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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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, neural crest. 14 15 Abstract (299, at 245 after editing) 16 During embryonic development, 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, Sox2, 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 genes [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 transcription factor 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.