Genome-wide analysis of early vascular tunic repair and regeneration for Botrylloides digenesis reveals striking similarities to human wound healing.

The early stages of regeneration after injury are similar to those of wound healing. The ascidian Botrylloides diegensis can regenerate an entire adult from a small fragment of vascular tunic following the removal of all zooids in an injury-induced regeneration model. We investigated the molecular and cellular changes following injury to determine the differences between the healing process and the initiation of whole-body regeneration (WBR). We conducted transcriptome analysis at specific time points during regeneration and wound healing to identify differentially expressed genes (DEGs) and the unique biological processes associated with each state. Our findings revealed 296 DEGs at 10 h post-injury (hpi), with 71 highly expressed in healed tissue and 225 expressed during the WBR process. These DEGs were predicted to play roles in tissue reorganization, integrin signaling, extracellular matrix organization, and the innate immune system. Pathway analysis of the upregulated genes in the healed tunic indicated functional enrichment related to tissue repair, as has been observed in other species. Additionally, we examined the cell types in the tunic and ampullae in both tissue states using histology and in situ hybridization for six genes identified by transcriptome analysis. We observed strong mRNA expression in cells within the WBR tunic, and in small RNA-positive granules near the tunic edge. We hypothesized that many of these genes function in the compaction of the ampullae tunic, which is a pivotal process for WBR and dormancy in B. diegensis, and in an immune response. These findings establish surprising similarities between ascidian regeneration and human wound healing, emphasizing the potential for future investigations into human regenerative and repair mechanisms. This study provides valuable insights into the gene sets specifically activated during regeneration compared to wound healing, shedding light on the divergent activities of these processes.

Deletion of a conserved genomic region associated with adolescent idiopathic scoliosis leads to vertebral rotation in mice.

Adolescent idiopathic scoliosis (AIS) is the most common form of scoliosis, in which spinal curvature develops in adolescence, and 90% of patients are female. Scoliosis is a debilitating disease that often requires bracing or surgery in severe cases. AIS affects 2%-5.2% of the population; however, the biological origin of the disease remains poorly understood. In this study, we aimed to determine the function of a highly conserved genomic region previously linked to AIS using a mouse model generated by CRISPR-CAS9 gene editing to knockout this area of the genome to understand better its contribution to AIS, which we named AIS_CRMΔ. We also investigated the upstream factors that regulate the activity of this enhancer in vivo, whether the spatial expression of the LBX1 protein would change with the loss of AIS-CRM function, and whether any phenotype would arise after deletion of this region. We found a significant increase in mRNA expression in the developing neural tube at E10.5, and E12.5, for not only Lbx1 but also other neighboring genes. Adult knockout mice showed vertebral rotation and proprioceptive deficits, also observed in human AIS patients. In conclusion, our study sheds light on the elusive biological origins of AIS, by targeting and investigating a highly conserved genomic region linked to AIS in humans. These findings provide valuable insights into the function of the investigated region and contribute to our understanding of the underlying causes of this debilitating disease.

Identification of novel microRNAs in the embryonic mouse brain using deep sequencing.

Many advances in small RNA-seq technology and bioinformatics pipelines have been made recently, permitting the discovery of novel miRNAs in the embryonic day 15.5 (E15.5) mouse brain. We aimed to improve miRNA discovery in this tissue to expand our knowledge of the regulatory networks that underpin normal neurodevelopment, find new candidates for neurodevelopmental disorder aetiology, and deepen our understanding of non-coding RNA evolution. A high-quality small RNA-seq dataset of 458 M reads was generated. An unbiased miRNA discovery pipeline identified fifty putative novel miRNAs, six of which were selected for further validation. A combination of conservation analysis and target functional prediction was used to determine the authenticity of novel miRNA candidates. These findings demonstrate that miRNAs remain to be discovered, particularly if they have the features of other small RNA species.

In vitro effects of wool-derived keratin on human dental pulp-derived stem cells for endodontic applications.

In this study we examine the influence of wool-derived keratin intermediate filament proteins (kIFPs) on human dental pulp-derived stem cells (hDPSCs). kIFPs were diluted (10 mg/mL to 0.001 mg/mL) in cell culture media. Effects on hDPSCs proliferation were measured using Alamar blue assay. Keratin concentrations of 1 mg/mL and 0.1 mg/mL were tested for odontogenic differentiation and mineralisation. Alkaline phosphatase (ALP) quantification (7th, 14th, and 21st days), alizarin red S (AR-S) staining and calcium quantification (21st day), reverse transcription polymerase chain reaction (RT-PCR, collagen expression), and immunocytochemical staining for dentin matrix protein (DMP) were performed. hDPSCs showed higher proliferation with kIFPs of 0.1 mg/mL or less (p < 0.0001). The 0.1 mg/mL keratin concentration promoted odontogenic differentiation, confirmed by increased ALP activity, significant calcium deposits (AR-S staining, p < 0.05), up-regulated collagen expression (RT-PCR, p < 0.05), and positive DMP staining. These results suggest that kIFPs could be a potential biomaterial for pulp-dentin regeneration.

Sex-biased gene and microRNA expression in the developing mouse brain is associated with neurodevelopmental functions and neurological phenotypes.

BACKGROUND: Sex differences pose a challenge and an opportunity in biomedical research. Understanding how sex chromosomes and hormones affect disease-causing mechanisms will shed light on the mechanisms underlying predominantly idiopathic sex-biased neurodevelopmental disorders such as ADHD, schizophrenia, and autism. Gene expression is a crucial conduit for the influence of sex on developmental processes; therefore, this study focused on sex differences in gene expression and the regulation of gene expression. The increasing interest in microRNAs (miRNAs), small, non-coding RNAs, for their contribution to normal and pathological neurodevelopment prompted us to test how miRNA expression differs between the sexes in the developing brain. METHODS: High-throughput sequencing approaches were used to identify transcripts, including miRNAs, that showed significantly different expression between male and female brains on day 15.5 of development (E15.5). RESULTS: Robust sex differences were identified for some genes and miRNAs, confirming the influence of biological sex on RNA. Many miRNAs that exhibit the greatest differences between males and females have established roles in neurodevelopment, implying that sex-biased expression may drive sex differences in developmental processes. In addition to highlighting sex differences for individual miRNAs, gene ontology analysis suggested several broad categories in which sex-biased RNAs might act to establish sex differences in the embryonic mouse brain. Finally, mining publicly available SNP data indicated that some sex-biased miRNAs reside near the genomic regions associated with neurodevelopmental disorders. CONCLUSIONS: Together, these findings reinforce the importance of cataloguing sex differences in molecular biology research and highlight genes, miRNAs, and pathways of interest that may be important for sexual differentiation in the mouse and possibly the human brain.

In biomedical research, understanding the differences between males and females is essential for understanding diseases that affect one sex more than the other. This study focused on gene expression and regulation differences between male and female mouse brains during development. We found that many microRNAs, small molecules that play a role in development were expressed differently between male and female brains. These differences could be important in understanding why males and females develop differently, particularly regarding neurodevelopmental disorders like ADHD, schizophrenia, and autism. We also found that some microRNAs that differed between males and females were located near genes associated with these disorders. Overall, the study highlights the importance of understanding sex differences in molecular biology research and provides insights into potential genes and pathways of interest for further study.

RNA sequencing and expression analysis reveal a role for Lhx9 in the haploinsufficient adult mouse ovary.

Understanding the molecular pathways that underpin ovarian development and function is vital for improving the research approaches to investigating fertility. Despite a significant improvement in our knowledge of molecular activity in the ovary, many questions remain unanswered in the quest to understand factors influencing fertility and ovarian pathologies such as cancer. Here, we present an investigation into the expression and function of the developmental transcription factor LIM Homeobox 9 (LHX9) in the adult mouse ovary. We have characterized Lhx9 expression in several cell types of the mature ovary across follicle stages. To evaluate possible LHX9 function in the adult ovary, we investigated ovarian anatomy and transcription in an Lhx9+/- knockout mouse model displaying subfertility. Despite a lack of gross anatomical differences between genotypes, RNA-sequencing found that 90 differentially expressed genes between Lhx9+/ - and Lhx9+/+ mice. Gene ontology analyses revealed a reduced expression of genes with major roles in ovarian steroidogenesis and an increased expression of genes associated with ovarian cancer. Analysis of the ovarian epithelium revealed Lhx9+/ - mice have a disorganized epithelial phenotype, corresponding to a significant increase in epithelial marker gene expression. These results provide an analysis of Lhx9 in the adult mouse ovary, suggesting a role in fertility and ovarian epithelial cancer.

The CNS-penetrating taxane drug TPI 287 potentiates antiglioma activity of the AURKA inhibitor alisertib in vivo.

INTRODUCTION: Glioblastoma (GBM) has a very poor prognosis despite current treatment. We previously found cytotoxic synergy between the AURKA inhibitor alisertib and the CNS-penetrating taxane TPI 287 against GBM tumor cells in vitro. METHODS: We used an orthotopic human GBM xenograft mouse model to test if TPI 287 potentiates alisertib in vivo. Western blotting, immunohistochemistry, siRNA knockdown, annexin V binding, and 3-dimensional Matrigel invasion assays were used to investigate potential mechanisms of alisertib and TPI 287 treatment interactions. RESULTS: Alisertib + TPI 287 combination therapy significantly prolonged animal survival compared to vehicle (p = 0.011), but only marginally compared to alisertib alone. Alisertib, TPI 287, and combined alisertib + TPI 287 reduced animal tumor volume compared to vehicle-treated controls. This was statistically significant for the combination therapy at 4 weeks (p < 0.0001). Alisertib + TPI 287 treatment decreased anti-apoptotic Bcl-2 protein levels in vivo and in vitro. Expression of the pro-apoptotic protein Bak was significantly increased by combination treatment (p < 0.0001). Pro-apoptotic Bim and Bak knockdown by siRNA decreased apoptosis by alisertib + TPI 287 in GB9, GB30, and U87 cells (p = 0.0005 to 0.0381). Although alisertib and TPI 287 significantly reduced GBM cell invasion (p < 0.0001), their combination was no more effective than TPI 287 alone. CONCLUSIONS: Results suggest that apoptosis is the dominant mechanism of potentiation of GBM growth inhibition by alisertib + TPI 287, in part through effects on Bcl-2 family proteins, providing a rationale for further laboratory testing of an AURKA inhibitor plus TPI 287 as a potential therapy against GBM.

Using RNA-Seq for Transcriptome Profiling of Botrylloides sp. Regeneration.

The decrease in sequencing costs and technology improvements has led to the adoption of RNA-sequencing to profile transcriptomes from further non-traditional regeneration model organisms such as the colonial ascidian Botrylloides leachii. The relatively unbiased way in which transcripts are identified and quantified makes this technique suitable to detect large-scale changes in expression, and the identification of novel transcripts and isoforms. Of particular interest to many researchers is the discovery of differentially expressed transcripts across different treatment conditions or stages of regeneration. This protocol describes a workflow starting from processing raw sequencing reads, mapping reads, assembly of transcripts, and measuring their abundance, creating lists of differentially expressed genes and their biological interpretation using gene ontologies. All programs used in this protocol are open-source software tools and freely available.

Characterization of a novel Lbx1 mouse loss of function strain.

Adolescent Idiopathic Scoliosis (AIS) is the most common type of spine deformity affecting 2-3% of the population worldwide. The etiology of this disease is still poorly understood. Several GWAS studies have identified single nucleotide polymorphisms (SNPs) located near the gene LBX1 that is significantly correlated with AIS risk. LBX1 is a transcription factor with roles in myocyte precursor migration, cardiac neural crest specification, and neuronal fate determination in the neural tube. Here, we further investigated the role of LBX1 in the developing spinal cord of mouse embryos using a CRISPR-generated mouse model expressing a truncated version of LBX1 (Lbx1Δ). Homozygous mice died at birth, likely due to cardiac abnormalities. To further study the neural tube phenotype, we used RNA-sequencing to identify 410 genes differentially expressed between the neural tubes of E12.5 wildtype and Lbx1Δ/Δ embryos. Genes with increased expression in the deletion line were involved in neurogenesis and those with broad roles in embryonic development. Many of these genes have also been associated with scoliotic phenotypes. In comparison, genes with decreased expression were primarily involved in skeletal development. Subsequent skeletal and immunohistochemistry analysis further confirmed these results. This study aids in understanding the significance of links between LBX1 function and AIS susceptibility.

Hierarchical Ordered Assembly of Genetically Modifiable Viruses into Nanoridge-in-Microridge Structures.

Hierarchically assembled nanomaterials can find a variety of applications in medicine, energy, and electronics. Here, an automatically controlled dip-pulling method is developed and optimized to generate an unprecedented ordered nano-to-micro hierarchical nanoridge-in-microridge (NiM) structure from a bacteria-specific human-safe virus, the filamentous phage with or without genetically displaying a foreign peptide. The NiM structure is pictured as a window blind with each lath (the microridge) made of parallel phage bundles (the nanoridges). It is independent of the substrate materials supporting it. Surprisingly, it can induce the bidirectional differentiation of stem cells into neurons and astrocytes within a short timeframe (only 8 d) not seen before, which is highly desired because both neurons and astrocytes are needed simultaneously in treating neurodegenerative diseases. Since phages can direct tissue regeneration, template materials formation, sense molecules, and build electrodes, the NiM structures displaying different peptides and on varying materials hold promise in many technologically important fields.

Age and Sex-Related Changes to Gene Expression in the Mouse Spinal Cord.

The spinal cord is essential for neuronal communication between the brain and rest of the body. To gain further insight into the molecular changes underpinning maturation of the mouse spinal cord, we analysed gene expression differences between 4 weeks of age (prior to puberty onset) and adulthood (8 weeks). We found 800 genes were significantly differentially expressed between juvenile and adult spinal cords. Gene ontology analysis revealed an overrepresentation of genes with roles in myelination and signal transduction among others. The expression of a further 19 genes was sexually dimorphic; these included both autosomal and sex-linked genes. Given the presence of steroid hormone receptors in the spinal cord, we also looked at the impact of two major steroid hormones, oestradiol and dihydrotestosterone (DHT) on spinal cord gene expression for selected genes. In gonadectomised male animals, implants with oestradiol and DHT produced significant changes to spinal cord gene expression. This study provides an overview of the global gene expression changes that occur as the spinal cord matures, over a key period of maturation. This confirms that both age and sex are important considerations in studies involving the spinal cord.

Histone deacetylase activity is required for Botrylloides leachii whole-body regeneration.

The colonial tunicate Botrylloides leachii is exceptional at regenerating from a piece of vascular tunic after loss of all adults from the colony. Previous transcriptome analyses indicate a brief period of healing before regeneration of a new adult (zooid) in as little as 8-10 days. However, there is little understanding of how the resulting changes to gene expression, required to drive regeneration, are initiated and how the overall process is regulated. Rapid changes to transcription often occur in response to chromatin changes, mediated by histone modifications such as histone acetylation. Here, we investigated a group of key epigenetic modifiers, histone deacetylases (HDAC), which are known to play an important role in many biological processes such as development, healing and regeneration. Through our transcriptome data, we identified and quantified the expression levels of HDAC and histone acetyltransferase enzymes during whole-body regeneration (WBR). To determine whether HDAC activity is required for WBR, we inhibited its action using valproic acid and trichostatin A. HDAC inhibition prevented the final morphological changes normally associated with WBR and resulted in aberrant gene expression. Botrylloides leachii genes including Slit2, TGF-ß, Piwi and Fzd4 all showed altered mRNA levels upon HDAC inhibition in comparison with the control samples. Additionally, atypical expression of Bl_Piwi was found in immunocytes upon HDAC inhibition. Together, these results show that HDAC function, specifically HDAC I/IIa class enzymes, are vital for B. leachii to undergo WBR successfully.

Whole-Body Regeneration in the Colonial Tunicate Botrylloides leachii.

The colonial marine invertebrate Botrylloides leachii belongs to the Tunicata subphylum, the closest invertebrate relatives to the vertebrate group and the only known class of chordates that can undergo whole-body regeneration (WBR). This dramatic developmental process allows a minute isolated fragment of B. leachii's vascular system, or a colony excised of all adults, to restore a functional animal in as little as 10 days. In addition to this exceptional regenerative capacity, B. leachii can reproduce both sexually, through a tadpole larval stage, and asexually, through palleal budding. Thus, three alternative developmental strategies lead to the establishment of filter-feeding adults. Consequently, B. leachii is particularly well suited for comparative studies on regeneration and should provide novel insights into regenerative processes in chordates.Here, after a short introduction on regeneration, we overview the biology of B. leachii as well as the current state of knowledge on WBR in this species and in related species of tunicates. Finally, we highlight the possible future directions that research might take in the study of WBR, including thoughts on technological approaches that appear most promising in this context. Overall, we provide a synthesis of the current knowledge on WBR in B. leachii to support research in this chordate species.

De novo draft assembly of the Botrylloides leachii genome provides further insight into tunicate evolution.

Tunicates are marine invertebrates that compose the closest phylogenetic group to the vertebrates. These chordates present a particularly diverse range of regenerative abilities and life-history strategies. Consequently, tunicates provide an extraordinary perspective into the emergence and diversity of these traits. Here we describe the genome sequencing, annotation and analysis of the Stolidobranchian Botrylloides leachii. We have produced a high-quality 159 Mb assembly, 82% of the predicted 194 Mb genome. Analysing genome size, gene number, repetitive elements, orthologs clustering and gene ontology terms show that B. leachii has a genomic architecture similar to that of most solitary tunicates, while other recently sequenced colonial ascidians have undergone genome expansion. In addition, ortholog clustering has identified groups of candidate genes for the study of colonialism and whole-body regeneration. By analysing the structure and composition of conserved gene linkages, we observed examples of cluster breaks and gene dispersions, suggesting that several lineage-specific genome rearrangements occurred during tunicate evolution. We also found lineage-specific gene gain and loss within conserved cell-signalling pathways. Such examples of genetic changes within conserved cell-signalling pathways commonly associated with regeneration and development that may underlie some of the diverse regenerative abilities observed in tunicates. Overall, these results provide a novel resource for the study of tunicates and of colonial ascidians.

Hematological Analysis of the Ascidian Botrylloides leachii (Savigny, 1816) During Whole-Body Regeneration.

Whole-body regeneration (WBR)-the formation of an entire adult from only a small fragment of its own tissue-is extremely rare among chordates. Exceptionally, in the colonial ascidian Botrylloides leachii (Savigny, 1816) a fully functional adult is formed from their common vascular system after ablation of all adults from the colony in just 10 d, thanks to their high blastogenetic potential. While previous studies have identified key genetic markers and morphological changes, no study has yet focused on the hematological aspects of regeneration despite the major involvement of the remaining vascular system and the contained hemocytes in this process. To dissect this process, we analyzed colony blood flow patterns using time-lapse microscopy to obtain a quantitative description of the velocity, reversal pattern, and average distance traveled by hemocytes. We also observed that flows present during regeneration are powered by temporally and spatially synchronized contractions of the terminal ampullae. In addition, we revised previous studies of B. leachii hematology as well as asexual development using histological sectioning and compared the role played by hemocytes during WBR. We found that regeneration starts with a rapid healing response characterized by hemocyte aggregation and infiltration of immunocytes, followed by increased activity of hemoblasts, recruitment of macrophage-like cells for clearing the tissues of debris, and their subsequent disappearance from the circulation concomitant with the maturation of a single regenerated adult. Overall, we provide a detailed account of the hematological properties of regenerating B. leachii colonies, providing novel lines of inquiry toward the decipherment of regeneration in chordates.

Selection and evaluation of reference genes for analysis of mouse (Mus musculus) sex-dimorphic brain development.

The development of the brain is sex-dimorphic, and as a result so are many neurological disorders. One approach for studying sex-dimorphic brain development is to measure gene expression in biological samples using RT-qPCR. However, the accuracy and consistency of this technique relies on the reference gene(s) selected. We analyzed the expression of ten reference genes in male and female samples over three stages of brain development, using popular algorithms NormFinder, GeNorm and Bestkeeper. The top ranked reference genes at each time point were further used to quantify gene expression of three sex-dimorphic genes (Wnt10b, Xist and CYP7B1). When comparing gene expression between the sexes expression at specific time points the best reference gene combinations are: Sdha/Pgk1 at E11.5, RpL38/Sdha E12.5, and Actb/RpL37 at E15.5. When studying expression across time, the ideal reference gene(s) differs with sex. For XY samples a combination of Actb/Sdha. In contrast, when studying gene expression across developmental stage with XX samples, Sdha/Gapdh were the top reference genes. Our results identify the best combination of two reference genes when studying male and female brain development, and emphasize the importance of selecting the correct reference genes for comparisons between developmental stages.

Uncovering the pathways underlying whole body regeneration in a chordate model, Botrylloides leachi using de novo transcriptome analysis.

BACKGROUND: Regenerative capacity differs greatly between animals. In vertebrates regenerative abilities are highly limited and tissue or organ specific. However the closest related chordate to the vertebrate clade, Botrylloides leachi, can undergo whole body regeneration (WBR). Therefore, research on WBR in B. leachi has focused on pathways known to be important for regeneration in vertebrates. To obtain a comprehensive vision of this unique process we have carried out the first de novo transcriptome sequencing for multiple stages of WBR occurring in B. leachi. The identified changes in gene expression during B. leachi WBR offer novel insights into this remarkable ability to regenerate. RESULTS: The transcriptome of B. leachi tissue undergoing WBR were analysed using differential gene expression, gene ontology and pathway analyses. We observed up-regulation in the expression of genes involved in wound healing and known developmental pathways including WNT, TGF-ß and Notch, during the earliest stages of WBR. Later in WBR, the expression patterns in several pathways required for protein synthesis, biogenesis and the organisation of cellular components were up-regulated. CONCLUSIONS: While the genes expressed early on are characteristic of a necessary wound healing response to an otherwise lethal injury, the subsequent vast increase in protein synthesis conceivably sustains the reestablishment of the tissue complexity and body axis polarity within the regenerating zooid. We have, for the first time, provided a global overview of the genes and their corresponding pathways that are modulated during WBR in B. leachi.

Evolution of the Sox gene family within the chordate phylum.

The ancient Sox gene family is a group of related transcription factors that perform a number of essential functions during embryonic development. During evolution, this family has undergone considerable expansion, particularly within the vertebrate lineage. In vertebrates SOX proteins are required for the specification, development and/or morphogenesis of most vertebrate innovations. Tunicates and lancelets are evolutionarily positioned as the closest invertebrate relatives to the vertebrate group. By identifying their Sox gene complement we can begin to reconstruct the gene set of the last common chordate ancestor before the split into invertebrates and vertebrate groups. We have identified core SOX family members from the genomes of six invertebrate chordates. Using phylogenetic analysis we determined their evolutionary relationships. We propose that the last common ancestor of chordates had at least seven Sox genes, including the core suite of SoxB, C, D, E and F as well as SoxH.