Sizzled (frzb3) physically interacts with non-canonical Wnt ligands to inhibit gastrulation cell movement.

The coordinated movement of germ layer progenitor cells reaches its peak at the dorsal side, where the Bmp signaling gradient is low, and minimum at the ventral side, where the Bmp gradient is high. This dynamic cell movement is regulated by the interplay of various signaling pathways. The non-canonical Wnt signaling cascade serves as a pivotal regulator of convergent and extension cellular movement, facilitated by the activation of small GTPases such as Rho, Rab, and Rac. However, the underlying cause of limited cell movement at the ventral side remains elusive. To explore the functional role of a key regulator in constraining gastrulation cell movement at the ventral side, we investigated the Bmp4-direct target gene, sizzled, to assess its potential role in inhibiting non-canonical Wnt signaling. In our current study, we demonstrated that ectopic expression of sizzled led to gastrulation defects in a dose-dependent manner, without altering cell fate specification. Overexpression of sizzled resulted in decreased elongation of Activin-treated animal cap and Keller explants. Furthermore, our immunoprecipitation assay unveiled the physical interaction of Sizzled with non-canonical Wnt ligand proteins (Wnt5 and Wnt11). Additionally, the activation of small GTPases involved in Wnt signaling mediation (RhoA and Rac1) was diminished upon sizzled overexpression. In summary, our findings suggest that Bmp4 signaling negatively modulates cell movement from the ventral side of the embryo by inducing sizzled expression during early Xenopus gastrulation.

Loss of Katnal2 leads to ependymal ciliary hyperfunction and autism-related phenotypes in mice.

Autism spectrum disorders (ASD) frequently accompany macrocephaly, which often involves hydrocephalic enlargement of brain ventricles. Katnal2 is a microtubule-regulatory protein strongly linked to ASD, but it remains unclear whether Katnal2 knockout (KO) in mice leads to microtubule- and ASD-related molecular, synaptic, brain, and behavioral phenotypes. We found that Katnal2-KO mice display ASD-like social communication deficits and age-dependent progressive ventricular enlargements. The latter involves increased length and beating frequency of motile cilia on ependymal cells lining ventricles. Katnal2-KO hippocampal neurons surrounded by enlarged lateral ventricles show progressive synaptic deficits that correlate with ASD-like transcriptomic changes involving synaptic gene down-regulation. Importantly, early postnatal Katnal2 re-expression prevents ciliary, ventricular, and behavioral phenotypes in Katnal2-KO adults, suggesting a causal relationship and a potential treatment. Therefore, Katnal2 negatively regulates ependymal ciliary function and its deletion in mice leads to ependymal ciliary hyperfunction and hydrocephalus accompanying ASD-related behavioral, synaptic, and transcriptomic changes.

Xbra modulates the activity of linker region phosphorylated Smad1 during Xenopus development.

The Bmp/Smad1 pathway plays a crucial role in developmental processes and tissue homeostasis. Mitogen-activated protein kinase (Mapk)/Erk mediated phosphorylation of Smad1 in the linker region leads to Smad1 degradation, cytoplasmic retention and inhibition of Bmp/Smad1 signaling. While Fgf/Erk pathway has been documented to inhibit Bmp/Smad1 signaling, several studies also suggests the cooperative interaction between these two pathways in different context. However, the precise role and molecular pathway of this collaborative interaction remain obscure. Here, we identified Xbra induced by Fgf/Erk signaling as a factor in a protective mechanism for Smad1. Xbra physically interacted with the linker region phosphorylated Smad1 to make Xbra/Smad1/Smad4 trimeric complex, leading to Smad1 nuclear localization and protecting it from ubiquitin-mediated proteasomal degradation. This interaction of Xbra/Smad1/Smad4 led to sustained nuclear localization of Smad1 and the upregulation of lateral mesoderm genes, while concurrently suppression of neural and blood forming genes. Taken together, the results suggests Xbra-dependent cooperative interplays between Fgf/Erk and Bmp/Smad1 signaling during lateral mesoderm specification in Xenopus embryos.

Deup1 Expression Interferes with Multiciliated Differentiation.

A recent study revealed that the loss of Deup1 expression does not affect either centriole amplification or multicilia formation. Therefore, the deuterosome per se is not a platform for amplification of centrioles. In this study, we examine whether gain-of-function of Deup1 affects the development of multiciliated ependymal cells. Our time-lapse study reveals that deuterosomes with an average diameter of 300 nm have two different fates during ependymal differentiation. In the first instance, deuterosomes are scattered and gradually disappear as cells become multiciliated. In the second instance, deuterosomes self-organize into a larger aggregate, called a deuterosome cluster (DC). Unlike scattered deuterosomes, DCs possess centriole components primarily within their large structure. A characteristic of DC-containing cells is that they tend to become primary ciliated rather than multiciliated. Our in utero electroporation study shows that DCs in ependymal tissue are mostly observed at early postnatal stages, but are scarce at late postnatal stages, suggesting the presence of DC antagonists within the differentiating cells. Importantly, from our bead flow assay, ectopic expression of Deup1 significantly impairs cerebrospinal fluid flow. Furthermore, we show that expression of mouse Deup1 in Xenopus embryos has an inhibitory effect on differentiation of multiciliated cells in the epidermis. Taken together, we conclude that the DC formation of Deup1 in multiciliated cells inhibits production of multiple centrioles.

ANKS1A-Deficiency Aberrantly Increases the Entry of the Protein Transport Machinery into the Ependymal Cilia.

In this study, we examine whether a change in the protein levels for FOP in Ankyrin repeat and SAM domain-containing protein 1A (ANKS1A)-deficient ependymal cells affects the intraflagellar transport (IFT) protein transport system in the multicilia. Three distinct abnormalities are observed in the multicilia of ANKS1A-deficient ependymal cells. First, there were a greater number of IFT88-positive trains along the cilia from ANKS1A deficiency. The results are similar to each isolated cilium as well. Second, each isolated cilium contains a significant increase in the number of extracellular vesicles (ECVs) due to the lack of ANKS1A. Third, Van Gogh-like 2 (Vangl2), a ciliary membrane protein, is abundantly detected along the cilia and in the ECVs attached to them for ANKS1A-deficient cells. We also use primary ependymal culture systems to obtain the ECVs released from the multicilia. Consequently, we find that ECVs from ANKS1A-deficient cells contain more IFT machinery and Vangl2. These results indicate that ANKS1A deficiency increases the entry of the protein transport machinery into the multicilia and as a result of these abnormal protein transports, excessive ECVs form along the cilia. We conclude that ependymal cells make use of the ECV-based disposal system in order to eliminate excessively transported proteins from basal bodies.

ANKS1A regulates LDL receptor-related protein 1 (LRP1)-mediated cerebrovascular clearance in brain endothelial cells.

Brain endothelial LDL receptor-related protein 1 (LRP1) is involved in the clearance of Aß peptides across the blood-brain barrier (BBB). Here we show that endothelial deficiency of ankyrin repeat and SAM domain containing 1 A (ANKS1A) reduces both the cell surface levels of LRP1 and the Aß clearance across the BBB. Association of ANKS1A with the NPXY motifs of LRP1 facilitates the transport of LRP1 from the endoplasmic reticulum toward the cell surface. ANKS1A deficiency in an Alzheimer's disease mouse model results in exacerbated Aß pathology followed by cognitive impairments. These deficits are reversible by gene therapy with brain endothelial-specific ANKS1A. In addition, human induced pluripotent stem cell-derived BBBs (iBBBs) were generated from endothelial cells lacking ANKS1A or carrying the rs6930932 variant. Those iBBBs exhibit both reduced cell surface LRP1 and impaired Aß clearance. Thus, our findings demonstrate that ANKS1A regulates LRP1-mediated Aß clearance across the BBB.

Oxidative stress in cerebrovascular disease and associated diseases.

Cellular aging is the most severe risk factor for neurodegenerative disease. Simultaneously, oxidative stress (OS) is a critical factor in the aging process, resulting from an imbalance between reactive oxygen and nitrogen species and the antioxidant defense system. Emerging evidence indicates that OS is a common cause of several age-related brain pathologies, including cerebrovascular diseases. Elevated OS disrupts endothelial functional ability by diminishing the bioavailability of nitric oxide (a vascular dilator), induces atherosclerosis, and impairs vasculature, which are all common characteristics of cerebrovascular disease. In this review, we summarize evidence supporting an active role of OS in cerebrovascular disease progression, focusing primarily on stroke pathogenesis. We briefly discuss hypertension, diabetes, heart disease, and genetic factors that are often linked to OS and are considered associated factors influencing stroke pathology. Finally, we discuss the current pharmaceutics/therapeutics available for treating several cerebrovascular diseases.

Systematic Multiomic Analysis of PKHD1L1 Gene Expression and Its Role as a Predicting Biomarker for Immune Cell Infiltration in Skin Cutaneous Melanoma and Lung Adenocarcinoma.

The identification of genetic factors that regulate the cancer immune microenvironment is important for understanding the mechanism of tumor progression and establishing an effective treatment strategy. Polycystic kidney and hepatic disease 1-like protein 1 (PKHD1L1) is a large transmembrane protein that is highly expressed in immune cells; however, its association with tumor progression remains unclear. Here, we systematically analyzed the clinical relevance of PKHD1L1 in the tumor microenvironment in multiple cancer types using various bioinformatic tools. We found that the PKHD1L1 mRNA expression levels were significantly lower in skin cutaneous melanoma (SKCM) and lung adenocarcinoma (LUAD) than in normal tissues. The decreased expression of PKHD1L1 was significantly associated with unfavorable overall survival (OS) in SKCM and LUAD. Additionally, PKHD1L1 expression was positively correlated with the levels of infiltrating B cells, cluster of differentiation (CD)-8+ T cells, and natural killer (NK) cells, suggesting that the infiltration of immune cells could be associated with a good prognosis due to increased PKHD1L1 expression. Gene ontology (GO) analysis also revealed the relationship between PKHD1L1-co-altered genes and the activation of lymphocytes, including B and T cells. Collectively, this study shows that PKHD1L1 expression is positively correlated with a good prognosis via the induction of immune infiltration, suggesting that PKHD1L1 has potential prognostic value in SKCM and LUAD.

Cerebral Cavernous Malformation (CCM)-like Vessel Lesion in the Aged ANKS1A-deficient Brain.

In this study, we show that ANKS1A is specifically expressed in the brain endothelial cells of adult mice. ANKS1A deficiency in adult mice does not affect the differentiation, growth, or patterning of the cerebrovascular system; however, its absence significantly impacts the cerebrovascular system of the aged brain. In aged ANKS1A knock-out (KO) brains, vessel lesions exhibiting cerebral cavernous malformations (CCMs) are observed. In addition, CCM-like lesions show localized peripheral blood leakage into the brain. The CCM-like lesions reveal immune cells infiltrating the parenchyma. The CCM-like lesions also contain significantly fewer astrocyte endfeets and tight junctions, indicating that the integrity of the BBB has been partially compromised. CCM-like lesions display increased fibronectin expression in blood vessels, which is also confirmed in cultured endothelial cells deficient for ANKS1A. Therefore, we hypothesize that ANKS1A may play a role in maintaining or stabilizing healthy blood vessels in the brain during aging.

The Organizer and Its Signaling in Embryonic Development.

Germ layer specification and axis formation are crucial events in embryonic development. The Spemann organizer regulates the early developmental processes by multiple regulatory mechanisms. This review focuses on the responsive signaling in organizer formation and how the organizer orchestrates the germ layer specification in vertebrates. Accumulated evidence indicates that the organizer influences embryonic development by dual signaling. Two parallel processes, the migration of the organizer's cells, followed by the transcriptional activation/deactivation of target genes, and the diffusion of secreting molecules, collectively direct the early development. Finally, we take an in-depth look at active signaling that originates from the organizer and involves germ layer specification and patterning.

Foxd4l1.1 Negatively Regulates Chordin Transcription in Neuroectoderm of Xenopus Gastrula.

Inhibition of the bone morphogenetic proteins (BMPs) is the primary step toward neuroectoderm formation in vertebrates. In this process, the Spemann organizer of the dorsal mesoderm plays a decisive role by secreting several extracellular BMP inhibitors such as Chordin (Chrd). Chrd physically interacts with BMP proteins and inhibits BMP signaling, which triggers the expression of neural-specific transcription factors (TFs), including Foxd4l1.1. Thus, Chrd induces in a BMP-inhibited manner and promotes neuroectoderm formation. However, the regulatory feedback mechanism of Foxd4l1.1 on mesodermal genes expression during germ-layer specification has not been fully elucidated. In this study, we investigated the regulatory mechanism of Foxd4l1.1 on chrd (a mesodermal gene). We demonstrate that Foxd4l1.1 inhibits chrd expression during neuroectoderm formation in two ways: First, Foxd4l1.1 directly binds to FRE (Foxd4l1.1 response elements) within the chrd promoter region to inhibit transcription. Second, Foxd4l1.1 physically interacts with Smad2 and Smad3, and this interaction blocks Smad2 and Smad3 binding to activin response elements (AREs) within the chrd promoter. Site-directed mutagenesis of FRE within the chrd(-2250) promoter completely abolished repressor activity of the Foxd4l1.1. RT-PCR and reporter gene assay results indicate that Foxd4l1.1 strongly inhibits mesoderm- and ectoderm-specific marker genes to maintain neural fate. Altogether, these results suggest that Foxd4l1.1 negatively regulates chrd transcription by dual mechanism. Thus, our study demonstrates the existence of precise reciprocal regulation of chrd transcription during neuroectoderm and mesoderm germ-layer specification in Xenopus embryos.

Functional Roles of FGF Signaling in Early Development of Vertebrate Embryos.

Fibroblast growth factors (FGFs) comprise a large family of growth factors, regulating diverse biological processes including cell proliferation, migration, and differentiation. Each FGF binds to a set of FGF receptors to initiate certain intracellular signaling molecules. Accumulated evidence suggests that in early development and adult state of vertebrates, FGFs also play exclusive and context dependent roles. Although FGFs have been the focus of research for therapeutic approaches in cancer, cardiovascular disease, and metabolic syndrome, in this review, we mainly focused on their role in germ layer specification and axis patterning during early vertebrate embryogenesis. We discussed the functional roles of FGFs and their interacting partners as part of the gene regulatory network for germ layer specification, dorsal-ventral (DV), and anterior-posterior (AP) patterning. Finally, we briefly reviewed the regulatory molecules and pharmacological agents discovered that may allow modulation of FGF signaling in research.

Physiological and photochemical evaluation of pepper methionine sulfoxide reductase B2 (CaMsrB2) expressing transgenic rice in saline habitat.

Two pepper methionine sulfoxide reductase B2 (CaMsrB2) gene expressing transgenic rice lines (L-8 and L-23) were interrogated with respect to their physiological and photochemical attributes along with control (WT, Ilmi) as a standard against varying levels of salt concentration which are 75 mM, 150 mM and 225 mM. Against various levels of salt (NaCl) concentration, recurring detrimental effects of extreme salt stress was observed and more pronounced in the wild type plants as compared to our transgenic lines. As the exacerbated effects of salinity is responsible for pushing the plants to their ecological tolerance, our transgenic lines performed well uplifted in different realms of physiology and photochemistry such as relative water content (RWC = 60-75%), stomatal conductance (gs = 70-190 mmolm-2s-1), performance index (PIABS = 1.0-4.5), maximal photochemical yield of PSII (FV/FM = 0.48-0.72) and chlorophyll content index (CCI = 5-7.2 au) in comparison to the control. Relative gene expression, ion analysis and antioxidants activity were analyzed in all treatments to ensure the hypothesis obtained from data of physiology and photochemistry. Photosynthetic apparatus is known to lose energy in various forms such as NPQ, DIO/CS, damages of reaction center (FV/FO) which are the markers of poor health were clearly decreased in the L-23 line as compared to L-8 and WT. Present study revealed the protruding tolerance of L-23 and L-8 transgenic lines with L-23 line in the lead in comparison to control and L-8 transgenic lines.

The regulatory roles of motile cilia in CSF circulation and hydrocephalus.

BACKGROUND: Cerebrospinal fluid (CSF) is an ultra-filtrated colorless brain fluid that circulates within brain spaces like the ventricular cavities, subarachnoid space, and the spine. Its continuous flow serves many primary functions, including nourishment, brain protection, and waste removal. MAIN BODY: The abnormal accumulation of CSF in brain cavities triggers severe hydrocephalus. Accumulating evidence had indicated that synchronized beats of motile cilia (cilia from multiciliated cells or the ependymal lining in brain ventricles) provide forceful pressure to generate and restrain CSF flow and maintain overall CSF circulation within brain spaces. In humans, the disorders caused by defective primary and/or motile cilia are generally referred to as ciliopathies. The key role of CSF circulation in brain development and its functioning has not been fully elucidated. CONCLUSIONS: In this review, we briefly discuss the underlying role of motile cilia in CSF circulation and hydrocephalus. We have reviewed cilia and ciliated cells in the brain and the existing evidence for the regulatory role of functional cilia in CSF circulation in the brain. We further discuss the findings obtained for defective cilia and their potential involvement in hydrocephalus. Furthermore, this review will reinforce the idea of motile cilia as master regulators of CSF movements, brain development, and neuronal diseases.

Transcriptomic Analysis of Rice Plants Overexpressing PsGAPDH in Response to Salinity Stress.

In plants, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a main enzyme in the glycolytic pathway. It plays an essential role in glycerolipid metabolism and response to various stresses. To examine the function of PsGAPDH (Pleurotus sajor-caju GAPDH) in response to abiotic stress, we generated transgenic rice plants with single-copy/intergenic/homozygous overexpression PsGAPDH (PsGAPDH-OX) and investigated their responses to salinity stress. Seedling growth and germination rates of PsGAPDH-OX were significantly increased under salt stress conditions compared to those of the wild type. To elucidate the role of PsGAPDH-OX in salt stress tolerance of rice, an Illumina HiSeq 2000 platform was used to analyze transcriptome profiles of leaves under salt stress. Analysis results of sequencing data showed that 1124 transcripts were differentially expressed. Using the list of differentially expressed genes (DEGs), functional enrichment analyses of DEGs such as Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed. KEGG pathway enrichment analysis revealed that unigenes exhibiting differential expression were involved in starch and sucrose metabolism. Interestingly, trehalose-6-phosphate synthase (TPS) genes, of which expression was enhanced by abiotic stress, showed a significant difference in PsGAPDH-OX. Findings of this study suggest that PsGAPDH plays a role in the adaptation of rice plants to salt stress.

The Rice GLYCINE-RICH PROTEIN 3 Confers Drought Tolerance by Regulating mRNA Stability of ROS Scavenging-Related Genes.

BACKGROUND: Plant glycine-rich proteins are categorized into several classes based on their protein structures. The glycine-rich RNA binding proteins (GRPs) are members of class IV subfamily possessing N-terminus RNA-recognition motifs (RRMs) and proposed to be involved in post-transcriptional regulation of its target transcripts. GRPs are involved in developmental process and cellular stress responses, but the molecular mechanisms underlying these regulations are still elusive. RESULTS: Here, we report the functional characterization of rice GLYCINE-RICH PROTEIN 3 (OsGRP3) and its physiological roles in drought stress response. Both drought stress and ABA induce the expression of OsGRP3. Transgenic plants overexpressing OsGRP3 (OsGRP3OE) exhibited tolerance while knock-down plants (OsGRP3KD) were susceptible to drought compared to the non-transgenic control. In vivo, subcellular localization analysis revealed that OsGRP3-GFP was transported from cytoplasm/nucleus into cytoplasmic foci following exposure to ABA and mannitol treatments. Comparative transcriptomic analysis between OsGRP3OE and OsGRP3KD plants suggests that OsGRP3 is involved in the regulation of the ROS related genes. RNA-immunoprecipitation analysis revealed the associations of OsGRP3 with PATHOGENESIS RELATED GENE 5 (PR5), METALLOTHIONEIN 1d (MT1d), 4,5-DOPA-DIOXYGENASE (DOPA), and LIPOXYGENASE (LOX) transcripts. The half-life analysis showed that PR5 transcripts decayed slower in OsGRP3OE but faster in OsGRP3KD, while MT1d and LOX transcripts decayed faster in OsGRP3OE but slower in OsGRP3KD plants. H2O2 accumulation was reduced in OsGRP3OE and increased in OsGRP3KD plants compared to non-transgenic plants (NT) under drought stress. CONCLUSION: OsGRP3 plays a positive regulator in rice drought tolerance and modulates the transcript level and mRNA stability of stress-responsive genes, including ROS-related genes. Moreover, OsGRP3 contributes to the reduction of ROS accumulation during drought stress. Our results suggested that OsGRP3 alleviates ROS accumulation by regulating ROS-related genes' mRNA stability under drought stress, which confers drought tolerance.

OsCRP1, a Ribonucleoprotein Gene, Regulates Chloroplast mRNA Stability That Confers Drought and Cold Tolerance.

Chloroplast ribonucleoproteins (cpRNPs) are nuclear-encoded and highly abundant proteins that are proposed to function in chloroplast RNA metabolism. However, the molecular mechanisms underlying the regulation of chloroplast RNAs involved in stress tolerance are poorly understood. Here, we demonstrate that CHLOROPLAST RNA-BINDING PROTEIN 1 (OsCRP1), a rice (Oryza sativa) cpRNP gene, is essential for stabilization of RNAs from the NAD(P)H dehydrogenase (NDH) complex, which in turn enhances drought and cold stress tolerance. An RNA-immunoprecipitation assay revealed that OsCRP1 is associated with a set of chloroplast RNAs. Transcript profiling indicated that the mRNA levels of genes from the NDH complex significantly increased in the OsCRP1 overexpressing compared to non-transgenic plants, whereas the pattern in OsCRP1 RNAi plants were opposite. Importantly, the OsCRP1 overexpressing plants showed a higher cyclic electron transport (CET) activity, which is essential for elevated levels of ATP for photosynthesis. Additionally, overexpression of OsCRP1 resulted in significantly enhanced drought and cold stress tolerance with higher ATP levels compared to wild type. Thus, our findings suggest that overexpression of OsCRP1 stabilizes a set of mRNAs from genes of the NDH complex involved in increasing CET activity and production of ATP, which consequently confers enhanced drought and cold tolerance.

The molecular dynamics of subdistal appendages in multi-ciliated cells.

The motile cilia of ependymal cells coordinate their beats to facilitate a forceful and directed flow of cerebrospinal fluid (CSF). Each cilium originates from a basal body with a basal foot protruding from one side. A uniform alignment of these basal feet is crucial for the coordination of ciliary beating. The process by which the basal foot originates from subdistal appendages of the basal body, however, is unresolved. Here, we show FGFR1 Oncogene Partner (FOP) is a useful marker for delineating the transformation of a circular, unpolarized subdistal appendage into a polarized structure with a basal foot. Ankyrin repeat and SAM domain-containing protein 1A (ANKS1A) interacts with FOP to assemble region I of the basal foot. Importantly, disruption of ANKS1A reduces the size of region I. This produces an unstable basal foot, which disrupts rotational polarity and the coordinated beating of cilia in young adult mice. ANKS1A deficiency also leads to severe degeneration of the basal foot in aged mice and the detachment of cilia from their basal bodies. This role of ANKS1A in the polarization of the basal foot is evolutionarily conserved in vertebrates. Thus, ANKS1A regulates FOP to build and maintain the polarity of subdistal appendages.

Physiological Aspects of Germination and Early Seedling Establishment of Pleurotus sajor-caju Glyceraldehyde-3-Phosphate Dehydrogenase Expressing Transgenic Rice in Saline Environment.

GPD encodes glyceraldehyde-3-phosphate dehydrogenase enzyme involved in sugar mobilization, particularly glycolysis and gluconeogenesis. The objective of this study was to determine physiological aspects of germination and early seedling establishment of PsGPD (Pleurotus sajor-caju glyceraldehyde-3-phosphate dehydrogenase) expressing transgenic rice (T5) against different salt concentrations. The T5 line that carried 2 copies of T-DNA and had the highest level of PsGPD expression was used in the investigation. Final germination percentage, amylase activity, reducing sugar accumulation, and chlorophyll biosynthesis were comparatively higher in PsGPD expressing transgenic rice against elevating saline conditions. A slow-paced conversion of porphyrin's precursors was seen through the matrix model and further elaborated by a graphical model. A sustained level of porphyrin was observed in PsGPD expressing transgenic rice. These data were concurrent with the relative gene expression and thermal imaging (thermography) of PsGPD expressing transgenic rice against salt stress. Morphological attributes also favored the salt tolerance exhibited by PsGPD-transformed rice.