NLK is required for Ras/ERK/SRF/ELK signaling to tune skeletal muscle development by phosphorylating SRF and antagonizing the SRF/MKL pathway

Serum response factor (SRF) regulates differentiation and proliferation by binding to RhoA-actin-activated MKL or Ras-MAPK-activated ELK transcriptional coactivators, but the molecular mechanisms responsible for SRF regulation remain unclear. Here, we show that Nemo-like kinase (NLK) is required for the promotion of SRF/ELK signaling in human and mouse cells. NLK was found to interact with and phosphorylate SRF at serine residues 101/103, which in turn enhanced the association between SRF and ELK. The enhanced affinity of SRF/ELK antagonized the SRF/MKL pathway and inhibited mouse myoblast differentiation in vitro. In a skeletal muscle-specific Nlk conditional knockout mouse model, forming muscle myofibers underwent hypertrophic growth, resulting in an increased muscle and body mass phenotype. We propose that both phosphorylation of SRF by NLK and phosphorylation of ELKs by MAPK are required for RAS/ELK signaling, confirming the importance of this ancient pathway and identifying an important role for NLK in modulating muscle development in vivo.

TALPID3/KIAA0586 Regulates Multiple Aspects of Neuromuscular Patterning During Gastrointestinal Development in Animal Models and Human

TALPID3/KIAA0586 is an evolutionary conserved protein, which plays an essential role in protein trafficking. Its role during gastrointestinal (GI) and enteric nervous system (ENS) development has not been studied previously. Here, we analyzed chicken, mouse and human embryonic GI tissues with TALPID3 mutations. The GI tract of TALPID3 chicken embryos was shortened and malformed. Histologically, the gut smooth muscle was mispatterned and enteric neural crest cells were scattered throughout the gut wall. Analysis of the Hedgehog pathway and gut extracellular matrix provided causative reasons for these defects. Interestingly, chicken intra-species grafting experiments and a conditional knockout mouse model showed that ENS formation did not require TALPID3, but was dependent on correct environmental cues. Surprisingly, the lack of TALPID3 in enteric neural crest cells (ENCC) affected smooth muscle and epithelial development in a non-cell-autonomous manner. Analysis of human gut fetal tissues with a KIAA0586 mutation showed strikingly similar findings compared to the animal models demonstrating conservation of TALPID3 and its necessary role in human GI tract development and patterning.

LncRNA-DRSGN (Down-Regulated in Spiral Ganglion Neurons) Competes with miR-27a for Binding to FAM172A, Supervises SGN Degeneration and ER (Endoplasmic Reticulum) Stress-Autophagy in CHARGE Syndrome

Background: Hearing loss is one of the most common disabilities in the world and brings a heavy burden to society. The current model is not stable enough, and it has caused serious model interference to clarify the pathogenesis of CHARGE syndrome. Methods: The knockout mouse model of FAM172A gene was constructed, and sits phenotype was identified. Besides, the next-genesequencing experiments of noncoding RNAs were performed utilizing the primary SGNs of model mice. The biofunctions of FAM172A in the relationships between ER (Endoplasmic reticulum) stress, autophagy, and intracellular calcium flux were investigated. Moreover, the above role associated with the competitive combination among LncRNA-DRSGN, miR-27a, and FAM172A were studied in the progression of SGN degeneration and autophagy in the model of CHARGE syndrome. Results: FAM172A(-/-) exhibited abnormal hearing, growth retardation, abnormal eye development, and dysgnosia. It was in line with the phenotype of CHARGE syndrome. Moreover, there was degeneration of SGNs in FAM172A(-/-) mice, and the differential expression of noncoding RNAs in primary SGNs were found and identified, including miR-27a and LncRNA-DRSGN. LncRNA-DRSGN regulated miR-27a as a ceRNA, and miR-27a inhibited FAM172A expression, LncRNA-DRSGN competed with miR-27a for binding to FAM172A, which participated in the regulation of ER stress-related calcium flux. LncRNA-DRSGN regulated the autophagy process of neurons by competing with miR-27a for binding to FAM172A. Conclusion: LncRNA-DRSGN competed with miR-27a for binding to FAM172A, participated in regulating ER stress-related calcium flux, then affected neuron degeneration and autophagy process of SGNs in the model of CHARGE syndrome.

Down-regulation of collagen XI during late post-natal corneal development is followed by up-regulation after injury

Collagen XI plays a role in nucleating collagen fibrils and in controlling fibril diameter. The aim of this research is to elucidate the role that collagen XI plays in corneal fibrillogenesis during development and following injury. The temporal and spatial expression of collagen XI was evaluated in C57BL/6 wild type (WT) mice. For wound healing studies in adult mice, stromal injuries were created using techniques that avoid caustic chemicals. The temporal expression and spatial localization of collagen XI was studied following injury in a Col11a1 inducible knockout mouse model. We found that collagen XI expression occurs during early maturation and is upregulated after stromal injury in areas of regeneration and remodeling. Abnormal fibrillogenesis with new fibrils of heterogenous size and shape occurs after injury in a decreased collagen XI matrix. In conclusion, we found that collagen XI is expressed in the stroma during development and following injury in adults. Collagen XI is a regulator of collagen fibrillogenesis in regenerating corneal tissue.

Arhgap21 Deficiency Results in Increase of Osteoblastic Lineage Cells in the Murine Bone Marrow Microenvironment

ARHGAP21 is a member of the RhoGAP family of proteins involved in cell growth, differentiation, and adhesion. We have previously shown that the heterozygous Arhgap21 knockout mouse model (Arhgap21+/−) presents several alterations in the hematopoietic compartment, including increased frequency of hematopoietic stem and progenitor cells (HSPC) with impaired adhesion in vitro, increased mobilization to peripheral blood, and decreased engraftment after bone marrow transplantation. Although these HSPC functions strongly depend on their interactions with the components of the bone marrow (BM) niche, the role of ARHGAP21 in the marrow microenvironment has not yet been explored. In this study, we investigated the composition and function of the BM microenvironment in Arhgap21+/− mice. The BM of Arhgap21+/− mice presented a significant increase in the frequency of phenotypic osteoblastic lineage cells, with no differences in the frequencies of multipotent stromal cells or endothelial cells when compared to the BM of wild type mice. Arhgap21+/− BM cells had increased capacity of generating osteogenic colony-forming units (CFU-OB) in vitro and higher levels of osteocalcin were detected in the Arhgap21+/− BM supernatant. Increased expression of Col1a1, Ocn and decreased expression of Trap1 were observed after osteogenic differentiation of Arhgap21+/− BM cells. In addition, Arhgap21+/− mice recipients of normal BM cells showed decreased leucocyte numbers during transplantation recovery. Our data suggest participation of ARHGAP21 in the balanced composition of the BM microenvironment through the regulation of osteogenic differentiation.

NMD abnormalities during brain development in the Fmr1-knockout mouse model of fragile X syndrome

Background Fragile X syndrome (FXS) is an intellectual disability attributable to loss of fragile X protein (FMRP). We previously demonstrated that FMRP binds mRNAs targeted for nonsense-mediated mRNA decay (NMD) and that FMRP loss results in hyperactivated NMD and inhibition of neuronal differentiation in human stem cells. Results We show here that NMD is hyperactivated during the development of the cerebral cortex, hippocampus, and cerebellum in the Fmr1-knockout (KO) mouse during embryonic and early postnatal periods. Our findings demonstrate that NMD regulates many neuronal mRNAs that are important for mouse brain development. Conclusions We reveal the abnormal regulation of these mRNAs in the Fmr1-KO mouse, a model of FXS, and highlight the importance of early intervention.

Deficiency of MicroRNA-181a Results in Transcriptome-Wide Cell-Specific Changes in the Kidney and Increases Blood Pressure

MicroRNA miR-181a is downregulated in the kidneys of hypertensive patients and hypertensive mice. In vitro, miR-181a is a posttranslational inhibitor of renin expression, but pleiotropic mechanisms by which miR-181a may influence blood pressure (BP) are unknown. Here, we determined whether deletion of miR-181a/b-1 in vivo changes BP and the molecular mechanisms involved at the single-cell level. We developed a KO (knockout) mouse model lacking miR-181a/b-1 genes using CRISPR/Cas9 technology. Radiotelemetry probes were implanted in 12-week-old C57BL/6J WT (wild type) and miR-181a/b-1 KO mice. Systolic and diastolic BP were 4- to 5-mm Hg higher in KO compared with WT mice over 24 hours ( P <0.01). Compared with WT mice, renal renin was higher in the juxtaglomerular cells of KO mice. BP was similar in WT mice on a high- (3.1%) versus low- (0.3%) sodium diet (+0.4±0.8 mm Hg), but KO mice showed salt sensitivity (+3.3±0.8 mm Hg; P <0.001). Since microRNAs can target several mRNAs simultaneously, we performed single-nuclei RNA sequencing in 6699 renal cells. We identified 12 distinct types of renal cells, all of which had genes that were dysregulated. This included genes involved in renal fibrosis and inflammation such as Stat4 , Col4a1 , Cd81 , Flt3l , Cxcl16 , and Smad4 . We observed upregulation of pathways related to the immune system, inflammatory response, reactive oxygen species, and nerve development, consistent with higher tyrosine hydroxylase in the kidney. In conclusion, downregulation of the miR-181a gene led to increased BP and salt sensitivity in mice. This is likely due to an increase in renin expression in juxtaglomerular cells, as well as microRNA-driven pleiotropic effects impacting renal pathways associated with hypertension.