Gli3 Regulates Vomeronasal Neurogenesis, Olfactory Ensheathing Cell Formation, and GnRH-1 Neuronal Migration

Gli3 is expressed in Hes-1⁺, but not in Ascl-1⁺, progenitors. A, B, E15.5 immunostaining against Hes-1 reveals Hes-1 expression in the cells located in the apical domains of the developing VNO (Black arrow). C, D, Immunostaining against Ascl-1 reveals expression of the neurogenic factor in cells in the basal territories of the developing VNO (White arrow), only background levels were found in the basal regions (Black arrow). E, F, Gli3 immunoreactivity was found in cells in the apical territories of the developing VNO (Black arrow). G, H, Double immunofluorescence against Gli3 and Hes-1 reveals strong Gli3 expression in the Hes-1⁺ cells (Black arrow) and lack of immunodetectable Gli3 in the most basal levels (white arrow). I, Proposed model with Gli3 expression in proliferative Hes-1⁺ cells and in daughter cells that exit the proliferative program and enter the Ascl-1⁺ controlled proneurogenic program in the basal regions of the developing VNO. J, X-Gal reaction on E15.5 sections of Gli1-LacZ Knock-in mouse shows active Shh signaling (Gli1 expression, blue, white arrows) in the palate (plt), nasal mesenchyme (NM), tongue (tng) but not in the olfactory epithelium (OE) nor in VNO (white arrowheads). K, magnification of the VNO showing lack of Gli1 expression. L, M, Serial sections; (L) Immunostaining against Gli3 shows strong Gli3 expression in the ventricular zone of the developing brain cortex (arrow), (M) X-Gal reaction showing lack of Gli1 expression in the ventricular zone. Scale bars: A, C, E, G, K, L, M, 100 μm; B, D, F, H, J, 50 μm.

Gli3 loss-of-function impairs the formation of Ascl-1⁺ neuronal progenitor cells and VSN neurogenesis but not VSN terminal differentiation. A, B, Immunostaining against the transcription factors Meis2 and AP-2ε highlights maturing apical and basal vomeronasal sensory neurons in controls (A) and Gli3^Xt/Xt mutants (B). C, Quantifications of the average number of AP-2ε differentiated VSNs and Meis2 differentiated apical VSNs reveal a significant reduction in the number of differentiated VSNs in Gli3^Xt/Xt mutants. D, E, PHH3 immunostaining and quantification (F) of VNO from WT and Gli3^Xt/Xt reveals a reduction in the total number of dividing cells with a significant reduction of basal progenitors (white arrowheads), but not apical progenitors (red arrows). G, H, Immunostaining against Ascl-1 in controls and Gli3^Xt/Xt mutants reveals (I) a significant reduction in the number of Ascl-1⁺ cells in the basal domains of the developing VNO. Scale bars, 100 μm.

Gli3^Xt/Xt mutants display severely impaired GnRH-1 neuronal migration. Representative images of immunostaining against GnRH-1 on WT (A–G) and Gli3^Xt/Xt (B–H) at E15.5. A–G, in WT GnRH-1ns migrate as a continuum from the vomeronasal organ (VNO) to the brain. GnRH-1ns from the nasal area (NA), cross the forebrain junction (FBJ), invade the brain ventral to the OB, migrate through the subpallium (SP) to eventually reach the hypothalamic area (Hyp). B, C, GnRH-1ns migrating in the nasal area and out of the VNO. D, Detail showing GnRH-1ns invading the subpallium (SP) ventral to the OB in the region corresponding to the putative accumbens (Ac), some GnRH-1ns migrate to the cortex (Ctx) and around the olfactory bulbs (OBs). E-H) In Gli3^Xt/Xt, a large portion of GnRH-1ns neurons form clusters of cells proximal to the VNO (E) while other migrate around the brain, occasional GnRH-1ns were found accessing the brain dorsal portion of the cortex (Ctx) (H). F, GnRH-1 immunostaining (brown) show less organized GnRH-1ns forming clumps while migrating toward the brain (cf. D). H, Detail showing GnRH-1ns were unable to invade the brain migrating along the meninges. I, Quantification of the distribution of GnRH-1ns in WT, Gli3^Xt/WT and Gli3^Xt/Xt. In Gli3^Xt/Xt the majority of the GnRH-1ns remain in the nasal area. Both Gli3^Xt/WT and Gli3^Xt/Xt have a significantly smaller number of GnRH-1ns in the brain compared with controls. Values are ±SE. Dots indicate numbers of embryos/genotype, unpaired t test, significant values p < 0.05.

Misrouted vomeronasal and terminal nerve axons in Gli3^Xt/Xt are associated with lack of ensheathing cells in the nasal mucosa. A, C, Immunostainings against peripherin and GnRH-1. B, D, peripherin and GnRH-1 immunofluorescence, in Gli3^Xt/Xt mutants the OSNs and VSNs form a fibrocellular cellular mass (FCM). The GnRH-1ns (D) and the TN do not remain in the FCM but migrate around the FB. C–I, WT E15.5, immunostaining against peripherin, Robo2, Nrp2; the TN fibers are negative for Robo2 expression and negative for Nrp2 (see merge in I). D–J, Gli3^Xt/Xt E15.5 immunostaining against peripherin, Robo2, and Nrp2 and merge. K, L, Schematic of the phenotypes and fiber immunoreactivity in controls and Gli3^Xt/Xt mutants. K1, K2, Immunofluorescence showing Neuropilin-1 (Nrp1) immunoreactivity in TN fibers invading the brain in WT embryos. L1, L2, peripherin and Neruopilin1⁺ fibers of the putative TN project around the brain. M1–M4, In control animals the GnRH-1ns migrate along peripherin⁺ bundles (M1), also immunoreactive for Robo2 (M2), Nrp2 (M3), Merge in M4. In Gli3^Xt/Xt, GnRH-1ns form large clumps of cells suggesting disorganized peripherin⁺ fibers. Some tangled fibers of the putative terminal nerve (N1) are negative for Nrp2 (N2) and Robo2 (N3). Merge in N4. O1–O4, In Gli3^Xt/Xt, tangles of putative TN fibers are positive for peripherin (O1) and Nrp1 (O2) and are associated with clusters of GnRH-1ns (O3). Merge in O4. P–S, GLI3^Xt/Xt lack of OECs associated with the vomeronasal nerve. peripherin IF and Sox10 IHC are combined. P, In controls, Sox10-immunoreactive OECs (magenta arrowheads) were found around the VNO, proximal to the basal lamina of the OE, along the peripherin⁺ vomeronasal and olfactory bundles and as part of the migratory mass (MM) in front of the brain. Sox10⁺ Schwann cells were found associated along nasal trigeminal projections (TG). Q, In Gli3^Xt/Xt mutants, Sox10-immunoreactive OECs were neither found proximal to the basal portions of the VNO, olfactory epithelium nor along the vomeronasal and olfactory projections (white arrowheads). Notably Sox10⁺ cells were found in the migratory mass (MM). As in controls Sox10⁺ Schwann cells were found associated along nasal trigeminal projections (TG). R, S, Magnifications of the VNO. R, In control animals Sox10⁺ OECs were found proximal to the basal lamina of the VNO, along the vomeronasal bundles. S, In Gli3^Xt/Xt Sox10⁺ OECs were rarely found in proximity of the basal lamina of the VNO and along the vomeronasal/terminal nerve bundles (white arrowheads). T, Quantification of Sox10⁺ OECs around the VNO, ****p < 0.0001. Scale bars, 100 μm.

Gli3^Xt/WT show no difference in GnRH-1ns numbers in the brain after birth. A, B, Immunostaining against GnRH-1 on postnatal WT and Gli3^Xt/WT heterozygous animals shows comparable immunoreactivity in GnRH-1 cell bodies in the preoptic area (arrows). C, Quantification for GnRH-1-immunoreactive cell bodies in the preoptic area indicates no statistical differences between controls and Gli3^Xt/WT heterozygous mutants. D, Quantification of area occupied by GnRH-1-immunoreactive fibers in the median eminence of WT controls and Gli3^Xt/WT heterozygous mutants. E, F, Representative images showing GnRH-1 immunoreactivity of GnRH-1 axons in the median eminence (ME) of WT and Gli3^Xt/WT heterozygous mutants.

Gli3^Xt/Xt mutants display an expansion of expression of forebrain ventral markers, form ectopic olfactory bulbs and lack of Sema3A expression in the forebrain. A, AP-2ε/GAD1 double immunostaining. Immunostaining against AP-2ε highlights mitral cells in the OB (magnified in C). GAD1 immunostaining highlights GABAergic neurons which are mostly located in the subpallium. B, In Gli3^Xt/Xt, AP-2ε highlight extopic mitral cells in the posterior portion of the forebrain (boxed, see magnification in (D) showing the ectopic AP-2ε⁺ OB in the posterior portion of the brain. GAD1 expression shows expansion of ventral forebrain maker. E, F, Illustration of GnRH-1ns migration in WT and Gli3-null animals. Area of Sema3A expression represented in light blue present in WT accumbens absent in the Gli3 KOs. G, H, In situ hybridization against Sema3A shows detectable Sema3A expression in the nasal area, basal brain (black arrows) and hindbrain of control and Gli3 mutants. No Sema3A expression was detected in Gli3^Xt mutants in the forebrain and cortex (compare red arrows in I with dotted arrows in H). Ac, Accumbens; Ctx, cortex; Hyp, hypothalamus; OB, olfactory bulb; pOB, putative olfactory bulb; SP, subpallium.

Ascl-1 KO have a comparable reduction in the number of vomeronasal and GnRH-1 neurons. A–C, Immunostaining against peripherin highlights neuronal projections (white arrows) emerging from the VNO of WT animals projecting toward the brain. B, D, In controls, immunostaining against GnRH-1 reveals migratory GnRH-1ns distributed between the VNO and the brain. E, G, In Ascl-1-null mutants, only sparse peripherin⁺ projections emerge from the OE and VNO (white arrows). F–H, In Ascl-1 KOs, GnRH-1ns emerge from the VNO migrating in the nasal area (white arrows). I, J, AP-2ε and Meis2 immunostaining highlights the reduced number of differentiated VSNs in Ascl-1 KOs (white arrow). K–N, Reduced olfactory and vomeronasal neurons in Ascl-1 KOs does not prevent ensheathing cell formation in the nasal mucosa. IF anti peripherin and Sox10 IHC are combined. K, M, In control animals, many Sox10-immunoreactive OECs (magenta arrows) are found around the VNO, along the peripherin⁺ vomeronasal bundles, (M) proximal to the developing OE and along the olfactory bundles. L, In Ascl-1^−/− mutants, Sox10-immunoreactive OECs were found associated with the sparse vomeronasal projections (magenta arrows) (N) proximal to the developing OE and along the olfactory bundles (magenta arrows). O, Quantification of GnRH-1ns in controls and Ascl-1 KOs shows dramatic reduction in the number of GnRH-1ns in Ascl-1 KOs. Forebrain junction (FBJ). Holm-Sidak's multiple-comparisons test. P, Quantification of VSNs, OECs, show similar defects in vomeronasal neurogenesis between Ascl-1^−/− and Gli3^Xt/Xt compared with WT controls however, more severe loss of OECs was found in Gli3^Xt/Xt compared with Ascl-1^−/−. White scale bars are 100 μm. Q, R, Diagrams summarizing key phenotypes observed in (Q) Gli3^Xt/Xt and (R) Ascl-1^null lines.

A, Pedigree of Kallmann syndrome proband (shown with arrow) with reproductive and nonreproductive phenotypes. B, KS proband underwent neuroendocrine profiling (Q 10 min × 12 h) to chart GnRH-induced LH secretion showing low amplitude, low-frequency pulses (inverted triangles indicate LH pulses). Shaded region represents the normal reference range. NA, Not available; T, total testosterone; LH, luteinizing hormone; FSH, follicle stimulating hormone; SHBG, sex-hormone binding globulin' TSH, thyroid-stimulating hormone.