 264 migratory neurons that were negative for GnRH-1, were actually positive for Islet1 tracing (Fig. 6 265 E). 266 267 Postnatal analyses of Isl1Cre lineage in the nasal cavity 268 During embryonic development, we observed Isl1 recombination in neurons in both olfactory and 269 vomeronasal epithelia. So, we analyzed the pattern of constitutive Isl1 Cre recombination in 270 postnatal animals. We performed double immunostaining against tdTomato and OMP at postnatal 271 day 8 and noticed that most olfactory epithelium neurons did not express OMP and Isl1. However, 272 we did find sparse neurons positive for Isl1 tracing in the most caudal ethmoid turbinates (0.12 273 (+/- 0.03 SEM) cells/1000m2). Isl1Cre positive sustentacular cells were found close to the border bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

274 between the respiratory epithelium and sensory olfactory epithelium in the ventral-lateral zones 275 of the OE and along the nasal septum (S) in all the sections from rostral to caudal. At visual gross 276 observation, we noticed more pronounced Isl1+ lineage in sustentacular cells in the caudal OE 277 along the lateral ethmoid turbinates. These data imply spatial/regional heterogeneity in gene 278 expression among sustentacular cells [65]. 279 Analysis of postnatal vomeronasal epithelia showed a different scenario from that in the OE. 280 Immunostaining against TdTom indicated Isl1 tracing in both non-sensory epithelium (NSE) and 281 sensory epithelium of the VNO. In the sensory epithelium, staining against OMP and tdTom 282 showed that sparse recombination in 3.74% (+/-1.06 SEM) of the neurons. The vomeronasal 283 neurons positive for tracing were variously distributed in apical and basal regions. Sox2 and 284 tdTomato immunostaining revealed, as observed in the OE, that Islet1 tracing was detectable in 285 sustentacular cells (Fig 7G). Since constitutive Isl1 lineage showed cells positive in olfactory and 286 vomeronasal epithelia, we further investigated if Isl1 recombination also occurred in neurons of 287 the septal organ and Gruenberg ganglion (GG). By performing immunostaining anti OMP in 288 postnatal animals, we found that both neuronal populations were negative for lineage tracing. 289 Immunostaining with anti-CART and -PDE2A antibodies also revealed an absence of Isl1 290 expression in Necklace glomeruli cells (NGCs) (data not shown). 291 292 Conditional Isl1 loss of function in GnRH-1ns 293 Isl1 is important for differentiation, cell migration, survival and axonal targeting of neurons of the 294 peripheral nervous system [53; 66; 67]. Our data revealed strong Isl1/2 expression in developing 295 migrating GnRH-1ns (Fig.3, 3,4,6). To test if Isl1 plays a role in GnRH-1ns migration or controls 296 GnRH expression, we generated GnRH-1Cre/Islflox/flox conditional mutants. Embryos were 297 analyzed at E15.5, which is the stage at which the majority of the GnRH-1ns have already 298 migrated in the brain [68]. Double immunostaining against Isl1/2 and GnRH confirmed detectable 299 Isl1 ablation in 38% (SE +/-5.28%) of GnRH-1 neurons (Fig. 9 A-B’). While Isl1 in controls was 300 detectable in virtually all migratory GnRH-1ns. In GnRH-1Cre/Islflox/flox conditional mutants, 301 GnRHns negative for Isl1 immunoreactivity were found in the nasal area, forebrain junction and 302 the brain. These data suggest that Isl1 expression is not required for GnRH-1 peptide expression 303 nor GnRH-1 neuronal migration. Quantification of GnRH-1ns distribution in nasal area, forebrain 304 junction and brain indicated an overall distribution of GnRH-1 neurons comparable with the one 305 of controls (Fig 9. E). 306 307 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

308 309 Discussion 310 Cellular derivatives of the olfactory placodes play central roles in respiration, chemosensory 311 detection, sexual development, fertility and inter and intra species social behaviors. 312 Understanding the basic mechanisms that define cellular diversity and underly normal and 313 pathological development of the olfactory placode derivatives is of high clinical importance. Isl1 314 expression can occur in various neurogenic placodes [69; 70]; however, a comprehensive 315 analysis of Isl1 expression remains unknown. Isl1, which is important for differentiation, cell 316 migration, survival and axonal targeting of neurons in the peripheral nervous system [53; 66; 67], 317 has a very high upregulation during GnRH-1 neuronal differentiation [52] . So, Isl1 remained a 318 potential suitable genetic marker to label derivatives of the olfactory placode [49]. Here, we sought 319 to trace Isl1 expression and lineage in the developing nasal area using in situ-hybridization, 320 immunohistochemistry (Fig.3), temporally controlled Is1CreRT recombination (Fig. 4) and 321 constitutive Isl1Cre genetic lineage tracing (Fig 5-8). 322 323 GnRH-1 lineage tracing in the developing nasal area 324 Using in situ hybridization and Immunohistochemistry, we detected Isl1 expression at E11.5 in 325 the putative GnRH neurogenic area, ventral to the developing VNO (Fig. 3A-D), in newly formed 326 GnRH-1 neurons and the developing olfactory epithelium (Fig3D-F’’’). At E11.5, Isl1 327 immunoreactivity occurred in 89%(+/-3.33%) of GnRH cells, in line with published reports [49]. 328 However, histochemistry at E13.5 and E15.5 showed strong Isl1/2 immunoreactivity in virtually all 329 GnRH-1 neurons, suggesting that Isl1 expression is not limited to specific subpopulations of 330 GnRH cells [49]. These data suggest a dynamic expression of Isl1 with more GnRH-1 cells 331 positive across development. 332 At E11.5 we also found the existence of proliferative cells positive for Isl1 in the developing 333 olfactory pit. Tamoxifen controlled Isl1CreERT tracing at E11.5 showed Isl1 lineage in putative 334 GnRH progenitors/newly formed GnRH-1 neurons, cells of the respiratory epithelium, few 335 proliferative progenitors in the developing olfactory pit, neurons at the border between respiratory 336 epithelium and developing olfactory epithelium and sparse neuronal (HuC/D+) cells in the OE and 337 VNO. Notably in this experiment we analyzed Isl1CreERT/R26R recombination 4 days after Tam 338 treatment. This means that, when we observed the histological samples, we identified either cells 339 that expressed Isl1 at the moment of Tam injection or cells that derived from progenitors 340 expressing Isl1 at time of Tam injection. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

341 In line with what observed after temporally Tam controlled recombination at E11.5, constitutively 342 active Isl1Cre recombination showed that, during embryonic development (Fig 5,6), Isl1 tracing 343 highlights neurons in OE proximal to the border with RE, neurons in the vomeronasal organ, cells 344 of the RE, GnRHns, migratory neurons negative for GnRH, and sustentacular cells in olfactory 345 and vomeronasal organ. 346 These data indicate that Isl1 expression/lineage is limited to specific subsets of olfactory placodal 347 derivatives. In contrast to prior observations during embryonic development, Isl1Cre 348 recombination analysis of postnatal Isl1Cre/R26R mice showed only sparse neurons positive for 349 Isl1 tracing in the most caudal regions of the olfactory sensory epithelium, while cells positive for 350 Isl1 tracing were still found in the VNO, respiratory epithelium and in sustentacular cells of both 351 OE and VNO (Fig.7). No Isl1 expression or lineage was in GC neurons and Grueneberg ganglion 352 (Fig.8). These data suggest that Isl1 positive neurons in the OE at the border with the RE during 353 embryonic development (Fig.4,5) either migrated out of the epithelium as a part of the migratory 354 mass or died, even though we could still detect Isl1 expression/lineage in some vomeronasal 355 organ neurons (Fig. 7 F-F’’). So, we further defined the fate of the Isl1 neurons detectable in the 356 OE during embryonic development. During embryonic development we could also detect axonal 357 projections positive for Isl1 traversing through the nasal area (Fig 4,5). Some axonal projections 358 likely belonged to the nasopalatine projections of the trigeminal nerve (Fig 3, 4,5). Although both 359 Isl1CreERT and Isl1Cre induced recombination in Peripherin+ fibers projected to the ventral OB, the 360 majority of OMP+ fibers projecting to the MOB and AOB appeared negative for Isl1 tracing (Fig 361 4,5). We did not pursue a detailed analysis of olfactory projections in postnatal animals, due to 362 technical challenges in tracing Isl1 recombination in mitral cells of the MOB. 363 364 Isl1 in the GnRH-1 system 365 During embryonic development GnRH-1ns migrate from the developing olfactory pit into the 366 hypothalamus. Once in the preoptic area, GnRH neurons (GnRH-1ns) control the hypothalamic– 367 pituitary–gonadal hormonal axis and reproductive development. Defective GnRH-1 release is the 368 cause of Hypogonadotropic Hypogonadism, a condition that negatively impacts normal body 369 development, social interactions, the ability to procreate, and physical performances [71; 72; 73; 370 74]. Hypogonadotropic Hypogonadism associated with congenital anosmia (impairment of the 371 sense of smell) is clinically defined as Kallmann Syndrome [75; 76; 77; 78; 79; 80; 81]. Though 372 postnatal development of the olfactory system has been extensively studied we still ignore many 373 aspects regarding early development of the olfactory pit derivatives. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

374 Previous studies in chick and mice indicate that soon after the olfactory placodes form they give 375 rise to migratory olfactory pioneer neurons, and to several other neuronal populations including 376 the terminal nerve cells and the GnRH-1ns. Placodal derived migratory neurons and the neural 377 crest derived Olfactory ensheathing cells form the migratory mass [45; 82]. 378 We previously shown that the fibers of the terminal nerve, upon which the GnRH-1ns migrate, are 379 distinct from the olfactory and vomeronasal sensory neurons [28; 36; 83]. What progenitors give 380 rise to the GnRH neurons, and what transcription factors control their onset are still open 381 questions. Moreover, if what we define as GnRH neurons is one homogenous neuronal 382 population is also a matter of debate. 383 Lineage tracing at different developmental stages suggest strong and consistent Isl1 expression 384 in GnRH-1ns and cells of the migratory mass (Fig.3,4,6). While the terminal nerve, that projects 385 from the vomeronasal area to the basal forebrain appeared to have heterogenous Isl1 expression/ 386 genetic lineage tracing. (Fig. 6) 387 Isl1 has been reported to be important in neuronal differentiation, cell migration, survival and 388 axonal targeting of various neuronal types [53; 66; 67]. The consistent Isl1/2 expression in 389 developing and migratory GnRH-1ns (Fig. 3,4,6), which is conserved from fish to humans 390 prompted us to test its developmental role. 391 We first made an attempt to conditionally KO Isl1, which as a whole KO is embryonic lethal, by 392 mating Isl1CreERT mice (Fig. 4), which are Isl1null heterozygous, with Isl1flox. After tamoxifen injection 393 at E11.5 we analyzed Isl1CreERT/flox mice and controls at E15.5 (as in Fig. 4). After Tamoxifen 394 treatment Isl1CreERT extensively recombines in the developing GnRH-1 neurons (Fig.4). 395 Surprisingly Tam+ Isl1CreERT/Isl1flox embryos did not show any obvious phenotype related to 396 GnRH-1ns (supplementary data). However, for this model, we could not verify the efficiency of 397 recombination because antibodies unable to detect the floxed Isl1 truncated protein were still 398 recognizing the Isl1CreERT protein product (see material and methods). For this reason, we report 399 these as supplementary observations. In order to further test the role of Isl1 in GnRH-1 neuronal 400 development we generated GnRH-1Cre/Isl1flox/flox conditional mutants. Here, we could document 401 complete loss of Is1 immunoreactvity in around ~40% of GnRH-1 neurons (Fig.9). Notably most 402 of the anti Isl1 Abs have some level of cross reactivity with Isl2, therefore we performed in-situ 403 hybridization against exon 4 of Isl1 paired with anti GnRH-1 immunofluorescent staining (not 404 shown). These experiments suggest similar penetrance of recombination as indicated by IF. 405 Notably, also in GnRH-1Cre/Isl1flox/flox mutants we did not observe obvious defects in GnRH-1 406 neuronal migration nor in GnRH-1 expression (Fig. 9). In fact, GnRH-1 neurons negative for Isl1 407 were found in both nasal area and brain. Total number and overall distribution of GnRH-1 was bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

408 comparable to controls. These data suggest that Isl1 alone is not necessary for GnRH-1 409 expression or neuronal migration. If Isl2 is also expressed in the GnRH-1ns and play a 410 compensatory effect is worth further analysis. 411 In conclusion, our data indicate very selective expression of Isl1 in neurons forming in the OP 412 including the GnRH-1 neurons, various migratory neuronal populations and some cells in the 413 VNO, terminal nerve and sustentacular cells on the more medial and lateral portions of the OE. 414 Isl-1 immunoreactivity and genetic tracing suggest that Isl-1 expression, though dynamic, is 415 common feature for virtually all GnRH-1 neurons [49] and several other neurons of the migratory 416 mass. If all these early migratory neurons derive from similar pre placodal subregions should be 417 further investigated. Performing GnRH-1ns specific Isl1 ablation in GnRHns we did not detect 418 obvious cell specification, differentiation, survival or migratory defects [49] at embryonic 419 development stages suggesting a dispensable role for Isl1 in GnRH neurons. 420 Our results leave open the question of what is the physiological role of this transcription factor in 421 murine GnRH-1 neurons development and function. Further studies focused on Isl1 role in 422 postnatal developmental and fertility can give definitive insights. 423 424 425 Ethics Statement 426 All mouse studies were approved by the University at Albany Institutional Animal Care and Use 427 Committee (IACUC). 428 429 Conflict of Interest 430 The authors declare that the research was conducted in the absence of any commercial or 431 financial relationships that could be construed as a potential conflict of interest.

432 Author Contributions

433 EZT, set the mouse mating, collected samples prepared histological samples, performed 434 immunostaining, imaging, and analyzed data at embryonic developmental stages. RRK prepared 435 histological samples, performed immunostaining and analyses of postnatal animals. EZT and PEF 436 designed the experiments wrote the article and prepared figure panels.

437 Funding

438 Research reported in this publication was supported by the Eunice Kennedy Shriver National 439 Institute of Child Health and Human Development of the National Institutes of Health under the 440 Awards R15-HD096411 (PEF), and R01-HD097331/HD/NICHD (PEF) and by the National 441 Institute of Deafness and other Communication Disorders of the National Institutes of Health 442 under the Award R01-DC017149 (PEF). bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

443

444 Fig.1) Formation of the olfactory pit. Drawings depict developing embryos between E9.5 and E11.5. The frontonasal areas where 445 the olfactory placodes/pits form are indicated with red arrows. Cartoons in the lower row illustrate the olfactory placode ingression 446 between E9.5 and E10.5 (chronal view) and a parasagittal view of the olfactory pit at E11.5. The lateral and rostral portions of the 447 invaginating olfactory placode (blue) act as the main source of FGF8. These Fgf8+ areas progressively differentiate into respiratory 448 epithelium. In the neurogenesis portion of the olfactory placode (yellow), proliferative Hes-1+ progenitors (green) give rise over time 449 to neurogenic Ascl-1 neuronal progenitors. Arrows show the vomeronasal organ (OE) and putative neurogenic area of the GnRHns 450 at E11.5 in the developing olfactory epithelium (OE),. The size of the pit and cells is arbitrary and does not reflect natural proportions 451 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

452

453 454 455 Fig. 2) E15.5 mouse embryo, neuronal populations in the nasal area. 456 Grueneberg ganglion (GG, green) and GC-D+/necklace (OSNs, magenta) project to the necklace glomeruli (NG). Olfactory neurons 457 of the main olfactory epithelium (MOE, yellow) projecting to the main olfactory bulb (MOB). Vomeronasal organ (VNO), composed of 458 basal (bVSNs, green) and apical (a-VSNs, red), projecting to anterior and posterior portions of the accessory olfactory bulb (AOB). 459 Septal Organ (SO, light blue) projecting to the ventral OB. Terminal nerve (TN, purple) projecting to the basal forebrain, GnRHns, 460 orange, scattered along the terminal nerve fibers from the vomeronasal area to the basal forebrain. Trigeminal nasopalatine (NPN) 461 and ethmoidal nerve projections (EN) drawn in dark gray. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

462

463 464 Figure 3. Isl1 expression in the OP is restricted to GnRH neurons and a few other cells types. A) E11.5 In-situ hybridization 465 anti-Isl1 mRNA shows Isl1 expression in the ventral portion of the developing olfactory pit proximal to the VNO. Arrows indicate 466 putative GnRH neurogenic area. Only spare cells positive for Ilset1 could be detected in the OE (arrowhead). B) Cartoon illustrating 467 the putative areas giving rise to RE, OE, VNO and the putative GnRH neurogenic area. Blue dots indicate GnRHns. C,C’) 468 Immunostaining anti-Isl1 (red) and the mitotic marker pHH3 (green) shows Ilset1 expression in sparse proliferative cells in the apical 469 portion of the OP (arrowheads) and in cells in the putative GnRH neurogenic area ventral to the VNO (boxed) compare to A,B. D) 470 Isl1 immunoreactivity in the putative GnRH neurogenic area (arrows, compare to A,B,C). C’-C”’) Isl1 expression (black) in newly 471 formed GnRH neurons(arrows) and in GnRH-1 negative cells (arrowheads). E-F”’ Isl1 expression (black) in migrating GnRHns in the 472 (arrows) in the nasal area at E13.5 (E-E”’) and in the brain at E15.5. (F-F’”). Olfactory epithelium (OE), Olfactory bulb (OB), Forebrain 473 junction (FBJ), Preoptic area (POA). bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

474 475 Fig. 4 Temporally controlled Isl1 lineage tracing at E11.5 primarily highlights cells of the respiratory epithelium and 476 GnRHns. A) Cartoon illustrating experiential strategy for Ilset-1CreERT/R26RTdTomato lineage tracing. Tamoxifen induction of Cre 477 recombiantion at E11.5, analysis at E.15.5. B,C) HuC/D/DsRed double immunostaining shows recombination (tdTomato) in the 478 Trigeminal Ganglion (TG), the inner ear as well as in cells of the Rathke’s Pouch (RP). D-D’’ In the developing nose recombination 479 appears to be mostly restricted to the respiratory epithelium (RE) and Ethmoidal (EN) trigeminal projections. D-D” Anti tdTomato; 480 OMP; Peripherin immunostaining showed that OMP positive olfactory projections of the main olfactory bulb. Peripherin positive 481 projections to the accessory olfactory bulb appear negative for td Tomato expression. However, tdTomato positive fibers (D’ white 482 arrow), likely belonging to the trigeminal/ethmoid nerve (See EN in D), appeared to project to the ventral OB. D”) OMP and 483 tdTomato immunoreactivity showed nearly complete absence of OSNs positive for Isl1 tracing. Neurons in the olfactory area 484 appeared mostly negative for Isl1 lineage D”, sparse OMP+ tdTomato + cells (Notched arrowhead) could be detected at the border 485 between olfactory and respiratory epithelium, boxed area see D’. E-E’’ E’’’) Pan neuronal marker HuC/D shows neurons of the 486 developing vomeronasal organ (VNO), mostly negative for Isl1 lineage (E’). HuC/D+ neurons at the border between OE and RE (E’) 487 migratory neurons, GnRH-1ns (E”) and HuC/D+only (E”’) were positive for Isl1 tracing. F) Quantification of recombination in 488 migratory GnRH-1+ and HuC/D GnRH-1 negative neurons. 489 490 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 Fig. 5 Constitutive Isl1 lineage tracing highlights: sparse neurons the OE, VNO, neuronal projections to the ventral OB, GnRH 524 neurons and some TN fibers A-B) Immunostaining against tdTomato of Isl1Cre/R26tdTomato lineage traced embryo’s at E14.5, 525 showing recombination the trigeminal ganglion (TG), inner ear, Rathke’s pouch (RP), the oral mucosa (OM), Respiratory epithelium 526 (RE) and in sparse cells in the OE and VNO. C) Anti-tdTomato; OMP and C’) anti td Tomato; HuC/D stainings shows that though 527 almost all the the OMP+ cells in the were negative for tracing (arrowheads) (C), those were neurons positive for HuC/D (arrowhead). 528 D-E’’’) Immunostaining against tdTomato, OMP, and Peripherin to highlight the projections of the developing olfactory system reveal 529 that Isl1 tracing (D’-D’’’’) is mostly absent in the OMP+ olfactory fibers (OF) and Peripherin+ vomeronasal fibers (VNF) innervating the 530 olfactory bulb (OB). GnRH-1 and other cells of the migratory mass positive for the tracing were detected invading the brain along the 531 terminal nerve (TN). 532 E-E”’ Peripherin positive TN fibers in the brain. Some fibers appear positive for the tracing (solid arrow) while other Peripherin fibers 533 appear to be negative (empty arrow). bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

534 535 Fig. 6 Isl1 constitutive lineage tracing highlights GnRHns, some cell bodies of the TN, and other migratory neurons. A-A’’) 536 HuC/D and tdTomato immunostaining of Isl1Cre+/-/R26tdTomato VNO at E14.5 show some neurons within the VNO positive (empty 537 arrowhead) and negative (notched arrowhead) for Isl1 recombination. Population of tdTomato+ non-neuronal cells positive for 538 tracing (arrow). B-B’’’) Anti- Robo3; tdTomato and Peripherin labeling shows that a portion of putative terminal nerve cell bodies 539 located within and proximal to the VNO are highlighted by Isl1 recombination (arrow) while a portion do not (arrowhead). C-C’) 540 GnRH and tdTomato staining shows recombination in the majority of the GnRHns (arrowheads). D-D’’) shows recombination in ~ 541 half of the (GnRH-;HuC/D+) migratory neurons (arrows. E) Quantification of Isl1Cre recombination in migratory GnRH+ and HuC/D 542 GnRH-1 negative neurons 543 Fig. 6 Isl1 constitutive lineage tracing highlights GnRHns, some cell bodies of the TN, and other migratory neurons. A-A’’) 544 HuC/D and tdTomato immunostaining of Isl1Cre+/-/R26tdTomato VNO at E14.5 show some neurons within the VNO positive (empty 545 arrowhead) and negative (notched arrowhead) for Isl1 recombination. Population of tdTomato+ non-neuronal cells positive for 546 tracing (arrow). B-B’’’) Anti- Robo3; tdTomato and Peripherin labeling shows that a portion of putative terminal nerve cell bodies 547 located within and proximal to the VNO are highlighted by Isl1 recombination (arrow) while a portion do not (arrowhead). C-C’) 548 GnRH and tdTomato staining shows recombination in the majority of the GnRHns (arrowheads). D-D’’) shows recombination in ~ 549 half of the (GnRH-;HuC/D+) migratory neurons (arrows. E) Quantification of Isl1Cre recombination in migratory GnRH+ and HuC/D 550 GnRH-1 negative neurons bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

551 552 Fig. 7 Lineage tracing of Isl1 positive cells postnatally highlights neuronal and non-neuronal cells in OE and VNE. A,B,C- 553 Immunofluorescence against tdTom showing Isl1 tracing in olfactory epithelium (OE) from rostral toward the caudal ethmoid 554 turbinates. Arrows (A,B,C) show tracing in OE. (B notched arrowheads) show tracing in VNO. D-D’’’) Double immunofluorescence 555 against OMP (green) and tdTom (red) show no colocalization in neurons of the OE. E-E’’’) Double immunofluorescence against 556 Sox2 (green) and tdTom (red) show Ils1Cre tracing (arrow marks) in the sustentacular cells of the OE. Sox2 is present in both 557 globose basal cells that are present in the base of the OE and sustentacular cells in the upper layers of the OE. F) Double 558 immunofluorescence against OMP (green) and tdTom (red) show Isl1 tracing in both non-sensory epithelium (NSE- arrow mark) and 559 sensory epithelium of the VNO. F’-F’’) Arrow marks show colocalized mature vomeronasal neurons (VSN) and notched arrow heads 560 show traced neurons that are not labelled with OMP. G-G’’) Double immunofluorescence against Sox2 (green) and tdTom (red) 561 show Isl1 tracing in Sustentacular cells in the VNO. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

562 Fig. 8 Post natal Isl1 constitutive lineage tracing is not expressed in other nasal sensory neurons. A) Isl1Cre/R26tdTomato 563 P8 parasagittal section of the nose immunostained against OMP and tdTomato show that Isl1 recombination is limited to the 564 respiratory epithelium even in post-natal stages. Neuronal sensory populations, such as the Gruenberg Ganglion GG B-B’’), is 565 negative for recombination. C) Coronal view of Isl1Cre/R26tdTomato at P8, stained against OMP and tdTomato shows Isl1 tracing 566 restricted to the non-sensory cells and cells of the respiratory epithelium. D-D’’) Magnification from C) of the border between 567 olfactory and respiratory epithelium shows Isl1Cre tracing restricted to the respiratory epithelium (arrowhead), while OMP+ neurons 568 are negative (arrow). E-E’’) Septal Organ (SO) from C) indicates Isl1 recombination is restricted to the non-sensory (arrowheads) 569 cells, while OMP+ neurons are negative (arrows). 570 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 Fig 9. Conditional Loss of Isl1 in GnRHns has no effect. A-B’) Isl1/2 and GnRH double IF on E15.5 WT (A-A’), where all GnRHns 603 express Isl1/2 (arrows) and cKO (B-B’) sections show that some GnRH-1ns lose the expression of Isl1 (arrowheads). C-D) 604 Immunohistochemistry of GnRH-1 on WT and cKO sections show comparable distribution of cells positive for GnRH-1. E) 605 Quantification of cKO show no changes in the number or distribution of GnRH-1ns. NS: Nasal Septum, OB: Olfactory Bulb, bFB: basal 606 Forebrain 607 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 Supp. Fig. 1 Conditional Loss of Isl1 at E11.5 does not affect GnRH1ns A-B) GnRH IHC on WT(A) and cKO (B) sections show 636 that between each genotype the distribution of GnRHns looks similar. C) Quantification of the number of GnRHns in whole embryo 637 heads show no significant regional changes or in total number 638 639 [1] G. Schlosser, Induction and specification of cranial placodes. Dev Biol 294 (2006) 303-51. 640 [2] S.A. Brugmann, and S.A. Moody, Induction and specification of the vertebrate ectodermal 641 placodes: precursors of the cranial sensory organs. Biol Cell 97 (2005) 303-19. 642 [3] A.P. Bailey, and A. Streit, Sensory organs: making and breaking the pre-placodal region. 643 Curr Top Dev Biol 72 (2006) 167-204. 644 [4] D. Zou, D. Silvius, B. Fritzsch, and P.X. Xu, Eya1 and Six1 are essential for early steps of 645 sensory neurogenesis in mammalian cranial placodes. Development 131 (2004) 5561-72. 646 [5] G. Schlosser, T. Awtry, S.A. Brugmann, E.D. Jensen, K. Neilson, G. Ruan, A. Stammler, D. 647 Voelker, B. Yan, C. Zhang, M.W. Klymkowsky, and S.A. Moody, Eya1 and Six1 648 promote neurogenesis in the cranial placodes in a SoxB1-dependent fashion. Dev Biol 649 320 (2008) 199-214. 650 [6] B. Chen, E.H. Kim, and P.X. Xu, Initiation of olfactory placode development and 651 neurogenesis is blocked in mice lacking both Six1 and Six4. Dev Biol 326 (2009) 75-85. 652 [7] N. Riddiford, and G. Schlosser, Dissecting the pre-placodal transcriptome to reveal 653 presumptive direct targets of Six1 and Eya1 in cranial placodes. Elife 5 (2016). 654 [8] A.L. Donner, V. Episkopou, and R.L. Maas, Sox2 and Pou2f1 interact to control lens and 655 olfactory placode development. Dev Biol 303 (2007) 784-99. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

656 [9] E. Cau, G. Gradwohl, C. Fode, and F. Guillemot, Mash1 activates a cascade of bHLH 657 regulators in olfactory neuron progenitors. Development 124 (1997) 1611-21. 658 [10] E. Cau, S. Casarosa, and F. Guillemot, Mash1 and Ngn1 control distinct steps of 659 determination and differentiation in the olfactory sensory neuron lineage. Development 660 129 (2002) 1871-80. 661 [11] P.E. Forni, K. Bharti, E.M. Flannery, T. Shimogori, and S. Wray, The indirect role of 662 fibroblast growth factor-8 in defining neurogenic niches of the olfactory/GnRH systems. 663 J Neurosci 33 (2013) 19620-34. 664 [12] M. Fornaro, S. Geuna, A. Fasolo, and M.G. Giacobini-Robecchi, HuC/D confocal imaging 665 points to olfactory migratory cells as the first cell population that expresses a post-mitotic 666 neuronal phenotype in the chick embryo. Neuroscience 122 (2003) 123-8. 667 [13] M. Fornaro, S. Raimondo, J.M. Lee, and M.G. Giacobini-Robecchi, Neuron-specific Hu 668 sub-cellular localization in primary sensory neurons. Ann Anat 189 (2007) 223- 669 8. 670 [14] S. Wray, P. Grant, and H. Gainer, Evidence that cells expressing luteinizing hormone- 671 releasing hormone mRNA in the mouse are derived from progenitor cells in the olfactory 672 placode. Proceedings of the National Academy of Sciences of the United States of 673 America 86 (1989) 8132-6. 674 [15] M. Schwanzel-Fukuda, and D.W. Pfaff, Origin of luteinizing hormone-releasing hormone 675 neurons. Nature 338 (1989) 161-4. 676 [16] E.M. Hilal, J.H. Chen, and A.J. Silverman, Joint migration of gonadotropin-releasing 677 hormone (GnRH) and neuropeptide Y (NPY) neurons from olfactory placode to central 678 nervous system. J Neurobiol 31 (1996) 487-502. 679 [17] M. Luo, The necklace olfactory system in mammals. J Neurogenet 22 (2008) 229-38. 680 [18] K. Mori, H. Manabe, and K. Narikiyo, Possible functional role of olfactory subsystems in 681 monitoring inhalation and exhalation. Front Neuroanat 8 (2014) 107. 682 [19] P.L. Greer, D.M. Bear, J.M. Lassance, M.L. Bloom, T. Tsukahara, S.L. Pashkovski, F.K. 683 Masuda, A.C. Nowlan, R. Kirchner, H.E. Hoekstra, and S.R. Datta, A Family of non- 684 GPCR Chemosensors Defines an Alternative Logic for Mammalian Olfaction. Cell 165 685 (2016) 1734-1748. 686 [20] S. Pfister, M.G. Dietrich, C. Sidler, J.M. Fritschy, I. Knuesel, and R. Elsaesser, 687 Characterization and turnover of CD73/IP(3)R3-positive microvillar cells in the adult 688 mouse olfactory epithelium. Chem Senses 37 (2012) 859-68. 689 [21] M. Ma, X. Grosmaitre, C.L. Iwema, H. Baker, C.A. Greer, and G.M. Shepherd, Olfactory 690 signal transduction in the mouse septal organ. J Neurosci 23 (2003) 317-24. 691 [22] F. Moine, J. Brechbuhl, M. Nenniger Tosato, M. Beaumann, and M.C. Broillet, Alarm 692 pheromone and kairomone detection via bitter taste receptors in the mouse Grueneberg 693 ganglion. BMC Biol 16 (2018) 12. 694 [23] T. Matsuo, D.A. Rossier, C. Kan, and I. Rodriguez, The wiring of Grueneberg ganglion 695 axons is dependent on neuropilin 1. Development 139 (2012) 2783-91. 696 [24] K. Mamasuew, N. Hofmann, H. Breer, and J. Fleischer, Grueneberg ganglion neurons are 697 activated by a defined set of odorants. Chem Senses 36 (2011) 271-82. 698 [25] A. Schmid, M. Pyrski, M. Biel, T. Leinders-Zufall, and F. Zufall, Grueneberg ganglion 699 neurons are finely tuned cold sensors. J Neurosci 30 (2010) 7563-8. 700 [26] H. Gruneberg, A ganglion probably belonging to the N. terminalis system in the nasal 701 mucosa of the mouse. Z Anat Entwicklungsgesch 140 (1973) 39-52. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

702 [27] Z.W. Jin, K.H. Cho, S. Shibata, M. Yamamoto, G. Murakami, and J.F. Rodriguez-Vazquez, 703 Nervus terminalis and nerves to the vomeronasal organ: a study using human fetal 704 specimens. Anat Cell Biol 52 (2019) 278-285. 705 [28] E.Z.M. Taroc, A. Prasad, J.M. Lin, and P.E. Forni, The terminal nerve plays a prominent 706 role in GnRH-1 neuronal migration independent from proper olfactory and vomeronasal 707 connections to the olfactory bulbs. Biol Open 6 (2017) 1552-1568. 708 [29] C.R. Wirsig-Wiechmann, Introduction to the anatomy and function of the nervus terminalis. 709 Microsc Res Tech 65 (2004) 1. 710 [30] M. Schwanzel-Fukuda, M.S. Garcia, J.I. Morrell, and D.W. Pfaff, Distribution of luteinizing 711 hormone-releasing hormone in the nervus terminalis and brain of the mouse detected by 712 immunocytochemistry. The Journal of comparative neurology 255 (1987) 231-44. 713 [31] H.A. Oelschlager, E.H. Buhl, and J.F. Dann, Development of the nervus terminalis in 714 mammals including toothed whales and humans. Ann N Y Acad Sci 519 (1987) 447-64. 715 [32] L. Jennes, The nervus terminalis in the mouse: light and electron microscopic 716 immunocytochemical studies. Ann N Y Acad Sci 519 (1987) 165-73. 717 [33] J.W. Brown, The nervus terminalis in insectivorous bat embryos and notes on its presence 718 during human ontogeny. Ann N Y Acad Sci 519 (1987) 184-200. 719 [34] O. Larsell, The nervus terminalis. Ann Otol Rhinol Laryngol 59 (1950) 414-38. 720 [35] A. Cuschieri, and L.H. Bannister, The development of the olfactory mucosa in the mouse: 721 light microscopy. J Anat 119 (1975) 277-86. 722 [36] E.Z.M. Taroc, A.S. Naik, J.M. Lin, N.B. Peterson, D.L. Keefe, Jr., E. Genis, G. Fuchs, R. 723 Balasubramanian, and P.E. Forni, Gli3 Regulates Vomeronasal Neurogenesis, Olfactory 724 Ensheathing Cell Formation, and GnRH-1 Neuronal Migration. J Neurosci 40 (2020) 725 311-326. 726 [37] E. Cau, G. Gradwohl, S. Casarosa, R. Kageyama, and F. Guillemot, Hes genes regulate 727 sequential stages of neurogenesis in the olfactory epithelium. Development 127 (2000) 728 2323-32. 729 [38] A. Cuschieri, and L.H. Bannister, The development of the olfactory mucosa in the mouse: 730 electron microscopy. J Anat 119 (1975) 471-98. 731 [39] P.E. Forni, and S. Wray, GnRH, anosmia and hypogonadotropic hypogonadism--where are 732 we? Front Neuroendocrinol 36 (2015) 165-77. 733 [40] B.M. Cattanach, C.A. Iddon, H.M. Charlton, S.A. Chiappa, and G. Fink, Gonadotrophin- 734 releasing hormone deficiency in a mutant mouse with hypogonadism. Nature 269 (1977) 735 338-40. 736 [41] R. Balasubramanian, A. Dwyer, S.B. Seminara, N. Pitteloud, U.B. Kaiser, and W.F. 737 Crowley, Jr., Human GnRH deficiency: a unique disease model to unravel the ontogeny 738 of GnRH neurons. Neuroendocrinology 92 (2010) 81-99. 739 [42] X. Zhou, W.W. Li, Q.Y. Wu, M.M. Yu, and X.Y. Xia, [Kallmann syndrome with deafness 740 caused by SOX10 mutation: Advances in research]. Zhonghua Nan Ke Xue 23 (2017) 741 838-841. 742 [43] F. Wang, S. Zhao, Y. Xie, W. Yang, and Z. Mo, De novo SOX10 Nonsense Mutation in a 743 Patient with Kallmann Syndrome, Deafness, Iris Hypopigmentation, and 744 Hyperthyroidism. Ann Clin Lab Sci 48 (2018) 248-252. 745 [44] V. Pingault, V. Bodereau, V. Baral, S. Marcos, Y. Watanabe, A. Chaoui, C. Fouveaut, C. 746 Leroy, O. Verier-Mine, C. Francannet, D. Dupin-Deguine, F. Archambeaud, F.J. Kurtz, J. 747 Young, J. Bertherat, S. Marlin, M. Goossens, J.P. Hardelin, C. Dode, and N. Bondurand, bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

748 Loss-of-function mutations in SOX10 cause Kallmann syndrome with deafness. Am J 749 Hum Genet 92 (2013) 707-24. 750 [45] P. Barraud, J.A. St John, C.C. Stolt, M. Wegner, and C.V. Baker, Olfactory ensheathing 751 are required for embryonic olfactory axon targeting and the migration of gonadotropin- 752 releasing hormone neurons. Biol Open 2 (2013) 750-9. 753 [46] P.E. Forni, C. Taylor-Burds, V.S. Melvin, T. Williams, and S. Wray, Neural crest and 754 ectodermal cells intermix in the nasal placode to give rise to GnRH-1 neurons, sensory 755 neurons, and olfactory ensheathing cells. J Neurosci 31 (2011) 6915-27. 756 [47] J. Suzuki, and N. Osumi, Neural crest and placode contributions to olfactory development. 757 Curr Top Dev Biol 111 (2015) 351-74. 758 [48] H. Katoh, S. Shibata, K. Fukuda, M. Sato, E. Satoh, N. Nagoshi, T. Minematsu, Y. 759 Matsuzaki, C. Akazawa, Y. Toyama, M. Nakamura, and H. Okano, The dual origin of the 760 peripheral olfactory system: placode and neural crest. Mol Brain 4 (2011) 34. 761 [49] Y. Shan, H. Saadi, and S. Wray, Heterogeneous Origin of Gonadotropin Releasing 762 Hormone-1 Neurons in Mouse Embryos Detected by Islet-1/2 Expression. Front Cell Dev 763 Biol 8 (2020) 35. 764 [50] R. Aguillon, J. Batut, A. Subramanian, R. Madelaine, P. Dufourcq, T.F. Schilling, and P. 765 Blader, Cell-type heterogeneity in the early zebrafish olfactory epithelium is generated 766 from progenitors within preplacodal ectoderm. Elife 7 (2018). 767 [51] T.K. Palaniappan, L. Slekiene, L. Gunhaga, and C. Patthey, Extensive apoptosis during the 768 formation of the terminal nerve ganglion by olfactory placode-derived cells with distinct 769 molecular markers. Differentiation 110 (2019) 8-16. 770 [52] C. Lund, V. Yellapragada, S. Vuoristo, D. Balboa, S. Trova, C. Allet, N. Eskici, K. Pulli, P. 771 Giacobini, T. Tuuri, and T. Raivio, Characterization of the human GnRH neuron 772 developmental transcriptome using a GNRH1-TdTomato reporter line in human 773 pluripotent stem cells. Dis Model Mech 13 (2020). 774 [53] Y. Sun, I.M. Dykes, X. Liang, S.R. Eng, S.M. Evans, and E.E. Turner, A central role for 775 Islet1 in sensory neuron development linking sensory and spinal gene regulatory 776 programs. Nat Neurosci 11 (2008) 1283-93. 777 [54] K.L. Laugwitz, A. Moretti, J. Lam, P. Gruber, Y. Chen, S. Woodard, L.Z. Lin, C.L. Cai, 778 M.M. Lu, M. Reth, O. Platoshyn, J.X. Yuan, S. Evans, and K.R. Chien, Postnatal + 779 cardioblasts enter fully differentiated cardiomyocyte lineages. Nature 433 (2005) 647-53. 780 [55] L. Yang, C.L. Cai, L. Lin, Y. Qyang, C. Chung, R.M. Monteiro, C.L. Mummery, G.I. 781 Fishman, A. Cogen, and S. Evans, Isl1Cre reveals a common Bmp pathway in heart and 782 limb development. Development 133 (2006) 1575-85. 783 [56] L. Madisen, T.A. Zwingman, S.M. Sunkin, S.W. Oh, H.A. Zariwala, H. Gu, L.L. Ng, R.D. 784 Palmiter, M.J. Hawrylycz, A.R. Jones, E.S. Lein, and H. Zeng, A robust and high- 785 throughput Cre reporting and characterization system for the whole mouse brain. Nat 786 Neurosci 13 (2010) 133-40. 787 [57] H. Yoon, L.W. Enquist, and C. Dulac, Olfactory inputs to hypothalamic neurons controlling 788 reproduction and fertility. Cell 123 (2005) 669-82. 789 [58] J.M. Lin, E.Z.M. Taroc, J.A. Frias, A. Prasad, A.N. Catizone, M.A. Sammons, and P.E. 790 Forni, The transcription factor Tfap2e/AP-2epsilon plays a pivotal role in maintaining the 791 identity of basal vomeronasal sensory neurons. Dev Biol (2018). bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

792 [59] H. Tanaka, Y. Nojima, W. Shoji, M. Sato, R. Nakayama, T. Ohshima, and H. Okamoto, 793 Islet1 selectively promotes peripheral axon outgrowth in Rohon-Beard primary sensory 794 neurons. Dev Dyn 240 (2011) 9-22. 795 [60] S.A. Hutchinson, and J.S. Eisen, Islet1 and Islet2 have equivalent abilities to promote 796 motoneuron formation and to specify motoneuron subtype identity. Development 133 797 (2006) 2137-47. 798 [61] M. Huang, A. Kantardzhieva, D. Scheffer, M.C. Liberman, and Z.Y. Chen, Hair cell 799 overexpression of Islet1 reduces age-related and noise-induced hearing loss. J Neurosci 800 33 (2013) 15086-94. 801 [62] M. Sjodal, and L. Gunhaga, Expression patterns of Shh, Ptc2, Raldh3, Pitx2, Isl1, Lim3 and 802 Pax6 in the developing chick hypophyseal placode and Rathke's pouch. Gene Expr 803 Patterns 8 (2008) 481-5. 804 [63] K. Radde-Gallwitz, L. Pan, L. Gan, X. Lin, N. Segil, and P. Chen, Expression of Islet1 805 marks the sensory and neuronal lineages in the mammalian inner ear. J Comp Neurol 477 806 (2004) 412-21. 807 [64] F. Castinetti, M.L. Brinkmeier, A.H. Mortensen, K.R. Vella, P. Gergics, T. Brue, A.N. 808 Hollenberg, L. Gan, and S.A. Camper, ISL1 Is Necessary for Maximal Thyrotrope 809 Response to Hypothyroidism. Mol Endocrinol 29 (2015) 1510-21. 810 [65] D.H. Brann, T. Tsukahara, C. Weinreb, M. Lipovsek, K. Van den Berge, B. Gong, R. 811 Chance, I.C. Macaulay, H.-J. Chou, R.B. Fletcher, D. Das, K. Street, H.R. de Bezieux, 812 Y.-G. Choi, D. Risso, S. Dudoit, E. Purdom, J. Mill, R.A. Hachem, H. Matsunami, D.W. 813 Logan, B.J. Goldstein, M.S. Grubb, J. Ngai, and S.R. Datta, Non-neuronal expression of 814 SARS-CoV-2 entry genes in the olfaory system suggests mechanisms underlying 815 COVID-19-associated anosmia. Science Advances (2020) eabc5801. 816 [66] S. Thor, J. Ericson, T. Brannstrom, and T. Edlund, The homeodomain LIM protein Isl-1 is 817 expressed in subsets of neurons and endocrine cells in the adult rat. Neuron 7 (1991) 881- 818 9. 819 [67] J.P. Thaler, S.J. Koo, A. Kania, K. Lettieri, S. Andrews, C. Cox, T.M. Jessell, and S.L. 820 Pfaff, A postmitotic role for Isl-class LIM homeodomain proteins in the assignment of 821 visceral spinal motor neuron identity. Neuron 41 (2004) 337-50. 822 [68] P.E. Forni, M. Fornaro, S. Guenette, and S. Wray, A role for FE65 in controlling GnRH-1 823 neurogenesis. J Neurosci 31 (2011) 480-91. 824 [69] J. Begbie, M. Ballivet, and A. Graham, Early steps in the production of sensory neurons by 825 the neurogenic placodes. Mol Cell Neurosci 21 (2002) 502-11. 826 [70] S. Zhuang, Q. Zhang, T. Zhuang, S.M. Evans, X. Liang, and Y. Sun, Expression of Isl1 827 during mouse development. Gene Expr Patterns 13 (2013) 407-12. 828 [71] W. Yin, and A.C. Gore, Neuroendocrine control of reproductive aging: roles of GnRH 829 neurons. Reproduction 131 (2006) 403-14. 830 [72] G. Zhang, J. Li, S. Purkayastha, Y. Tang, H. Zhang, Y. Yin, B. Li, G. Liu, and D. Cai, 831 Hypothalamic programming of systemic ageing involving IKK-beta, NF-kappaB and 832 GnRH. Nature 497 (2013) 211-6. 833 [73] S.S. Burmeister, E.D. Jarvis, and R.D. Fernald, Rapid behavioral and genomic responses to 834 social opportunity. PLoS Biol 3 (2005) e363. 835 [74] K.P. Maruska, and R.D. Fernald, Social regulation of in the hypothalamic- 836 pituitary-gonadal axis. Physiology (Bethesda) 26 (2011) 412-23. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.276360; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

837 [75] B.S. Kallmann FJ, The genetic aspects of primary eunuchoidism. J Ment Defic 48 (1944) 838 203-236. 839 [76] R. Legouis, J.P. Hardelin, J. Levilliers, J.M. Claverie, S. Compain, V. Wunderle, P. 840 Millasseau, D. Le Paslier, D. Cohen, D. Caterina, and et al., The candidate gene for the 841 X-linked Kallmann syndrome encodes a protein related to adhesion molecules. Cell 67 842 (1991) 423-35. 843 [77] C. Dode, J. Levilliers, J.M. Dupont, A. De Paepe, N. Le Du, N. Soussi-Yanicostas, R.S. 844 Coimbra, S. Delmaghani, S. Compain-Nouaille, F. Baverel, C. Pecheux, D. Le Tessier, C. 845 Cruaud, M. Delpech, F. Speleman, S. Vermeulen, A. Amalfitano, Y. Bachelot, P. 846 Bouchard, S. Cabrol, J.C. Carel, H. Delemarre-van de Waal, B. Goulet-Salmon, M.L. 847 Kottler, O. Richard, F. Sanchez-Franco, R. Saura, J. Young, C. Petit, and J.P. Hardelin, 848 Loss-of-function mutations in FGFR1 cause autosomal dominant Kallmann syndrome. 849 Nature genetics 33 (2003) 463-5. 850 [78] S. Guioli, B. Incerti, E. Zanaria, B. Bardoni, B. Franco, K. Taylor, A. Ballabio, and G. 851 Camerino, Kallmann syndrome due to a translocation resulting in an X/Y fusion gene. 852 Nature genetics 1 (1992) 337-40. 853 [79] B. Incerti, S. Guioli, A. Pragliola, E. Zanaria, G. Borsani, R. Tonlorenzi, B. Bardoni, B. 854 Franco, D. Wheeler, A. Ballabio, and et al., Kallmann syndrome gene on the X and Y 855 : implications for evolutionary divergence of human sex chromosomes. 856 Nature genetics 2 (1992) 311-4. 857 [80] J.P. Hardelin, J. Levilliers, I. del Castillo, M. Cohen-Salmon, R. Legouis, S. Blanchard, S. 858 Compain, P. Bouloux, J. Kirk, C. Moraine, and et al., X -linked Kallmann 859 syndrome: stop mutations validate the candidate gene. Proc Natl Acad Sci U S A 89 860 (1992) 8190-4. 861 [81] N. Pitteloud, C. Zhang, D. Pignatelli, J.D. Li, T. Raivio, L.W. Cole, L. Plummer, E.E. 862 Jacobson-Dickman, P.L. Mellon, Q.Y. Zhou, and W.F. Crowley, Jr., Loss-of-function 863 mutation in the prokineticin 2 gene causes Kallmann syndrome and normosmic idiopathic 864 hypogonadotropic hypogonadism. Proceedings of the National Academy of Sciences of 865 the United States of America 104 (2007) 17447-52. 866 [82] P. Barraud, A.A. Seferiadis, L.D. Tyson, M.F. Zwart, H.L. Szabo-Rogers, C. Ruhrberg, K.J. 867 Liu, and C.V. Baker, Neural crest origin of olfactory ensheathing glia. Proc Natl Acad 868 Sci U S A 107 (2010) 21040-5. 869 [83] E.Z.M. Taroc, J.M. Lin, A.J. Tulloch, A. Jaworski, and P.E. Forni, GnRH-1 Neural 870 Migration From the Nose to the Brain Is Independent From Slit2, Robo3 and NELL2 871 Signaling. Front Cell Neurosci 13 (2019) 70. 872 873