Retinoic acid, an essential component of the roof plate organizer, promotes the spatiotemporal segregation of dorsal neural fates.

Dorsal neural tube-derived retinoic acid promotes the end of neural crest production and transition into a definitive roof plate. Here, we analyze how this impacts the segregation of central and peripheral lineages, a process essential for tissue patterning and function. Localized in ovo inhibition in quail embryos of retinoic acid activity followed by single-cell transcriptomics unraveled a comprehensive list of differentially expressed genes relevant to these processes. Importantly, progenitors co-expressed neural crest, roof plate and dI1 interneuron markers, indicating a failure in proper lineage segregation. Furthermore, separation between roof plate and dI1 interneurons is mediated by Notch activity downstream of retinoic acid, highlighting their crucial role in establishing the roof plate-dI1 boundary. Within the peripheral branch, where absence of retinoic acid resulted in neural crest production and emigration extending into the roof plate stage, sensory progenitors failed to separate from melanocytes, leading to formation of a common glia-melanocyte cell with aberrant migratory patterns. In summary, the implementation of single-cell RNA sequencing facilitated the discovery and characterization of a molecular mechanism responsible for the segregation of dorsal neural fates during development.

From neural tube to spinal cord: The dynamic journey of the dorsal neuroepithelium.

In a developing embryo, formation of tissues and organs is remarkably precise in both time and space. Through cell-cell interactions, neighboring progenitors coordinate their activities, sequentially generating distinct types of cells. At present, we only have limited knowledge, rather than a systematic understanding, of the underlying logic and mechanisms responsible for cell fate transitions. The formation of the dorsal aspect of the spinal cord is an outstanding model to tackle these dynamics, as it first generates the peripheral nervous system and is later responsible for transmitting sensory information from the periphery to the brain and for coordinating local reflexes. This is reflected first by the ontogeny of neural crest cells, progenitors of the peripheral nervous system, followed by formation of the definitive roof plate of the central nervous system and specification of adjacent interneurons, then a transformation of roof plate into dorsal radial glia and ependyma lining the forming central canal. How do these peripheral and central neural branches segregate from common progenitors? How are dorsal radial glia established concomitant with transformation of the neural tube lumen into a central canal? How do the dorsal radial glia influence neighboring cells? This is only a partial list of questions whose clarification requires the implementation of experimental paradigms in which precise control of timing is crucial. Here, we outline some available answers and still open issues, while highlighting the contributions of avian models and their potential to address mechanisms of neural patterning and function.

Rspo1 and Rspo3 are required for sensory lineage neural crest formation in mouse embryos.

BACKGROUND: R-spondins (Rspos) are secreted proteins that modulate Wnt/ß-catenin signaling. At the early stages of spinal cord development, Wnts (Wnt1, Wnt3a) and Rspos (Rspo1, Rspo3) are co-expressed in the roof plate, suggesting that Rspos are involved in development of dorsal spinal cord and neural crest cells in cooperation with Wnt ligands. RESULTS: Here, we found that Rspo1 and Rspo3, as well as Wnt1 and Wnt3a, maintained roof-plate-specific expression until late embryonic stages. Rspo1- and Rspo3-double-knock-out (dKO) embryos partially exhibited the phenotype of Wnt1 and Wnt3a dKO embryos. While the number of Ngn2-positive sensory lineage neural crest cells is reduced in Rspo-dKO embryos, development of dorsal spinal cord, including its size and dorso-ventral patterning in early development, elongation of the roof plate, and proliferation of ependymal cells, proceeded normally. Consistent with these slight defects, Wnt/ß-catenin signaling was not obviously changed in developing spinal cord of dKO embryos. CONCLUSIONS: Our results show that Rspo1 and Rspo3 are dispensable for most developmental processes involving roof plate-derived Wnt ligands, except for specification of a subtype of neural crest cells. Thus, Rspos may modulate Wnt/ß-catenin signaling in a context-dependent manner.

Novel recombinant R-spondin1 promotes hair regeneration by targeting the Wnt/ß-catenin signaling pathway.

Roof plate-specific spondin 1 (R-spondin1, RSPO1) is a Wnt/ß-catenin signaling pathway activator that binds with Wnt ligands to stimulate the Wnt/ß-catenin signaling pathway, which is key to hair regeneration. However, it is not clear whether recombinant RSPO1 (rRSPO1) affects hair regeneration. Here, we treat C57BL/6 male mice with rRSPO1 and investigate the expression of the Wnt/ß-catenin signaling pathway and the activation of hair follicle stem cells in the dorsal skin. The mouse skin color score and hair-covered area are determined to describe hair growth, and the skin samples are subjected to H&E staining, western blot analysis and immunofluorescence staining to evaluate hair follicle development and the expressions of Wnt/ß-catenin signaling pathway-related proteins. We find that rRSPO1 activates mouse hair follicle stem cells (mHFSCs) and accelerates hair regeneration. rRSPO1 increases the hair-covered area, the number of hair follicles, and the hair follicle diameter and length. Moreover, rRSPO1 enhances the activity of Wnt/ß-catenin signaling pathway-related proteins and the expressions of HFSC markers, as well as mHFSC viability. These results indicate that subcutaneous injection of rRSPO1 can improve hair follicle development by activating the Wnt/ß-catenin signaling pathway, thereby promoting hair regeneration. This study demonstrates that rRSPO1 has the potential to treat hair loss by activating the Wnt/ß-catenin signaling pathway.

Origin and development of circumventricular organs in living vertebrate.

The circumventricular organs (CVOs) function by mediating chemical communication between blood and brain across the blood-brain barrier. Their origin and developmental mechanisms involved are not understood in enough detail due to a lack of molecular markers common for CVOs. These rather small and inconspicuous organs are found in close vicinity to the third and fourth brain ventricles suggestive of ancient evolutionary origin. Recently, an integrated approach based on analysis of CVOs development in the enhancer-trap transgenic zebrafish led to an idea that almost all of CVOs could be highlighted by GFP expression in this transgenic line. This in turn suggested that an enhancer along with a set of genes it regulates may illustrate the first common element of developmental regulation of CVOs. It seems to be related to a mechanism of suppression of the canonical Wnt/ ß-catenin signaling that functions in development of fenestrated capillaries typical for CVOs. Based on that observation the common molecular elements of the putative developmental mechanism of CVOs will be discussed in this review.

Wnt produced by stretched roof-plate cells is required for the promotion of cell proliferation around the central canal of the spinal cord.

Cell morphology changes dynamically during embryogenesis, and these changes create new interactions with surrounding cells, some of which are presumably mediated by intercellular signaling. However, the effects of morphological changes on intercellular signaling remain to be fully elucidated. In this study, we examined the effect of morphological changes in Wnt-producing cells on intercellular signaling in the spinal cord. After mid-gestation, roof-plate cells stretched along the dorsoventral axis in the mouse spinal cord, resulting in new contact at their tips with the ependymal cells that surround the central canal. Wnt1 and Wnt3a were produced by the stretched roof-plate cells and delivered to the cell process tip. Whereas Wnt signaling was activated in developing ependymal cells, Wnt activation in dorsal ependymal cells, which were close to the stretched roof plate, was significantly suppressed in embryos with roof plate-specific conditional knockout of Wls, which encodes a factor that is essential for Wnt secretion. Furthermore, proliferation of these cells was impaired in Wls conditional knockout mice during development and after induced spinal cord injury in adults. Therefore, morphological changes in Wnt-producing cells appear to generate new Wnt signal targets.

Altering metabolite distribution at Xenopus cleavage stages affects left-right gene expression asymmetries.

The left-right (L-R) axis of most bilateral animals is established during gastrulation when a transient ciliated structure creates a directional flow of signaling molecules that establish asymmetric gene expression in the lateral plate mesoderm. However, in some animals, an earlier differential distribution of molecules and cell division patterns initiate or at least influence L-R patterning. Using single-cell high-resolution mass spectrometry, we previously reported a limited number of small molecule (metabolite) concentration differences between left and right dorsal-animal blastomeres of the eight-cell Xenopus embryo. Herein, we examined whether altering the distribution of some of these molecules influenced early events in L-R patterning. Using lineage tracing, we found that injecting right-enriched metabolites into the left cell caused its descendant cells to disperse in patterns that varied from those in control gastrulae; this did not occur when left-enriched metabolites were injected into the right cell. At later stages, injecting left-enriched metabolites into the right cell perturbed the expression of genes known to: (a) be required for the formation of the gastrocoel roof plate (foxj1); (b) lead to the asymmetric expression of Nodal (dand5/coco); or (c) result from asymmetrical nodal expression (pitx2). Despite these perturbations in gene expression, we did not observe heterotaxy in heart or gut looping at tadpole stages. These studies indicate that altering metabolite distribution at cleavage stages at the concentrations tested in this study impacts the earliest steps of L-R gene expression that then can be compensated for during organogenesis.

Apoptosis is involved in maintaining the character of the midbrain and the diencephalon roof plate after neural tube closure.

Apoptosis, a major form of programmed cell death, is massively observed in neural plate border and subsequently in the roof plate (RP). While deficiency of apoptosis often results in brain malformations including exencephaly and hydrocephalus, the impact of apoptosis on RP formation and maintenance remains unclear. Here we described that mouse embryos deficient in Apaf1, a gene crucial for the intrinsic apoptotic pathway, in C57BL/6 genetic background exhibited narrow and discontinuous expression of RP marker genes in the midline of the midbrain and the diencephalon. Instead, cells positive for the neuroectodermal gene SOX1 ectopically accumulated in the midline. A lineage-tracing experiment suggests that these ectopic SOX1-positive cells began to accumulate in the midline of apoptosis-deficient embryos after E9.5. These embryos further displayed malformation of the subcommissural organ, which has been discussed in the etiology of hydrocephalus. Thus, the apoptosis machinery prevents ectopic emergence of SOX1-positive cells in the midbrain and the diencephalon RP, and helps in maintaining the character of the RP in the diencephalon and midbrain, thereby ensuring proper brain development.

Camel regulates development of the brain ventricular system.

Development of the brain ventricular system of vertebrates and the molecular mechanisms involved are not fully understood. The developmental genes expressed in the elements of the brain ventricular system such as the ependyma and circumventricular organs act as molecular determinants of cell adhesion critical for the formation of brain ventricular system. They control brain development and function, including the flow of cerebrospinal fluid. Here, we describe the novel distantly related member of the zebrafish L1-CAM family of genes-camel. Whereas its maternal transcripts distributed uniformly, the zygotic transcripts demonstrate clearly defined expression patterns, in particular in the axial structures: floor plate, hypochord, and roof plate. camel expresses in several other cell lineages with access to the brain ventricular system, including the midbrain roof plate, subcommissural organ, organum vasculosum lamina terminalis, median eminence, paraventricular organ, flexural organ, and inter-rhombomeric boundaries. This expression pattern suggests a role of Camel in neural development. Several isoforms of Camel generated by differential splicing of exons encoding the sixth fibronectin type III domain enhance cell adhesion differentially. The antisense oligomer morpholino-mediated loss-of-function of Camel affects cell adhesion and causes hydrocephalus and scoliosis manifested via the tail curled down phenotype. The subcommissural organ's derivative-the Reissner fiber-participates in the flow of cerebrospinal fluid. The Reissner fiber fails to form upon morpholino-mediated Camel loss-of-function. The Camel mRNA-mediated gain-of-function causes the Reissner fiber misdirection. This study revealed a link between Chl1a/Camel and Reissner fiber formation, and this supports the idea that CHL1 is one of the scoliosis factors.

From Neural Crest to Definitive Roof Plate: The Dynamic Behavior of the Dorsal Neural Tube.

Research on the development of the dorsal neural tube is particularly challenging. In this highly dynamic domain, a temporal transition occurs between early neural crest progenitors that undergo an epithelial-to-mesenchymal transition and exit the neural primordium, and the subsequent roof plate, a resident epithelial group of cells that constitutes the dorsal midline of the central nervous system. Among other functions, the roof plate behaves as an organizing center for the generation of dorsal interneurons. Despite extensive knowledge of the formation, emigration and migration of neural crest progenitors, little is known about the mechanisms leading to the end of neural crest production and the transition into a roof plate stage. Are these two mutually dependent or autonomously regulated processes? Is the generation of roof plate and dorsal interneurons induced by neural tube-derived factors throughout both crest and roof plate stages, respectively, or are there differences in signaling properties and responsiveness as a function of time? In this review, we discuss distinctive characteristics of each population and possible mechanisms leading to the shift between the above cell types.

Loss of the small GTPase Arl8b results in abnormal development of the roof plate in mouse embryos.

Lysosomes are acidic organelles responsible for degrading both exogenous and endogenous materials. The small GTPase Arl8 localizes primarily to lysosomes and is involved in lysosomal function. In the present study, using Arl8b gene-trapped mutant (Arl8b-/- ) mice, we show that Arl8b is required for the development of dorsal structures of the neural tube, including the thalamus and hippocampus. In embryonic day (E) 10.5 Arl8b-/- embryos, Sox1 (a neuroepithelium marker) was ectopically expressed in the roof plate, whereas the expression of Gdf7 and Msx1 (roof plate markers) was reduced in the dorsal midline of the midbrain. Ectopic expression of Sox1 in Arl8b-/- embryos was detected also at E9.0 in the neural fold, which gives rise to the roof plate. In addition, the levels of Bmp receptor IA and phosphorylated Smad 1/5/8 (downstream of BMP signaling) were increased in the neural fold of E9.0 Arl8b-/- embryos. These results suggest that Arl8b is involved in the development of the neural fold and the subsequently formed roof plate, possibly via control of BMP signaling.

Multiple steps characterise ventricular layer attrition to form the ependymal cell lining of the adult mouse spinal cord central canal.

The ventricular layer of the spinal cord is remodelled during embryonic development and ultimately forms the ependymal cell lining of the adult central canal, which retains neural stem cell potential. This anatomical transformation involves the process of dorsal collapse; however, accompanying changes in tissue organisation and cell behaviour as well as the precise origin of cells contributing to the central canal are not well understood. Here, we describe sequential localised cell rearrangements which accompany the gradual attrition of the spinal cord ventricular layer during development. This includes local breakdown of the pseudostratified organisation of the dorsal ventricular layer prefiguring dorsal collapse and evidence for a new phenomenon, ventral dissociation, during which the ventral-most floor plate cells separate from a subset that are retained around the central canal. Using cell proliferation markers and cell-cycle reporter mice, we further show that following dorsal collapse, ventricular layer attrition involves an overall reduction in cell proliferation, characterised by an intriguing increase in the percentage of cells in G1/S. In contrast, programmed cell death does not contribute to ventricular layer remodelling. By analysing transcript and protein expression patterns associated with key signalling pathways, we provide evidence for a gradual decline in ventral sonic hedgehog activity and an accompanying ventral expansion of initial dorsal bone morphogenetic protein signalling, which comes to dominate the forming the central canal lining. This study identifies multiple steps that may contribute to spinal cord ventricular layer attrition and adds to increasing evidence for the heterogeneous origin of the spinal cord ependymal cell population, which includes cells from the floor plate and the roof plate as well as ventral progenitor domains.

Rspos and malignant tumors of digestive system. / Rsposå’Œæ¶ˆåŒ–ç³»ç»Ÿè‚¿ç˜¤çš„å ³ç³».

Roof plate specific spondins (Rspos) are important activators of the Wnt signaling pathway discovered recently. Rspos include four secreted proteins: Rspo1, Rspo2, Rspo3, and Rspo4.They are mainly involved in the regulation of cell proliferation and differentiation via regulating the canonical Wnt/ß-catenin signaling pathway.The physiological functions of Rspos include regulating sex selection, limb development, organ formation and development. Rspos are involved in the pathogenesis of some malignant tumors, and the roles of Rspos vary in different types of tumors.

Neural crest emigration: From start to stop.

Within the dynamic context of a developing embryo, the multicellular patterns formed are extraordinarily precise. Through cell-cell communication, neighboring progenitors coordinate their activities, sequentially generating distinct tissues. The development of the dorsal neural tube remarkably illustrates this principle. It first generates neural crest (NC) cells, precursors of most of the peripheral nervous system, and then becomes the roof plate (RP) of the central nervous system. While the molecular network regulating emigration of NC progenitors has been extensively studied, the mechanisms by which dorsal neural tube precursors transit from an initial NC fate to a definitive RP identity remain widely open to investigation. Critical differences exist between premigratory NC and RP cells. Whereas the former extensively proliferate and undergo an epithelial-to-mesenchymal transition that generates cellular migrations, the latter progressively exit the cell cycle and regain epithelial traits including apico-basal polarity and regeneration of a laminin-containing basement membrane. To understand this transition, the nature of the cross-talk between these two sequentially forming progenitor subsets should be unraveled, including the identity and mode of action of signals that, on the one hand, induce the arrest of NC emigration, and, on the other hand, promote formation of a definitive RP.

Retinoic acid signaling regulates development of the dorsal forebrain midline and the choroid plexus in the chick.

The developing forebrain roof plate (RP) contains a transient signaling center, perturbations in which have been linked to holoprosencephaly (HPE). Here, we describe a novel domain of retinoic acid (RA) signaling that is specific to the chick RP and demonstrate that RA signaling is sufficient for inducing characteristics of the RP in ectopic locations. We further demonstrate that, unlike what has been observed in the mouse, RA signaling is essential for invagination of the RP in chick, failure of which leads to an HPE-like phenotype. In addition, we found that RA exerts a negative influence on choroid plexus differentiation. Thus, our findings identify RA as a novel regulator of chick forebrain RP development.

Roof plate mediated morphogenesis of the forebrain: New players join the game.

The roof plate is a crucial signaling center located at the dorsal midline of the developing central nervous system (CNS) along its rostro-caudal axis. By virtue of secreting multiple signaling molecules, it regulates diverse processes such as specification of dorsal fate, proliferation and axon guidance. In the forebrain, the roof plate is not only involved in patterning but is also involved in the division of the single forebrain vesicle into the two cerebral hemispheres, the failure of which leads to certain forms of holoprosencephaly. Although several molecular players such as Fgfs, BMPs, Wnts and Shh have been identified as crucial regulators of development of the forebrain, little is known about how they interact to bring about the morphological changes associated with the division of the forebrain vesicle into the cerebral hemispheres. Recent studies have now identified the dorsal mesenchyme as an additional source of signaling cues, which is likely to influence the division of the forebrain vesicle into cerebral hemispheres. In this review, we discuss the current understanding about the molecular mechanisms of roof plate mediated patterning and morphogenesis of the forebrain including some recently identified factors that influence this process and also highlight the gaps in our knowledge that remain.

MafB is required for development of the hindbrain choroid plexus.

The choroid plexus (ChP) is a non-neural epithelial tissue that produces cerebrospinal fluid (CSF). The ChP differentiates from the roof plate, a dorsal midline structure of the neural tube. However, molecular mechanisms underlying ChP development are poorly understood compared to neural development. MafB is a bZip transcription factor that is known to be expressed in the roof plate. Here we investigated the role of MafB in embryonic development of the hindbrain ChP (hChP) using Mafb-deficient mice. Immunohistochemical analyses revealed that MafB is expressed in the roof plate and early hChP epithelial cells but its expression disappears at a later embryonic stage. We also found that the Mafb-deficient hChP exhibits delayed differentiation and results in hypoplasia compared to the wild-type hChP. Furthermore, the Mafb-deficient hChP exhibits increased apoptotic cell death and decreased proliferating cells at E12.5, an early stage of hChP development. Collectively, our findings reveal that MafB play an important role in promoting hChP development during embryogenesis.

A dynamic code of dorsal neural tube genes regulates the segregation between neurogenic and melanogenic neural crest cells.

Understanding when and how multipotent progenitors segregate into diverse fates is a key question during embryonic development. The neural crest (NC) is an exemplary model system with which to investigate the dynamics of progenitor cell specification, as it generates a multitude of derivatives. Based on 'in ovo' lineage analysis, we previously suggested an early fate restriction of premigratory trunk NC to generate neural versus melanogenic fates, yet the timing of fate segregation and the underlying mechanisms remained unknown. Analysis of progenitors expressing a Foxd3 reporter reveals that prospective melanoblasts downregulate Foxd3 and have already segregated from neural lineages before emigration. When this downregulation is prevented, late-emigrating avian precursors fail to upregulate the melanogenic markers Mitf and MC/1 and the guidance receptor Ednrb2, generating instead glial cells that express P0 and Fabp. In this context, Foxd3 lies downstream of Snail2 and Sox9, constituting a minimal network upstream of Mitf and Ednrb2 to link melanogenic specification with migration. Consistent with the gain-of-function data in avians, loss of Foxd3 function in mouse NC results in ectopic melanogenesis in the dorsal tube and sensory ganglia. Altogether, Foxd3 is part of a dynamically expressed gene network that is necessary and sufficient to regulate fate decisions in premigratory NC. Their timely downregulation in the dorsal neural tube is thus necessary for the switch between neural and melanocytic phases of NC development.

Multipotent caudal neural progenitors derived from human pluripotent stem cells that give rise to lineages of the central and peripheral nervous system.

The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the glycogen synthase kinase 3ß and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named "caudal neural progenitors" (CNPs). CNPs coexpress caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential. Exposure of CNPs to BMP2/4, sonic hedgehog, or FGF2 signaling efficiently directs their fate to neural crest/roof plate cells, floor plate cells, and caudally specified neuroepithelial cells, respectively. Neural crest derived from CNPs differentiated to neural crest derivatives and demonstrated extensive migratory properties in vivo. Importantly, we also determined the key extrinsic factors specifying CNPs from human embryonic stem cell include FGF8, canonical WNT, and IGF1. Our studies are the first to identify a multipotent neural progenitor derived from hPSCs, that is the precursor for major neural lineages of the embryonic caudal neural tube.

Are there conserved roles for the extracellular matrix, cilia, and junctional complexes in left-right patterning?

Many different types of molecules have essential roles in patterning the left-right axis and directing asymmetric morphogenesis. In particular, the relationship between signaling molecules and transcription factors has been explored extensively. Another group of proteins implicated in left-right patterning are components of the extracellular matrix, apical junctions, and cilia. These structural molecules have the potential to participate in the conversion of morphogenetic cues from the extracellular environment into morphogenetic patterning via their interactions with the actin cytoskeleton. Although it has been relatively easy to temporally position these proteins within the hierarchy of the left-right patterning pathway, it has been more difficult to define how they mechanistically fit into these pathways. Consequently, our understanding of how these factors impart patterning information to influence the establishment of the left-right axis remains limited. In this review, we will discuss those structural molecules that have been implicated in early phases of left-right axis development.