Sox2 is required for olfactory pit formation and olfactory neurogenesis through BMP restriction and Hes5 upregulation.

The transcription factor Sox2 is necessary to maintain pluripotency of embryonic stem cells, and to regulate neural development. Neurogenesis in the vertebrate olfactory epithelium persists from embryonic stages through adulthood. The role Sox2 plays for the development of the olfactory epithelium and neurogenesis within has, however, not been determined. Here, by analysing Sox2 conditional knockout mouse embryos and chick embryos deprived of Sox2 in the olfactory epithelium using CRISPR-Cas9, we show that Sox2 activity is crucial for the induction of the neural progenitor gene Hes5 and for subsequent differentiation of the neuronal lineage. Our results also suggest that Sox2 activity promotes the neurogenic domain in the nasal epithelium by restricting Bmp4 expression. The Sox2-deficient olfactory epithelium displays diminished cell cycle progression and proliferation, a dramatic increase in apoptosis and finally olfactory pit atrophy. Moreover, chromatin immunoprecipitation data show that Sox2 directly binds to the Hes5 promoter in both the PNS and CNS. Taken together, our results indicate that Sox2 is essential to establish, maintain and expand the neuronal progenitor pool by suppressing Bmp4 and upregulating Hes5 expression.

The facial neural crest controls fore- and midbrain patterning by regulating Foxg1 expression through Smad1 activity.

The facial neural crest (FNC), a pluripotent embryonic structure forming craniofacial structures, controls the activity of brain organisers and stimulates cerebrum growth. To understand how the FNC conveys its trophic effect, we have studied the role of Smad1, which encodes an intracellular transducer, to which multiple signalling pathways converge, in the regulation of Foxg1. Foxg1 is a transcription factor essential for telencephalic specification, the mutation of which leads to microcephaly and mental retardation. Smad1 silencing, based on RNA interference (RNAi), was performed in pre-migratory FNC cells. Soon after electroporation of RNAi molecules, Smad1 inactivation abolished the expression of Foxg1 in the chick telencephalon, resulting in dramatic microcephaly and partial holoprosencephaly. In addition, the depletion of Foxg1 activity altered the expression Otx2 and Foxa2 in di/mesencephalic neuroepithelium. However, when mutated forms of Smad1 mediating Fgf and Wnt signalling were transfected into FNC cells, these defects were overcome. We also show that, downstream of Smad1 activity, Dkk1, a Wnt antagonist produced by the FNC, initiated the specification of the telencephalon by regulating Foxg1 activity. Additionally, the activity of Cerberus in FNC-derived mesenchyme synergised with Dkk1 to control Foxg1 expression and maintain the balance between Otx2 and Foxa2.

Distinct Opsin 3 (Opn3) Expression in the Developing Nervous System during Mammalian Embryogenesis.

Opsin 3 (Opn3) is highly expressed in the adult brain, however, information for spatial and temporal expression patterns during embryogenesis is significantly lacking. Here, an Opn3-eGFP reporter mouse line was used to monitor cell body expression and axonal projections during embryonic and early postnatal to adult stages. By applying 2D and 3D fluorescence imaging techniques, we have identified the onset of Opn3 expression, which predominantly occurred during embryonic stages, in various structures during brain/head development. In addition, this study defines over twenty Opn3-eGFP-positive neural structures never reported before. Opn3-eGFP was first observed at E9.5 in neural regions, including the ganglia that will ultimately form the trigeminal, facial and vestibulocochlear cranial nerves (CNs). As development proceeds, expanded Opn3-eGFP expression coincided with the formation and maturation of critical components of the central and peripheral nervous systems (CNS, PNS), including various motor-sensory tracts, such as the dorsal column-medial lemniscus (DCML) sensory tract, and olfactory, acoustic, and optic tracts. The widespread, yet distinct, detection of Opn3-eGFP already at early embryonic stages suggests that Opn3 might play important functional roles in the developing brain and spinal cord to regulate multiple motor and sensory circuitry systems, including proprioception, nociception, ocular movement, and olfaction, as well as memory, mood, and emotion. This study presents a crucial blueprint from which to investigate autonomic and cognitive opsin-dependent neural development and resultant behaviors under physiological and pathophysiological conditions.

Elucidation of Cellular Mechanisms That Regulate the Sustained Contraction and Relaxation of the Mammalian Iris.

Purpose: In mammals, pupil constriction and dilation form the pupillary light reflex (PLR), which is mediated by both brain-regulated (parasympathetic) and local iris-driven reflexes. To better understand the cellular mechanisms that regulate pupil physiological dynamics via central and local photoreception, we have examined the regulation of the PLR via parasympathetic and local activation, respectively. Methods: In this study, the PLR was examined in mouse enucleated eyes ex vivo in real-time under different ionic conditions in response to acetylcholine and/or blue light (480 nm). The use of pupillometry recordings captured the relaxation, contraction, and pupil escape (redilation) processes for 10 minutes up to 1 hour. Results: Among others, our results show that ryanodine receptor channels are the main driver for iridal stimulation-contraction coupling, in which extracellular influx of Ca2+ is required for amplification of pupil constriction. Both local and parasympathetic iridal activations are necessary, but not sufficient for sustained pupil constriction. Moreover, the degree of membrane potential repolarization in the dark is correlated with the latency and velocity of iridal constriction. Furthermore, pupil escape is driven by membrane potential hyperpolarization where voltage-gated potassium channels play a crucial role. Conclusions: Together, this study presents new mechanisms regulating synchronized pupil dilation and contraction, sustained pupil constriction, iridal stimulation-contraction coupling, and pupil escape.

Temporal Requirement of Mab21l2 During Eye Development in Chick Reveals Stage-Dependent Functions for Retinogenesis.

Purpose: Different missense mutations in the single exon gene Mab21l2 have been identified in unrelated families with various bilateral eye malformations, including microphthalmia, anophthalmia, and coloboma, but the molecular function of Mab21l2 during eye development still remains largely unknown. Methods: We have established an in vivo Mab21l2-deficient eye development model in chick, by using a Mab21l2 RNA interference construct that we electroporated in ovo in prospective retinal cells. In addition, we designed a Mab21l2 gain-of-function electroporation vector. Mab21l2-modulated retinas were analyzed on consecutive sections in terms of morphology, and molecular markers for apoptosis, cell proliferation, and retinogenesis. Results: Our Mab21l2-deficient chick model mimics human ocular phenotypes. When Mab21l2 is downregulated prior to optic vesicle formation, the embryos develop anophthalmia, and Mab21l2 inhibition by optic cup stages results in a microphthalmic colobomatous phenotype. Our results show that inhibition of Mab21l2 affects cell proliferation, cell cycle exit, and the expression of Atoh7/Ath5, NeuroD4/Ath3, Isl1, Pax6, AP-2α, and Prox1. In addition, Mab21l2 overexpression hampers cell cycle exit and differentiation of retinal progenitor cells (RPCs). Conclusions: Our results highlight the importance of a regulated temporal expression of Mab21l2 during eye development: At early stages, Mab21l2 is required to maintain RPC proliferation and expansion of cell number; before retinogenesis, a decrease in Mab21l2 expression in proliferating RPCs is required for cell cycle exit and differentiation; during retinogenesis, Mab21l2 is chronologically upregulated in RGCs, followed by differentiated horizontal and amacrine cells and cone photoreceptor cells.