Paolo Emanuele Forni

The vomeronasal organ (VNO) is a specialized chemosensory structure that detects chemical cues involved in predator avoidance, social interactions, and reproductive behaviors. In mice, the VNO contains distinct vomeronasal sensory neurons (VSNs) that express vomeronasal receptors (VRs) and formyl peptide receptors. Apical VSNs express Meis2, V1Rs, and Gαi2, whereas basal VSNs express Tfap2ɛ/AP-2ɛ, V2Rs, and Gαo. V2Rs are classified into families A–E, and basal neurons co-express a family-C receptor with a VR from another family. Single-cell RNA sequencing identified ∼980 genes differentially expressed between V1R and V2R neurons, many of which are linked to endoplasmic reticulum (ER) functions. Notably, Canopy1 (Cnpy1) is highly enriched in V2R neurons. The VNO of Cnpy1 knockout mice develops normally but undergoes progressive loss of V2R neurons, associated with increased ER stress gene expression, upregulation of family-C V2R mRNAs, and reduced V2R protein levels. V2R VSNs in Cnpy1 KOs fail to respond to pheromones, show altered guidance and adhesion gene expression, and display disrupted connectivity with the accessory olfactory bulb. These findings emphasize the role of cell-specific ER protein repertoires in maintaining neuronal function.

The olfactory placode (OP) generates a broad array of chemosensory neurons in the nasal region, including olfactory sensory neurons, vomeronasal sensory neurons, neurons of the septal organ, and Grueneberg ganglion. During invagination, the OP also generates migratory neuronal populations, including gonadotropin‐releasing hormone‐1 (GnRH‐1) neurons, somatostatin‐expressing neurons, and Prokineticin Receptor 2 (Prokr2) pioneer/terminal nerve (TN) neurons. Despite extensive research, the genetic lineage and molecular identity of many OP‐derived neurons remain unclear. GnRH‐1 neurons are vital for reproductive functions and chemosensory processing but are genetically distinct from olfactory and vomeronasal sensory neurons. The regulator Goofy/Gfy is expressed in placode‐derived chemosensory neurons. Our study aimed to determine whether its expression is restricted to these neurons or shared among OP derivatives. The 123Cre mouse expresses Cre under the Gfy1 promoter. Cre tracing analyses across embryonic stages showed widespread 123Cre recombination in chemosensory neurons but not in migrating GnRH‐1. However, at late development and postnatal stages, some Gfy‐traced cells appear in the basal forebrain, including a subset of GnRH‐positive neurons. These findings demonstrate heterogeneity among migratory nasal neurons and suggest that, besides GnRH‐1, other nasal‐origin neurons also populate the forebrain. Postnatal studies of vomeronasal neurons revealed distinct developmental trajectories for the two main VSN subtypes.

The olfactory placode (OP) gives rise to a wide array of chemosensory neurons in the nasal region, including olfactory sensory neurons, vomeronasal sensory neurons, and those of the septal organ and Grueneberg ganglion. During placodal invagination, the OP also produces migratory neurons, including gonadotropin-releasing hormone-1 (GnRH-1) neurons, somatostatin-expressing neurons, Prokineticin Receptor 2 (Prokr2) pioneer/terminal nerve (TN) neurons, which are thought to initiate olfactory bulb development. Despite decades of research, the genetic lineage and molecular identity of OP-derived neuronal types remain under investigation. GnRH-1 neurons play essential roles in reproduction and chemodetection but appear genetically distinct from olfactory and vomeronasal chemosensory neurons. The Golgi-associated olfactory signaling regulator Goofy/Gfy is broadly expressed across the placode-derived nasal chemosensory neurons. To determine whether its expression is specific to nasal chemosensory neurons or it is a shared genetic feature across OP derivatives we characterized Gfy expression and lineage at embryonic and postnatal stages. Our results confirm broad Gfy expression in developing chemosensory neurons and subsets of olfactory pioneer/TN neurons. However, we found that while Gfy was not expressed in GnRH-1 neurons while migrating, analysis at late development and postnatal stages, revealed the existence of Gfy-traced neuronal populations in the basal forebrain, some of which also express GnRH. These findings uncover previously unrecognized genetic heterogeneity among migratory nasal neurons and reinforce previous studies suggesting the existence of additional neurons with nasal origin in the brain. In addition to this, analysis of Gfy expression along the developmental trajectory of vomeronasal sensory neurons at postnatal stages revealed intriguing differences in the developmental dynamics across the two main types of vomeronasal sensory neurons.

The vomeronasal organ (VNO) is a specialized chemosensory structure in the nasal cavity that detects pheromones and mediates social and reproductive behaviors. The VNO of rodents is populated by different types of vomeronasal sensory neurons (VSNs). Apical VSNs, located near the lumen, express the transcription factor (TF) Meis2, V1R family receptors, and the G protein subunit Gao; the VSNs distributed closer to the basal lamina express the TF Tfap2e/AP-2ε, V2R receptors, and the G protein subunit Gai2. In addition, sparse cells in the VNO express the Formyl Peptide Receptors (FPRs). Single-cell mRNA sequencing (scRNA Seq) identified over 980 differentially expressed genes between these cell types, with many linked to the endoplasmic reticulum (ER). Among these ER proteins, Canopy1 (Cnpy1), was found to be among the most enriched genes in V2R+ VSNs. Previously studied only in zebrafish, Cnpy1 was found to affect Fgfr1 signaling and is thus also known as “FGF signaling regulator-1”. In a previous study, we discovered that AP-2e upregulates Cnpy1 expression. Although Cnpy1 knockout mice are viable and have normal VNO development at birth, they experience a progressive degeneration and loss of V2R-expressing VSNs. Prior to symptoms, the basal VSNs of KO mice display reduced V2R protein immunoreactivity in the soma and a complete absence of the protein at the lumen of the VNO, rendering the neurons non-functional. Cnpy1 KOs exhibit altered guidance cues and adhesion molecule expression, along with disrupted connectivity to the accessory olfactory bulb (AOB). Our study shows that distinct neuronal types depend on unique ER protein repertoires to maintain proper proteostasis. The loss of Cnpy1 highlights the importance of cell–type–specific ER factors in the differentiation and function of specific neurons, revealing mechanisms that drive neuronal diversity and vulnerability to ER gene disruption.

Olfactory Ensheathing Cells (OECs) are glial cells originating from the neural crest and are critical for bundling olfactory axons to the brain. Their development is crucial for the migration of Gonadotropin‐Releasing Hormone‐1 (GnRH‐1) neurons, which are essential for puberty and fertility. OECs have garnered interest as potential therapeutic targets for central nervous system lesions, although their development is not fully understood. Our single‐cell RNA sequencing of mouse embryonic nasal tissues suggests that OECs and Schwann cells share a common origin from Schwann cell precursors yet exhibit significant genetic differences. The transcription factors Yap and Taz have previously been shown to play a crucial role in Schwann cell development. We used Sox10‐Cre mice to conditionally ablate Yap and Taz in the migrating neural crest and its derivatives. Our analyses showed reduced Sox10+ glial cells along nerves in the nasal region, altered gene expression in Schwann cells (SCs), melanocytes, and OECs, and a significant reduction in olfactory sensory neurons and vascularization in the vomeronasal organ. However, despite these changes, GnRH‐1 neuronal migration remained unaffected. Our findings highlight the importance of the Hippo pathway in OEC development and how changes in cranial neural crest derivatives indirectly impact the development of olfactory epithelia.

Neuronal migration during embryonic development is a fundamental process. In the developing nose of rodents, neurons that form during early neurogenic waves in the olfactory placode leave this structure to migrate toward or into the developing brain as part of the migratory mass. This mass includes gonadotropin‐releasing hormone‐1 (GnRH‐1) neurons, pioneer/terminal nerve (TN) neurons, as well as neural crest‐derived olfactory glial cells called olfactory ensheathing cells. There have been a limited number of molecular markers available to effectively trace and functionally manipulate the early migratory neurons that originate in the olfactory region. Contactin‐2 (Cntn2), also known as transiently expressed axonal surface glycoprotein‐1 (TAG‐1), has been used to label various developing neuronal populations, including the commissural neurons of the spinal cord, motor neurons, and TN neurons. Previous single‐cell RNA sequencing analyses of the developing olfactory system have identified Cntn2 expression in the TN, suggesting that Cntn2 is a suitable molecular marker for studying nasal migratory neurons. To trace Cntn2 expression in the developing olfactory system, we generated an inducible Cntn2CreERT2 mouse line. In this study, we outline how this mouse line can serve as an effective tool for time‐controlled chimeric manipulation of specific neuronal populations of interest.

Neuronal migration during embryonic development is a fundamental process. In the developing nose of rodents, neurons that form during early neurogenic waves in the olfactory placode leave this structure to migrate toward or into the developing brain as part of the migratory mass. This mass includes Gonadotropin-releasing hormone-1 (GnRH-1) neurons, pioneer/terminal nerve (TN) neurons, as well as neural crest-derived olfactory glial cells called olfactory ensheathing cells. There have been a limited number of molecular markers available to effectively trace and functionally manipulate the early migratory neurons that originate in the olfactory region. Contactin-2 (Cntn2), also known as transiently expressed axonal surface glycoprotein-1 (TAG-1), has been used to label various developing neuronal populations, including the commissural neurons of the spinal cord, motor neurons, and TN neurons. Previous single-cell RNA sequencing analyses of the developing olfactory system have identified Cntn2 expression in the TN, suggesting that Cntn2 is a suitable molecular marker for studying nasal migratory neurons. To trace Cntn2 expression in the developing olfactory system, we generated an inducible Cntn2CreERT2 mouse line. In this study, we outline how this mouse line can serve as an effective tool for time-controlled chimeric manipulation of specific neuronal populations of interest.

Olfactory Ensheathing Cells (OECs) are glial cells originating from the neural crest, critical for bundling olfactory axons to the brain. Their development is crucial for the migration of Gonadotropin-Releasing Hormone-1 (GnRH-1) neurons, which are essential for puberty and fertility. OECs have garnered interest as potential therapeutic targets for central nervous system lesions, although their development is not fully understood. Our single-cell RNA sequencing of mouse embryonic nasal tissues suggests that OECs and Schwann cells share a common origin from Schwann cell precursors yet exhibit significant genetic differences. The transcription factors Yap and Taz have previously been shown to play a crucial role in Schwann cell development. We used Sox10 -Cre mice to conditionally ablate Yap and Taz in migrating the neural crest and its derivatives. Our analyses showed reduced Sox10+ glial cells along nerves in the nasal region, altered gene expression of SCs, melanocytes, and OECs, and a significant reduction in olfactory sensory neurons and vascularization in the vomeronasal organ. However, despite these changes, GnRH-1 neuronal migration remained unaffected. Our findings highlight the importance of the Hippo pathway in OEC development and how changes in cranial neural crest derivatives indirectly impact the development of olfactory epithelia.