Combination of blockade of endothelin signalling and compensation of IGF1 expression protects the retina from degeneration.

Retinitis pigmentosa (RP) and macular dystrophy (MD) cause severe retinal dysfunction, affecting 1 in 4000 people worldwide. This disease is currently assumed to be intractable, because effective therapeutic methods have not been established, regardless of genetic or sporadic traits. Here, we examined a RP mouse model in which the Prominin-1 (Prom1) gene was deficient and investigated the molecular events occurring at the outset of retinal dysfunction. We extracted the Prom1-deficient retina subjected to light exposure for a short time, conducted single-cell expression profiling, and compared the gene expression with and without stimuli. We identified the cells and genes whose expression levels change directly in response to light stimuli. Among the genes altered by light stimulation, Igf1 was decreased in rod photoreceptor cells and astrocytes under the light-stimulated condition. Consistently, the insulin-like growth factor (IGF) signal was weakened in light-stimulated photoreceptor cells. The recovery of Igf1 expression with the adeno-associated virus (AAV) prevented photoreceptor cell death, and its treatment in combination with the endothelin receptor antagonist led to the blockade of abnormal glial activation and the promotion of glycolysis, thereby resulting in the improvement of retinal functions, as assayed by electroretinography. We additionally demonstrated that the attenuation of mammalian/mechanistic target of rapamycin (mTOR), which mediates IGF signalling, leads to complications in maintaining retinal homeostasis. Together, we propose that combinatorial manipulation of distinct mechanisms is useful for the maintenance of the retinal condition.

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.

Regulation of progenitor cell survival by a novel chromatin remodeling factor during neural tube development.

During development, progenitor cell survival is essential for proper tissue functions, but the underlying mechanisms are not fully understood. Here we show that ERCC6L2, a member of the Snf2 family of helicase-like proteins, plays an essential role in the survival of developing chick neural cells. ERCC6L2 expression is induced by the Sonic Hedgehog (Shh) signaling molecule by a mechanism similar to that of the known Shh target genes Ptch1 and Gli1. ERCC6L2 blocks programmed cell death induced by Shh inhibition and this inhibition is independent of neural tube patterning. ERCC6L2 knockdown by siRNA resulted in the aberrant appearance of apoptotic cells. Furthermore, ERCC6L2 cooperates with the Shh signal and plays an essential role in the induction of the anti-apoptotic factor Bcl-2. Taken together, ERCC6L2 acts as a key factor in ensuring the survival of neural progenitor cells.

Erratum: Acquisition of neural fate by combination of BMP blockade and chromatin modification.

[This corrects the article DOI: 10.1016/j.isci.2023.107887.].

Acquisition of neural fate by combination of BMP blockade and chromatin modification.

Neural induction is a process where naive cells are converted into committed cells with neural characteristics, and it occurs at the earliest step during embryogenesis. Although the signaling molecules and chromatin remodeling for neural induction have been identified, the mutual relationships between these molecules are yet to be fully understood. By taking advantage of the neural differentiation system of mouse embryonic stem (ES) cells, we discovered that the BMP signal regulates the expression of several polycomb repressor complex (PRC) component genes. We particularly focused on Polyhomeotic Homolog 1 (Phc1) and established Phc1-knockout (Phc1-KO) ES cells. We found that Phc1-KO failed to acquire the neural fate, and the cells remained in pluripotent or primitive non-neural states. Chromatin accessibility analysis suggests that Phc1 is essential for chromatin packing. Aberrant upregulation of the BMP signal was confirmed in the Phc1 homozygotic mutant embryos. Taken together, Phc1 is required for neural differentiation through epigenetic modification.

Intercellular exchange of Wnt ligands reduces cell population heterogeneity during embryogenesis.

Wnt signaling is required to maintain bipotent progenitors for neural and paraxial mesoderm cells, the neuromesodermal progenitor (NMP) cells that reside in the epiblast and tailbud. Since epiblast/tailbud cells receive Wnt ligands produced by one another, this exchange may average out the heterogeneity of Wnt signaling levels among these cells. Here, we examined this possibility by replacing endogenous Wnt3a with a receptor-fused form that activates signaling in producing cells, but not in neighboring cells. Mutant mouse embryos show a unique phenotype in which maintenance of many NMP cells is impaired, although some cells persist for long periods. The epiblast cell population of these embryos increases heterogeneity in Wnt signaling levels as embryogenesis progresses and are sensitive to retinoic acid, an endogenous antagonist of NMP maintenance. Thus, mutual intercellular exchange of Wnt ligands in the epiblast cell population reduces heterogeneity and achieves robustness to environmental stress.

Manipulation of Signal Gradient and Transcription Factors Recapitulates: Multiple Hypothalamic Identities.

During development, the hypothalamus emerges from the ventral diencephalon and is regionalized into several distinct functional domains. Each domain is characterized by a different combination of transcription factors, including Nkx2.1, Nkx2.2, Pax6, and Rx, which are expressed in the presumptive hypothalamus and its surrounding regions, and play critical roles in defining each area. Here, we recapitulated the molecular networks formed by the gradient of Sonic Hedgehog (Shh) and the aforementioned transcription factors. Using combinatorial experimental systems of directed neural differentiation of mouse embryonic stem (ES) cells, as well as a reporter mouse line and gene overexpression in chick embryos, we deciphered the regulation of transcription factors by different Shh signal intensities. We then used CRISPR/Cas9 mutagenesis to demonstrate the mutual repression between Nkx2.1 and Nkx2.2 in a cell-autonomous manner; however, they induce each other in a non-cell-autonomous manner. Moreover, Rx resides upstream of all these transcription factors and determines the location of the hypothalamic region. Our findings suggest that Shh signaling and its downstream transcription network are required for hypothalamic regionalization and establishment.

The Rx transcription factor is required for determination of the retinal lineage and regulates the timing of neuronal differentiation.

Understanding the molecular mechanisms leading to retinal development is of great interest for both basic scientific and clinical applications. Several signaling molecules and transcription factors involved in retinal development have been isolated and analyzed; however, determining the direct impact of the loss of a specific molecule is problematic, due to difficulties in identifying the corresponding cellular lineages in different individuals. Here, we conducted genome-wide expression analysis with embryonic stem (ES) cells devoid of the Rx gene, which encodes one of several homeobox transcription factors essential for retinal development. We performed three-dimensional differentiation of wild-type and mutant cells and compared their gene-expression profiles. The mutant tissue failed to differentiate into the retinal lineage and exhibited precocious expression of genes characteristic of neuronal cells. Together, these results suggest that Rx expression is an important biomarker of the retinal lineage and that it helps regulates appropriate differentiation stages.

Morphological and Functional Changes of Roof Plate Cells in Spinal Cord Development.

The most dorsal region, or roof plate, is the dorsal organizing center of developing spinal cord. This region is also involved in development of neural crest cells, which are the source of migratory neural crest cells. During early development of the spinal cord, roof plate cells secrete signaling molecules, such as Wnt and BMP family proteins, which regulate development of neural crest cells and dorsal spinal cord. After the dorso-ventral pattern is established, spinal cord dynamically changes its morphology. With this morphological transformation, the lumen of the spinal cord gradually shrinks to form the central canal, a cavity filled with cerebrospinal fluid that is connected to the ventricular system of the brain. The dorsal half of the spinal cord is separated by a glial structure called the dorsal (or posterior) median septum. However, underlying mechanisms of such morphological transformation are just beginning to be understood. Recent studies reveal that roof plate cells dramatically stretch along the dorso-ventral axis, accompanied by reduction of the spinal cord lumen. During this stretching process, the tips of roof plate cells maintain contact with cells surrounding the shrinking lumen, eventually exposed to the inner surface of the central canal. Interestingly, Wnt expression remains in stretched roof plate cells and activates Wnt/ß-catenin signaling in ependymal cells surrounding the central canal. Wnt/ß-catenin signaling in ependymal cells promotes proliferation of neural progenitor and stem cells in embryonic and adult spinal cord. In this review, we focus on the role of the roof plate, especially that of Wnt ligands secreted by roof plate cells, in morphological changes occurring in the spinal cord.

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.

Differences in the secretion and transport of Wnt proteins.

During the last three decades, our understanding about Wnt signaling has progressed greatly, especially with regards to the molecular mechanism of intracellular transmission of this signaling, as well as its physiological roles. In parallel, the molecular nature of Wnt proteins has gradually but surely been clarified. Wnt proteins are post-translationaly modified with fatty acid and glycosaminoglycans, resulting in constraint of the 3D structure and behavior of the proteins. Specific binding proteins or extracellular vesicles, which appear to shield the lipid moiety from the aquatic environment, enable Wnt proteins to be transported in the extracellular space. Equally, Wnt-interacting proteins in the extracellular space, including heparan sulfate proteoglycan, are also involved in its spreading. Recent studies also show that intercellular transmission of Wnt proteins occurs by cell migration and extension of cell protrusions. Here, we will show the molecular and cellular bases of the trafficking of Wnt proteins and discuss questions that remain to be answered.

Androgen receptor of the frog Rana rugosa: molecular cloning and its characterization.

The androgen receptor(AR) gene is located on the Z and W sex chromosomes in the frog Rana rugosa, designated Z- and W-AR, respectively. Among various tissues of an adult frog, AR expression levels were highest in the testis and brain. In the testis, AR was expressed in germ cells. AR expression occured in developing embryos from stage 21 and was very high in the gonad of a male tadpole before the onset of sex determination. When Z- and W-AR were expressed in Xenopus A6 cells, they activated androgen-dependent transcription of a luciferase reporter gene. By contrast, estrogen receptor (ER) alpha and beta showed no sexually dimorphic expression during sex determination, but their expressions became much higher in the gonad of a female tadpole after sex determination. In addition, AR transcripts in the ZZ-tadpoles were twice as abundant as in the ZW genotype. In contrast, W-AR expression was extremely low although when W-AR was expressed in A6 cells, it activated transcription in the luciferase assay. In this regard it is worth noting that the promoter regions of Z- and W-AR are not identical. The results suggest that Z-AR plays an important role in the testis formation in a R. rugosa tadpole, whereas ERbeta is involved in ovary differentiation. Very low expression of W-AR may be due to its promoter region having mutations in key transcription factor binding sites, although these remain to be identified. Thus, it is proposed that AR could be a candidate for a male-determining gene in R. rugosa.