Probiotics Enhance Bone Growth and Rescue BMP Inhibition: New Transgenic Zebrafish Lines to Study Bone Health

Zebrafish larvae, especially gene-specific mutants and transgenic lines, are increasingly used to study vertebrate skeletal development and human pathologies such as osteoporosis, osteopetrosis and osteoarthritis. Probiotics have been recognized in recent years as a prophylactic treatment for various bone health issues in humans. Here, we present two new zebrafish transgenic lines containing the coding sequences for fluorescent proteins inserted into the endogenous genes for sp7 and col10a1a with larvae displaying fluorescence in developing osteoblasts and the bone extracellular matrix (mineralized or non-mineralized), respectively. Furthermore, we use these transgenic lines to show that exposure to two different probiotics, Bacillus subtilis and Lactococcus lactis, leads to an increase in osteoblast formation and bone matrix growth and mineralization. Gene expression analysis revealed the effect of the probiotics, particularly Bacillus subtilis, in modulating several skeletal development genes, such as runx2, sp7, spp1 and col10a1a, further supporting their ability to improve bone health. Bacillus subtilis was the more potent probiotic able to significantly reverse the inhibition of bone matrix formation when larvae were exposed to a BMP inhibitor (LDN212854)

Empirical Evaluation of Deep Learning Approaches for Landmark Detection in Fish Bioimages

In this paper we perform an empirical evaluation of variants of deep learning methods to automatically localize anatomical landmarks in bioimages of fishes acquired using different imaging modalities (microscopy and radiography). We compare two methodologies namely heatmap based regression and multivariate direct regression, and evaluate them in combination with several Convolutional Neural Network (CNN) architectures. Heatmap based regression approaches employ Gaussian or Exponential heatmap generation functions combined with CNNs to output the heatmaps corresponding to landmark locations whereas direct regression approaches output directly the (x, y) coordinates corresponding to landmark locations. In our experiments, we use two microscopy datasets of Zebrafish and Medaka fish and one radiography dataset of gilthead Seabream. On our three datasets, the heatmap approach with Exponential function and U-Net architecture performs better. Datasets and open-source code for training and prediction are made available to ease future landmark detection research and bioimaging applications.2. Zero hunger3. Good health and well-being9. Industry, innovation and infrastructur

WNT11, a new gene associated with early-onset osteoporosis, is required for osteoblastogenesis.

Monogenic early-onset osteoporosis (EOOP) is a rare disease defined by low bone mineral density (BMD) that results in increased risk of fracture in children and young adults. Although several causative genes have been identified, some of the EOOP causation remains unresolved. Whole-exome sequencing revealed a de novo heterozygous loss-of-function mutation in WNT11 (NM_004626.2:c.677_678dup p.Leu227Glyfs*22) in a 4-year-old boy with low BMD and fractures. We identified two heterozygous WNT11 missense variants (NM_004626.2:c.217G > A p.Ala73Thr) and (NM_004626.2:c.865G > A p.Val289Met) in a 51-year-old woman and in a 61-year-old woman respectively, both with bone fragility. U2OS cells with heterozygous WNT11 mutation (NM_004626.2:c.690_721delfs*40) generated by CRISPR-Cas9 showed reduced cell proliferation (30%) and osteoblast differentiation (80%) as compared with wild-type U2OS cells. The expression of genes in the Wnt canonical and non-canonical pathways was inhibited in these mutant cells, but recombinant WNT11 treatment rescued the expression of Wnt pathway target genes. Furthermore, the expression of RSPO2, a WNT11 target involved in bone cell differentiation, and its receptor LGR5, was decreased in WNT11 mutant cells. Treatment with WNT5A and WNT11 recombinant proteins reversed LGR5 expression, but WNT3A recombinant protein treatment had no effect on LGR5 expression in mutant cells. Moreover, treatment with recombinant RSPO2 but not WNT11 or WNT3A activated the canonical pathway in mutant cells. In conclusion, we have identified WNT11 as a new gene responsible for EOOP, with loss-of-function variant inhibiting bone formation via Wnt canonical and non-canonical pathways. WNT11 may activate Wnt signaling by inducing the RSPO2-LGR5 complex via the non-canonical Wnt pathway

A study of serum uric acid and atrial fibrillation in hypertensive patients

Background: The aim is to study the correlation of serum uric acid and atrial fibrillation in hypertensive individuals and the effect of duration of hypertension on atrial fibrillation & serum uric acid (SUA). Materials and Methods: Patients (age between 35-65years) were selected from outpatient OPD & IPD. A control group of 100 non hypertensive individuals and another group of 100 hypertensive patients were enrolled. Serum uric acid, Echocardiography: A Trans Thoracic Echocardiography (TTE) measurement of Left atrium diameter (LVST), interventricular septal thickness (LVPWT), posterior wall thickness, left ventricular end diastolic diameter (LV) and LV ejection fraction (LVEF) was recorded. Results: Hyperuricemia incidence in controls was 11% and hyperuricemia incidence in cases was 65 %. The incidence of hyperuricemia in cases with phase 1 of hypertension was 6.27±1.22 mg/dl and those with phase 2 of hypertension was 7.59±1mg/dl which was significant. Atrial fibrillation incidence was 4% in the hypertensive patients and the atrial fibrillation incidence in normotensive patients was 1%. Conclusion: Hypertension duration had a significant effect on the SUA levels and revealed that there was noteworthy increase in the SUA level in individuals with atrial fibrillation than those without atrial fibrillation

A reporter zebrafish line for live fluorescent visualisation of bone extracellular matrix

Using the CRISP/Cas9 technology, we targeted the zebrafish col10a1a gene to insert the Gfp coding sequence into its genome, generating a transgenic zebrafish line expressing a secreted Gfp fusion protein in the developing skeleton that reveals the bone extracellular matrix.BIOMEDAQ

Isolation of Osteoblast RNA from Danio rerio larvae – presenting a novel method or an update to the existing one !

The arrangement and distribution of cartilages, dermal bones, and muscles that organize the musculoskeletal system takes place during the embryonic development. Bones are of great significance for the human body, which is known to provide skeletal support, form, strength, movement to the body, it serves as a home for hematopoiesis, reservoiring calcium and other important minerals. While intensely investigated in mammals, zebrafish or Danio rerio has been somewhat a late entrant as a model organism in the area of bone research. In Developmental biology, zebrafish, along with another small teleost species medaka, have long been used as model organisms. Their popularity results from the rapid external development of their larvae, their amenability to genetic manipulation and also, importantly, to the translucency of the larvae, which allows detailed observation of organogenesis in the living fish. The craniofacial skeleton of zebrafish is of comparable complexity to that of terrestrial vertebrates, and is known to contain bones of both dermal and chondral origins, which form from the neural crest-derived cells relatively early in the course of development. Also, of crucial importance is that both the key regulators of skeletal development and the control of the major signaling pathways are known to be highly conserved between mammals and teleosts. Therefore, findings in fish are likely to have an application in mammalian osteogenesis or bone development studies. Bone Morphogenic Proteins (BMPs) have been widely studied for their important role in many developmental and physiological processes, including skeleton formation and homeostasis. Former studies in zebrafish have highlighted the crucial role of BMP signaling before 48 h post-fertilization (hpf) for cartilage formation in the skull, while further results from our lab revealed that for the onset of bone formation, proper BMP signaling between 48 and 72 hpf is crucial to ensure osteoblast function and ossification. Transgenic lines such as the Tg(osterix:mCherry) allow not only to observe bone-forming cells (osteoblasts) in vivo with live imaging, along with several staining procedure, we are now interested in isolating RNA from osteoblasts at 5 dpf from zebrafish using FACS and look at the transcriptome of these cells in the presence or absence of BMP inhibitors. In this study we show new method to isolate mRNA from these bone forming cells.BIOMEDAQ

Osteoblast populations and bone extracellular matrix proteins during skeletal development in zebrafish.

peer reviewedUnderstanding osteoblast differentiation and their function in bone matrix deposition and mineralization is central to the comprehension of bone development and of various bone pathologies. It is crucial to not only follow the expression of some landmark transcription factors or ECM proteins, but to also investigate the status of signaling pathways and factors regulating these processes. This can only be achieved by assessing the entire transcriptome of these cells. We employed developing Tg(sp7:gfp-cr) zebrafish larvae to study the osteoblast transcriptome by isolating the entire osteoblast population at 4dpf, focusing on the possible existence of specific subpopulations. RNA-seq. Analysis revealed two populations at this stage and the differentially expressed gene list that showed genes related to bone and cartilage development. Further, GO term analysis gave us hits very specific to bone extracellular matrix, skeletal development, cartilage and bone specific genes, transcription factors and Calcium regulating hormones etc. to name a few. The expression and function of specific genes of bone extracellular matrix, revealed by these analyses, were investigated using mutant lines and state of the art technologies. Thereafter, characterized for bone and cartilage staining’s at various stages of development. Taken together, these different approaches should allow us to better understand the function of the different genes and the regulatory circuits that are involved in skeletal differentiation, morphogenesis and mineralization, specifically in a non-mammalian vertebrate, but potentially also in humans

wnt11f2 Zebrafish, an Animal Model for Development and New Insights in Bone Formation.

peer reviewedWnt signaling is a key regulator of osteoblast differentiation and mineralization in humans and animals, mediated by the canonical Wnt/β-catenin and noncanonical signaling pathways. Both pathways are crucial in regulating osteoblastogenesis and bone formation. The zebrafish silberblick (slb) carries a mutation in wnt11f2, a gene that contributes to embryonic morphogenesis; however, its role in bone morphology is unknown. wnt11f2 was originally known as wnt11; it was recently reclassified to avoid confusion in comparative genetics and disease modeling. The goal of this review is to summarize the characterization of the wnt11f2 zebrafish mutant and to deliver some new insights concerning its role in skeletal development. In addition to the previously described defects in early development in this mutant as well as craniofacial dysmorphia, we show an increase in tissue mineral density in the heterozygous mutant that points to a possible role of wnt11f2 in high bone mass phenotypes

3D biomimetic platform reveals the first interactions of the embryo and the maternal blood vessels.

peer reviewedThe process of implantation and the cellular interactions at the embryo-maternal interface are intrinsically difficult to analyze, as the implanting embryo is concealed by the uterine tissues. Therefore, the mechanisms mediating the interconnection of the embryo and the mother are poorly understood. Here, we established a 3D biomimetic culture environment that harbors the key features of the murine implantation niche. This culture system enabled direct analysis of trophoblast invasion and revealed the first embryonic interactions with the maternal vasculature. We found that implantation is mediated by the collective migration of penetrating strands of trophoblast giant cells, which acquire the expression of vascular receptors, ligands, and adhesion molecules, assembling a network for communication with the maternal blood vessels. In particular, Pdgf signaling cues promote the establishment of the heterologous contacts. Together, the biomimetic platform and our findings thereof elucidate the hidden dynamics of the early interactions at the implantation site

Zebrafish Osteoblast Transcriptome reveals genes that encode bone extra cellular matrix proteins which on mutating affect bone development

peer reviewedUnderstanding osteoblast differentiation and their function in bone matrix deposition and mineralization is central to the comprehension of bone development and of various bone pathologies. It is, therefore, crucial to not only follow the expression of some landmark transcription factors or ECM proteins but to also investigate the status of signaling pathways and factors regulating these processes. This can only be achieved by assessing the entire transcriptome of these cells. The aim is to study the osteoblast transcriptome, focusing on the possible existence of specific subpopulations at 4dpf and to investigate the role of the fbln1 and col10a1a genes that encode bone ECM proteins in zebrafish skeletal development indicated in the osteoblast transcriptomic data