Ed Zandro M. Taroc

Embryonic ectoderm patterning in the cranial region establishes the neural plate, neural crest, sensory placodes, and non-neural ectoderm in coordination with the underlying mesoderm and endoderm. Although many studies have defined the signaling pathways and transcriptional regulators driving individual lineage specification, the temporal and spatial coordination among adjacent tissues remains poorly understood. To address this, we analyzed a single-cell RNA sequencing (scRNAseq) data set of a developmental series from the chick midbrain axial level, spanning stages from gastrulation to post-neurulation. Focusing on a defined region enabled high-resolution analysis of transcriptional dynamics and subpopulation transitions. Transcription factor downstream activity inference identified several ubiquitously expressed chromatin and histone modifiers, previously not associated with ectodermal patterning, that exhibit canonical downstream activity spatiotemporally restricted to specific developing domains. Moreover, we uncovered genes that remain canonical downstream activity throughout the progression of individual ectodermal domains, while others displayed negative activity scores, reflecting a repressed or alternatively a non-canonical downstream activity status that changed between cell types and developmental stage but was independent of transcription factor binding availability status on open chromatin. Finally, ligand-receptor interaction analyses across germ layers highlighted global signaling networks coordinating early embryogenesis, which were verified by in situ hybridization-based gene expression data. In sum, our study provides novel insights into the gene regulatory inputs that shape ectodermal cell fate decisions during neurulation and establishes a new framework for analyzing scRNAseq data to understand tissue patterning over developmental time.

Neurocristopathies account for half of all birth defects and several cancers highlighting the need to understand early neural crest (NC) development, for which suitable human models don’t exist. Here, we present a pluripotent-stem-cell-based 3D ectodermal organoid model that faithfully recapitulates early ectodermal patterning of future central nervous system, epidermis and cranial and trunk NC, as well as a diverse selection of NC derivatives —offering a comprehensive platform to study neurocristopathies from early induction of pluripotent-like stem cells at the neural plate border to differentiated cells. DiGeorge syndrome (DGS) is caused by a hemizygous microdeletion of ∼fifty genes, many of which play broad roles during embryogenesis. While DGS is traditionally considered to originate from all germ layers, its clinical manifestations—including craniofacial anomalies, cardiac outflow tract defects, thymic hypoplasia and thyroid dysfunction—are consistent with tissues requiring NC contributions. This raises the question why DGS mainly manifest in organs of NC origin? Using patient-derived iPSCs, we show that DGS organoids display reduced pluripotency gene expression and impaired maintenance of ectodermal stem cells, leading to defective NC specification, posteriorization of cranial NC, and failure to form cartilage. We identify a small subset of genes within the DGS deletion as potential drivers of these early NC defects. Consequently, impaired NC cells are further affected during cranial and vagal mesenchyme maturation, likely worsened by hemizygosity of additional DGS genes that individually, without the initial NC defect, are insufficient to cause the disease. We hypothesize that DGS is primarily, or possibly entirely, a neurocristopathy.

Sexual development and puberty are two key developmental processes that are crucial for the propagation and evolution of species. The hormone Gonadotropin releasing hormone-1 (GnRH-1) is a key hormone in regulating both processes by controlling the hormonal Hypothalamic-Pituitary-Gonadal axis in the release of sex hormones (Testosterone, Estrogen, Progesterone) at the correct developmental time points initiate puberty and progress sexual development. A subset of hypothalamic neurons known as the GnRH-1 neurons (GnRH-1ns) release GnRH-1 in a pulsatile fashion during embryonic and post-natal development to specifically regulate these processes. The GnRH-1ns are an interesting group of neurons because they are formed in the periphery, specifically the nose, and must migrate to the hypothalamus during mid-gestation. Disruptions in GnRH-1ns formation, migration, or signaling can lead to a human disorder known as hypogonadotropic hypogonadism (HH) that is characterized as an individual with low levels of GnRH-1 followed by decreased levels of pituitary gonadotropins and low sex hormones, which phenotypically leads to delayed or absent puberty. HH has two versions, normosmic idiopathic HH where only pubertal defects are seen or Kallmann Syndrome (KS) where pubertal defects co-segregate with olfaction defects. The close association of olfaction and puberty phenotypes as well as the developmental origin of the GnRH-1ns have led many to believe that GnRH-1ns migrate on the axons of the olfactory system. However, the relationship of GnRH-1ns and the olfactory system had not been thoroughly investigated. Furthermore, the migratory partner of the GnRH-1ns known as the Olfactory Ensheathing Cells (OECs) has not been thoroughly characterized during development.My doctoral research focused on understanding better the relationship of GnRH-1ns and the olfactory/vomeronasal/terminal nerve system and characterizing the developmental progression of the OECs. In the second and third chapters of my dissertation I discovered that the migration of the GnRH-1ns from the nose and the brain are independent of olfactory bulb formation and correct olfactory connections. I propose instead that the GnRH-1ns migrate on the axons of long forgotten nerve known as terminal nerve/nervus terminalis/cranial nerve 0. In the fourth and fifth chapters of my dissertation I identified two transcription factors, Ascl1 and Isl1, to be expressed by the neuronal progenitors of the GnRH-1ns, providing for the first-time markers to identify GnRH-1ns during early development. And lastly, in chapter 4 and chapter 6, I discovered the importance of a transcription factor, Gli3, in forming the OECs. Specifically, my data suggests that OECs are coming from a multipotent cell

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.

During embryonic development, the olfactory placode (OP) generates migratory neurons, including olfactory pioneer neurons, cells of the terminal nerve (TN), gonadotropin‐releasing hormone‐1 (GnRH‐1) neurons, and other uncharacterized neurons. Pioneer neurons from the OP induce olfactory bulb (OB) morphogenesis. In mice, GnRH‐1 neurons appear in the olfactory system around mid‐gestation and migrate via the TN axons to different brain regions. The GnRH‐1 neurons are crucial in controlling the hypothalamic‐pituitary‐gonadal axis. Kallmann syndrome is characterized by impaired olfactory system development, defective OBs, secretion of GnRH‐1, and infertility. The precise mechanistic link between the olfactory system and GnRH‐1 development remains unclear. Studies in humans and mice highlight the importance of the prokineticin‐2/prokineticin‐receptor‐2 (Prokr2) signaling pathway in OB morphogenesis and GnRH‐1 neuronal migration. Prokr2 loss‐of‐function mutations can cause Kallmann syndrome (KS), and hence the Prokr2 signaling pathway represents a unique model to decipher the olfactory/GnRH‐1 connection. We discovered that Prokr2 is expressed in the TN neurons during the critical period of GnRH‐1 neuron formation, migration, and induction of OB morphogenesis. Single‐cell RNA sequencing identified that the TN is formed by neurons distinct from the olfactory neurons. The TN neurons express multiple genes associated with KS. Our study suggests that the aberrant development of pioneer/TN neurons might cause the KS spectrum.

During embryonic development, the olfactory placode (OP) generates migratory neurons, including olfactory pioneer neurons, cells of the terminal nerve (TN), Gonadotropin-releasing hormone-1 (GnRH-1) neurons, and other uncharacterized neurons. Pioneer neurons from the olfactory placode induce olfactory bulb morphogenesis. In mice, GnRH-1 neurons appear in the olfactory system around mid-gestation and migrate via the terminal nerve axons to different brain regions. The GnRH-1 neurons are crucial in controlling the hypothalamic-pituitary-gonadal (HPG) axis. Kallmann syndrome is characterized by impaired olfactory system development, defective olfactory bulbs, defective secretion of GnRH-1, and infertility. The precise mechanistic link between the olfactory system and GnRH-1 development remains unclear. Studies in humans and mice highlight the importance of the Prokineticin-2/Prokineticin-Receptor-2 (Prokr2) signaling pathway in olfactory bulb morphogenesis and GnRH-1 neuronal migration. Prokr2 loss-of-function mutations can cause Kallmann syndrome, and hence the Prokr2 signaling pathway represents a unique model to decipher the olfactory/GnRH-1 connection. We discovered that Prokr2 is expressed in the TN neurons during the critical period of GnRH-1 neuron formation, migration, and induction of olfactory bulb morphogenesis. Single-cell RNA sequencing identified that the TN is formed by neurons that are distinct from the olfactory neurons. The TN neurons express multiple genes associated with KS. Our study suggests that the aberrant development of pioneer/TN neurons might cause the KS spectrum. Key Points: 1) Pioneer or terminal nerve neurons play a crucial role in initiating the development of the olfactory bulbs. We found that the Prokineticin Receptor-2 gene, associated with Kallmann syndrome, is expressed by the olfactory pioneer/terminal nerve neurons. 2) We genetically traced, isolated, and conducted Single-cell RNA sequencing on terminal nerve neurons of rodents. This analysis revealed a significant enrichment of gene expression related to Kallmann syndrome. 3) Our study indicates that the investigation of Pioneer/terminal nerve neurons should be a pivotal focal point for comprehending developmental defects affecting olfactory and GnRH-1 systems.

The ability of terrestrial vertebrates to find food and mating partners, and to avoid predators, relies on the detection of chemosensory information. Semiochemicals responsible for social and sexual behaviors are detected by chemosensory neurons of the vomeronasal organ (VNO), which transmits information to the accessory olfactory bulb. The vomeronasal sensory epithelium of most mammalian species contains a uniform vomeronasal system; however, rodents and marsupials have developed a more complex binary vomeronasal system, containing vomeronasal sensory neurons (VSNs) expressing receptors of either the V1R or V2R family. In rodents, V1R/apical and V2R/basal VSNs originate from a common pool of progenitors. Using single cell RNA-sequencing, we identified differential expression of Notch1 receptor and Dll4 ligand between the neuronal precursors at the VSN differentiation dichotomy. Our experiments show that Notch signaling is required for effective differentiation of V2R/basal VSNs. In fact, Notch1 loss of function in neuronal progenitors diverts them to the V1R/apical fate, whereas Notch1 gain of function redirects precursors to V2R/basal. Our results indicate that Notch signaling plays a pivotal role in triggering the binary differentiation dichotomy in the VNO of rodents.

Neuronal identity dictates the position in an epithelium, and the ability to detect, process, and transmit specific signals to specified targets. Transcription factors (TFs) determine cellular identity via direct modulation of genetic transcription and recruiting chromatin modifiers. However, our understanding of the mechanisms that define neuronal identity and their magnitude remains a critical barrier to elucidate the etiology of congenital and neurodegenerative disorders. The rodent vomeronasal organ provides a unique system to examine in detail the molecular mechanisms underlying the differentiation and maturation of chemosensory neurons. Here we demonstrated that the identity of postmitotic/maturing VSNs and vomeronasal dependent behaviors can be reprogrammed through the rescue of AP-2ε expression in the AP-2ε^Null mice and by inducing ectopic AP-2ε expression in mature apical VSNs. We suggest that the transcription factor AP-2ε can reprogram VSNs bypassing cellular plasticity restrictions, and that it directly controls the expression of batteries of vomeronasal genes.

The ability of terrestrial vertebrates to find food, mating partners and to avoid predators heavily relies on the detection of chemosensory information from the environment. The olfactory system of most vertebrate species comprises two distinct chemosensory systems usually referred to as the main and the accessory olfactory system. Olfactory sensory neurons of the main olfactory epithelium detect and transmit odor information to main olfactory bulb (MOB), while the chemosensory neurons of the vomeronasal organ detect semiochemicals responsible for social and sexual behaviors and transmit information to the accessory olfactory bulb (AOB). The vomeronasal sensory epithelium (VNE) of most mammalian species contains uniform vomeronasal (VN) system with vomeronasal sensory neurons (VSNs) expressing vomeronasal receptors of the V1R family. However, rodents and some marsupials have developed a more complex binary VN system, where VNO containing a second main type of VSNs expressing vomeronasal receptors of the V2R family is identified. In mice, V1R and V2R VSNs form from a common pool of progenitors but have distinct differentiation programs. As they mature, they segregate in different regions of the VNE and connect with different parts of the AOB. How these two main types of VSNs are formed has never been addressed. In this study, using single cell RNA sequencing data, we identified differential expression of Notch1 receptor and Dll4 ligand among the neuronal precursors at the VSN dichotomy. We further demonstrated with loss of function (LOF) and gain of function (GOF) studies that Dll4-Notch1 signaling plays a crucial role in triggering the binary dichotomy between the two main types of VSNs in mice. Graphical

During embryonic development, symmetric ectodermal thickenings [olfactory placodes (OP)] give rise to several cell types that comprise the olfactory system, such as those that form the terminal nerve ganglion (TN), gonadotropin releasing hormone-1 neurons (GnRH-1ns), and other migratory neurons in rodents. Even though the genetic heterogeneity among these cell types is documented, unidentified cell populations arising from the OP remain. One candidate to identify placodal derived neurons in the developing nasal area is the transcription factor Isl1, which was recently identified in GnRH-3 neurons of the terminal nerve in fish, as well as expression in neurons of the nasal migratory mass (MM). Here, we analyzed the Isl1 genetic lineage in chemosensory neuronal populations in the nasal area and migratory GnRH-1ns in mice using in situ hybridization, immunolabeling a Tamoxifen inducible Isl1Cre^ERT and a constitutive Isl1^Cre knock-in mouse lines. In addition, we also performed conditional Isl1 ablation in developing GnRH neurons. We found Isl1 lineage across non-sensory cells of the respiratory epithelium and sustentacular cells of OE and VNO. We identified a population of transient embryonic Isl1 + neurons in the olfactory epithelium and sparse Isl1 + neurons in postnatal VNO. Isl1 is expressed in almost all GnRH neurons and in approximately half of the other neuron populations in the MM. However, Isl1 conditional ablation alone does not significantly compromise GnRH-1 neuronal migration or GnRH-1 expression, suggesting compensatory mechanisms. Further studies will elucidate the functional and mechanistic role of Isl1 in development of migratory endocrine neurons.