Core planar cell polarity genes VANGL1 and VANGL2 in predisposition to congenital vertebral malformations.

Congenital scoliosis (CS), affecting approximately 0.5 to 1 in 1,000 live births, is commonly caused by congenital vertebral malformations (CVMs) arising from aberrant somitogenesis or somite differentiation. While Wnt/ß-catenin signaling has been implicated in somite development, the function of Wnt/planar cell polarity (Wnt/PCP) signaling in this process remains unclear. Here, we investigated the role of Vangl1 and Vangl2 in vertebral development and found that their deletion causes vertebral anomalies resembling human CVMs. Analysis of exome sequencing data from multiethnic CS patients revealed a number of rare and deleterious variants in VANGL1 and VANGL2, many of which exhibited loss-of-function and dominant-negative effects. Zebrafish models confirmed the pathogenicity of these variants. Furthermore, we found that Vangl1 knock-in (p.R258H) mice exhibited vertebral malformations in a Vangl gene dose- and environment-dependent manner. Our findings highlight critical roles for PCP signaling in vertebral development and predisposition to CVMs in CS patients, providing insights into the molecular mechanisms underlying this disorder.

Orchestration of tissue shape changes and gene expression patterns in development.

In development, tissue shape changes and gene expression patterns give rise to morphogenesis. Understanding tissue shape changes requires the analysis of mechanical properties of the tissue such as tissue rigidity, cell influx from neighboring tissues, cell shape changes and cell proliferation. Local and global gene expression patterns can be influenced by neighbor exchange and tissue shape changes. Here we review recent studies on the mechanisms for tissue elongation and its influences on dynamic gene expression patterns by focusing on vertebrate somitogenesis. We first introduce mechanical and biochemical properties of the segmenting tissue that drive tissue elongation. Then, we discuss patterning in the presence of cell mixing, scaling of signaling gradients, and dynamic phase waves of rhythmic gene expression under tissue shape changes. We also highlight the importance of theoretical approaches to address the relation between tissue shape changes and patterning.

Jagged 2b induces intercellular signaling within somites to establish hematopoietic stem cell fate in zebrafish.

During development, the somites play a key role in the specification of hematopoietic stem cells (HSCs). In zebrafish, the somitic Notch ligands Delta-c (Dlc) and Dld, both of which are regulated by Wnt16, directly instruct HSC fate in a shared vascular precursor. However, it remains unclear how this signaling cascade is spatially and temporally regulated within somites. Here, we show in zebrafish that an additional somitic Notch ligand, Jagged 2b (Jag2b), induces intercellular signaling to drive wnt16 expression. Jag2b activated Notch signaling in segmented somites at the early stage of somitogenesis. Loss of jag2b led to a reduction in the expression of wnt16 in the somites and an HSC marker, runx1, in the dorsal aorta, whereas overexpression of jag2b increased both. However, Notch-activated cells were adjacent to, but did not overlap with, wnt16-expressing cells within the somites, suggesting that an additional signaling molecule mediates this intercellular signal transduction. We uncover that Jag2b-driven Notch signaling induces efna1b expression, which regulates wnt16 expression in neighboring somitic cells. Collectively, we provide evidence for previously unidentified spatiotemporal regulatory mechanisms of HSC specification by somites.

A dual involvement of Protocadherin 18a in stromal cell development guides the formation of a functional hematopoietic niche.

Hematopoietic stem and progenitor cells emerge from the aorta and migrate to the caudal hematopoietic tissue (CHT) of zebrafish larvae, the hematopoietic equivalent of the mammalian fetal liver, for their proliferation and differentiation. We previously reported that somite-derived stromal cells were a key component of the CHT niche. Here, we found that the cell adhesion protein Protocadherin 18a (Pcdh18a) is expressed in the stromal cell progenitors (SCPs) emigrating from somites toward the future CHT. Deletion of most of the Pcdh18a intracellular domain caused a decrease in the number of SCPs, the directionality of their migration, and the cell-contact mediated repulsion that normally occurs between migrating SCPs. These defects were followed by abnormal morphogenesis of the venous plexus that forms the CHT framework, and the inability of the CHT to function as a niche for hematopoietic stem and progenitor cells. Finally, we found that the extracellular domain of Pcdh18a mediates trans heterophilic adhesion of stromal cells to endothelial cells in vivo and thereby the reticular versus perivascular fate of SCPs. Thus, Pcdh18a expression in SCPs is essential for the proper development of the hematopoietic niche.

Somite development and regionalisation of the vertebral axial skeleton.

A critical stage in the development of all vertebrate embryos is the generation of the body plan and its subsequent patterning and regionalisation along the main anterior-posterior axis. This includes the formation of the vertebral axial skeleton. Its organisation begins during early embryonic development with the periodic formation of paired blocks of mesoderm tissue called somites. Here, we review axial patterning of somites, with a focus on studies using amniote model systems - avian and mouse. We summarise the molecular and cellular mechanisms that generate paraxial mesoderm and review how the different anatomical regions of the vertebral column acquire their specific identity and thus shape the body plan. We also discuss the generation of organoids and embryo-like structures from embryonic stem cells, which provide insights regarding axis formation and promise to be useful for disease modelling.

An emerging role for tissue plasticity in developmental precision.

Reproducible tissue morphology is a fundamental feature of embryonic development. To ensure such robustness during tissue morphogenesis, inherent noise in biological processes must be buffered. While redundant genes, parallel signaling pathways and intricate network topologies are known to reduce noise, over the last few years, mechanical properties of tissues have been shown to play a vital role. Here, taking the example of somite shape changes, I will discuss how tissues are highly plastic in their ability to change shapes leading to increased precision and reproducibility.

Transcription factor support for the dual embryological origin of the sternocleidomastoid and trapezius muscles.

The embryological origin of the trapezius and sternocleidomastoid muscles has been debated for over a century. To shed light on this issue, the present anatomical study was performed. Five fresh frozen human cadavers, three males and two females, were used for this study. Samples from each specimen's trapezius and sternocleidomastoid were fixed in 10% formalin and placed in paraffin blocks. As Paired like homeodomain 2 (Pitx2) and T-box factor 1(Tbx1) have been implicated in the region and muscle type regulation, we performed Tbx1 and Pitx2 Immunohistochemistry (IHC) on these muscle tissue samples to identify the origin of the trapezius and sternocleidomastoid muscles. We have used the latest version of QuPath, v0.4.3, software to quantify the Tbx and Pitx2 staining. For the sternocleidomastoid muscle, for evaluated samples, the average amount of positively stained Tbx1 and Pitx2 was 25% (range 16%-30%) and 18% (range 12%-23%), respectively. For the trapezius muscles, for evaluated samples, the average amount of positively stained Tbx1 and Pitx2 parts of the samples was 17% (range 15%-20%) and 15% (14%-17%), respectively. Our anatomical findings suggest dual origins of both the trapezius and sternocleidomastoid muscles. Additionally, as neither Pitx2 nor Tbx1 made up all the staining observed for each muscle, other contributions to these structures are likely. Future studies with larger samples are now necessary to confirm these findings.

Oscillatory control of Delta-like1 in cell interactions regulates dynamic gene expression and tissue morphogenesis.

Notch signaling regulates tissue morphogenesis through cell-cell interactions. The Notch effectors Hes1 and Hes7 are expressed in an oscillatory manner and regulate developmental processes such as neurogenesis and somitogenesis, respectively. Expression of the mRNA for the mouse Notch ligand Delta-like1 (Dll1) is also oscillatory. However, the dynamics of Dll1 protein expression are controversial, and their functional significance is unknown. Here, we developed a live-imaging system and found that Dll1 protein expression oscillated in neural progenitors and presomitic mesoderm cells. Notably, when Dll1 expression was accelerated or delayed by shortening or elongating the Dll1 gene, Dll1 oscillations became severely dampened or quenched at intermediate levels, as modeled mathematically. Under this condition, Hes1 and Hes7 oscillations were also dampened. In the presomitic mesoderm, steady Dll1 expression led to severe fusion of somites and their derivatives, such as vertebrae and ribs. In the developing brain, steady Dll1 expression inhibited proliferation of neural progenitors and accelerated neurogenesis, whereas optogenetic induction of Dll1 oscillation efficiently maintained neural progenitors. These results indicate that the appropriate timing of Dll1 expression is critical for the oscillatory networks and suggest the functional significance of oscillatory cell-cell interactions in tissue morphogenesis.

A Spatio-Temporal-Dependent Requirement of Sonic Hedgehog in the Early Development of Sclerotome-Derived Vertebrae and Ribs.

Derived from axial structures, Sonic Hedgehog (Shh) is secreted into the paraxial mesoderm, where it plays crucial roles in sclerotome induction and myotome differentiation. Through conditional loss-of-function in quail embryos, we investigate the timing and impact of Shh activity during early formation of sclerotome-derived vertebrae and ribs, and of lateral mesoderm-derived sternum. To this end, Hedgehog interacting protein (Hhip) was electroporated at various times between days 2 and 5. While the vertebral body and rib primordium showed consistent size reduction, rib expansion into the somatopleura remained unaffected, and the sternal bud developed normally. Additionally, we compared these effects with those of locally inhibiting BMP activity. Transfection of Noggin in the lateral mesoderm hindered sternal bud formation. Unlike Hhip, BMP inhibition via Noggin or Smad6 induced myogenic differentiation of the lateral dermomyotome lip, while impeding the growth of the myotome/rib complex into the somatic mesoderm, thus affirming the role of the lateral dermomyotome epithelium in rib guidance. Overall, these findings underscore the continuous requirement for opposing gradients of Shh and BMP activity in the morphogenesis of proximal and distal flank skeletal structures, respectively. Future research should address the implications of these early interactions to the later morphogenesis and function of the musculo-skeletal system and of possible associated malformations.

Neural tube development depends on notochord-derived sonic hedgehog released into the sclerotome.

Sonic hedgehog (Shh), produced in the notochord and floor plate, is necessary for both neural and mesodermal development. To reach the myotome, Shh has to traverse the sclerotome and a reduction of sclerotomal Shh affects myotome differentiation. By investigating loss and gain of Shh function, and floor-plate deletions, we report that sclerotomal Shh is also necessary for neural tube development. Reducing the amount of Shh in the sclerotome using a membrane-tethered hedgehog-interacting protein or Patched1, but not dominant active Patched, decreased the number of Olig2+ motoneuron progenitors and Hb9+ motoneurons without a significant effect on cell survival or proliferation. These effects were a specific and direct consequence of Shh reduction in the mesoderm. In addition, grafting notochords in a basal but not apical location, vis-à-vis the tube, profoundly affected motoneuron development, suggesting that initial ligand presentation occurs at the basal side of epithelia corresponding to the sclerotome-neural tube interface. Collectively, our results reveal that the sclerotome is a potential site of a Shh gradient that coordinates the development of mesodermal and neural progenitors.

2,4-di-tert-butylphenol exposure impairs osteogenic differentiation.

2,4-di-tert-butylphenol (2,4-DTBP) is a synthetic antioxidant used in polyethylene crosspolymer (PEX) water distribution pipes and food-related plastics. 2,4-DTBP can leach from plastic materials and has been found in breast milk, cord blood, and placental tissue, giving rise to the concern that this compound may interfere with fetal development. The objective of this study is to assess the impacts of 2,4-DTBP on cellular differentiation. Human induced pluripotent stem (HiPS) cells were differentiated into osteoblasts or myoblasts over 40 days, and analyzed for markers of somite, dermomyotome, sclerotome, myoblast, and osteoblast development. When cultured as stem cells, 2,4-DTBP did not alter cell viability and expression of markers (NANOG, OCT4). However, upon differentiation into somite-like cells, 2,4-DTBP had reduced levels of MEOX1 and TBX6 transcripts, while NANOG and OCT4 were in turn upregulated in a dose-dependent manner. At the sclerotome-like stage, PAX9 mRNA decreased by 2-fold in the 0.5 µM and 1.0 µM 2,4-DTBP exposure groups. After 40 days of differentiation into an osteoblast-like lineage, exposure to 2,4-DTBP significantly reduced expression of the osteogenesis transcripts RUNX2 and OSX in a dose-dependent manner. Further, Alizarin Red staining of calcium deposits was decreased in the 0.5 µM and 1.0 µM treatment groups. In contrast, myogenesis was not affected by 2,4-DTBP exposure. Interestingly, KEAP1 expression was significantly increased in the sclerotomal-like cells, but decreased in the dermomytomal-like cells, which may suggest a mechanism of action. Overall, this study shows that 2,4-DTBP can delay key processes during sclerotome and osteoblast development, leading to a potential for bone developmental issues in exposed individuals.

Downregulation of Zebrafish Cytosolic Sialidase Neu3.2 Affects Skeletal Muscle Development.

Sialidases remove terminal sialic acids residues from the non-reducing ends of glycoconjugates. They have been recognized as catabolic enzymes that work within different subcellular compartments and can ensure the proper turn-over of glycoconjugates. Four mammalian sialidases (NEU1-4) exist, with different subcellular localization, pH optimum and substrate specificity. In zebrafish, seven different sialidases, with high homology to mammalian counterparts, have been identified. Zebrafish Neu3.2 is similar to the human cytosolic sialidase NEU2, which is involved in skeletal muscle differentiation and exhibits a broad substrate specificity toward gangliosides and glycoproteins. In zebrafish neu3.2, mRNA is expressed during somite development, and its enzymatic activity has been detected in the skeletal muscle and heart of adult animals. In this paper, 1-4-cell-stage embryos injected with neu3.2 splice-blocking morpholino showed severe embryonic defects, mainly in somites, heart and anterior-posterior axis formation. Myog and myod1 expressions were altered in morphants, and impaired musculature formation was associated with a defective locomotor behavior. Finally, the co-injection of Neu2 mouse mRNA in morphants rescued the phenotype. These data are consistent with the involvement of cytosolic sialidase in pathologies related to muscle formation and support the validity of the model to investigate the pathogenesis of the diseases.

Characteristics of Shisa Family Genes in Zebrafish.

Shisa represents a type of single-transmembrane adaptor protein containing an N-terminal cysteine-rich domain and a proline-rich C-terminal region. Nine shisa subfamily genes have been proposed in most vertebrates; however, some might be species-specific. The number of shisa genes present in zebrafish remains unclear. This study aimed to investigate the evolutionary relationships among shisa family genes in zebrafish (TU strain) using phylogenetic and syntenic analyses. The function of shisa-2 was preliminarily examined via CRISPR/Cas13d-mediated knockdown. Following identification in zebrafish, 10 shisa family genes, namely shisa-1, 2, 3, 4, 5, 6, 7, 8, 9a, and 9b, were classified into three main clades and six subclades. Their encoding proteins contained a cysteine-rich N-terminal domain and a proline-rich C-terminal region containing different motifs. A specific syntenic block containing atp8a2 and shisa-2 was observed to be conserved across all species. Furthermore, all these genes were expressed during embryogenesis. Shisa-2 was expressed in the presomitic mesoderm, somites, and so on. Shisa-2 was identified as a regulator of the expression of the somite formation marker mesp-ab. Overall, our study provides new insights into the evolution of shisa family genes and the control of shisa-2 over the convergent extension cells of somitic precursors in zebrafish.

Assessing Myf5 and Lbx1 contribution to carapace development by reproducing their turtle-specific signatures in mouse embryos.

BACKGROUND: The turtle carapace is an evolutionary novelty resulting from changes in the processes that build ribs and their associated muscles in most tetrapod species. Turtle embryos have several unique features that might play a role in this process, including the carapacial ridge, a Myf5 gene with shorter coding region that generates an alternative splice variant lacking exon 2, and unusual expression patterns of Lbx1 and HGF. RESULTS: We investigated these turtle-specific expression differences using genetic approaches in mouse embryos. At mid-gestation, mouse embryos producing Myf5 transcripts lacking exon 2 replicated some early properties of turtle somites, but still developed into viable and fertile mice. Extending Lbx1 expression into the hypaxial dermomyotomal lip of trunk somites to mimic the turtle Lbx1 expression pattern, produced fusions in the distal part of the ribs. CONCLUSIONS: Turtle-like Myf5 activity might generate a plastic state in developing trunk somites under which they can either enter carapace morphogenetic routes, possibly triggered by signals from the carapacial ridge, or still engage in the development of a standard tetrapod ribcage in the absence of those signals. In addition, trunk Lbx1 expression might play a later role in the formation of the lateral border of the carapace.

Fine-tuning of the PAX-SIX-EYA-DACH network by multiple microRNAs controls embryo myogenesis.

MicroRNAs (miRNAs), short non-coding RNAs, which act post-transcriptionally to regulate gene expression, are of widespread significance during development and disease, including muscle disease. Advances in sequencing technology and bioinformatics led to the identification of a large number of miRNAs in vertebrates and other species, however, for many of these miRNAs specific roles have not yet been determined. LNA in situ hybridisation has revealed expression patterns of somite-enriched miRNAs, here we focus on characterising the functions of miR-128. We show that antagomiR-mediated knockdown (KD) of miR-128 in developing chick somites has a negative impact on skeletal myogenesis. Computational analysis identified the transcription factor EYA4 as a candidate target consistent with the observation that miR-128 and EYA4 display similar expression profiles. Luciferase assays confirmed that miR-128 interacts with the EYA4 3'UTR. In vivo experiments also suggest that EYA4 is regulated by miR-128. EYA4 is a member of the PAX-SIX-EYA-DACH (PSED) network of transcription factors. Therefore, we identified additional candidate miRNA binding sites in the 3'UTR of SIX1/4, EYA1/2/3 and DACH1. Using the miRanda algorithm, we found sites for miR-128, as well as for other myogenic miRNAs, miR-1a, miR-206 and miR-133a, some of these were experimentally confirmed as functional miRNA target sites. Our results reveal that miR-128 is involved in regulating skeletal myogenesis by directly targeting EYA4 with indirect effects on other PSED members, including SIX4 and PAX3. Hence, the inhibitory effect on myogenesis observed after miR-128 knockdown was rescued by concomitant knockdown of PAX3. Moreover, we show that the PSED network of transcription factors is co-regulated by multiple muscle-enriched microRNAs.

Single cell dynamics of embryonic muscle progenitor cells in zebrafish.

Muscle stem cells hold a great therapeutic potential in regenerating damaged muscles. However, the in vivo behavior of muscle stem cells during muscle growth and regeneration is still poorly understood. Using zebrafish as a model, we describe the in vivo dynamics and function of embryonic muscle progenitor cells (MPCs) in the dermomyotome. These cells are located in a superficial layer external to muscle fibers and express many extracellular matrix (ECM) genes, including collagen type 1 α2 (col1a2). Utilizing a new col1a2 transgenic line, we show that col1a2+ MPCs display a ramified morphology with dynamic cellular processes. Cell lineage tracing demonstrates that col1a2+ MPCs contribute to new myofibers in normal muscle growth and also during muscle regeneration. A combination of live imaging and single cell clonal analysis reveals a highly choreographed process of muscle regeneration. Activated col1a2+ MPCs change from the quiescent ramified morphology to a polarized and elongated morphology, generating daughter cells that fuse with existing myofibers. Partial depletion of col1a2+ MPCs severely compromises muscle regeneration. Our work provides a dynamic view of embryonic muscle progenitor cells during zebrafish muscle growth and regeneration.

Transcriptional autoregulation of zebrafish tbx6 is required for somite segmentation.

The presumptive somite boundary in the presomitic mesoderm (PSM) is defined by the anterior border of the expression domain of Tbx6 protein. During somite segmentation, the expression domain of Tbx6 is regressed by Ripply-meditated degradation of Tbx6 protein. Although the expression of zebrafish tbx6 remains restricted to the PSM, the transcriptional regulation of tbx6 remains poorly understood. Here, we show that the expression of zebrafish tbx6 is maintained by transcriptional autoregulation. We find that a proximal-located cis-regulatory module, TR1, which contains two putative T-box sites, is required for somite segmentation in the intermediate body and for proper expression of segmentation genes. Embryos with deletion of TR1 exhibit significant reduction of tbx6 expression at the 12-somite stage, although its expression is initially observed. Additionally, Tbx6 is associated with TR1 and activates its own expression in the anterior PSM. Furthermore, the anterior expansion of tbx6 expression in ripply gene mutants is suppressed in a TR1-dependent manner. The results suggest that the autoregulatory loop of zebrafish tbx6 facilitates immediate removal of Tbx6 protein through termination of its own transcription at the anterior PSM.

Imaging and manipulating the segmentation clock.

During embryogenesis, the processes that control how cells differentiate and interact to form particular tissues and organs with precise timing and shape are of fundamental importance. One prominent example of such processes is vertebrate somitogenesis, which is governed by a molecular oscillator called the segmentation clock. The segmentation clock system is initiated in the presomitic mesoderm in which a set of genes and signaling pathways exhibit coordinated spatiotemporal dynamics to establish regularly spaced boundaries along the body axis; these boundaries provide a blueprint for the development of segment-like structures such as spines and skeletal muscles. The highly complex and dynamic nature of this in vivo event and the design principles and their regulation in both normal and abnormal embryogenesis are not fully understood. Recently, live-imaging has been used to quantitatively analyze the dynamics of selected components of the circuit, particularly in combination with well-designed experiments to perturb the system. Here, we review recent progress from studies using live imaging and manipulation, including attempts to recapitulate the segmentation clock in vitro. In combination with mathematical modeling, these techniques have become essential for disclosing novel aspects of the clock.

Size-reduced embryos reveal a gradient scaling-based mechanism for zebrafish somite formation.

Little is known about how the sizes of animal tissues are controlled. A prominent example is somite size, which varies widely both within an individual and across species. Despite intense study of the segmentation clock governing the timing of somite generation, how it relates to somite size is poorly understood. Here, we examine somite scaling and find that somite size at specification scales with the length of the presomitic mesoderm (PSM) despite considerable variation in PSM length across developmental stages and in surgically size-reduced embryos. Measurement of clock period, axis elongation speed and clock gene expression patterns demonstrate that existing models fail to explain scaling. We posit a 'clock and scaled gradient' model, in which somite boundaries are set by a dynamically scaling signaling gradient across the PSM. Our model not only explains existing data, but also makes a unique prediction that we confirm experimentally - the formation of periodic 'echoes' in somite size following perturbation of the size of one somite. Our findings demonstrate that gradient scaling plays a central role in both progression and size control of somitogenesis.

miR-133-mediated regulation of the Hedgehog pathway orchestrates embryo myogenesis.

Skeletal myogenesis serves as a paradigm to investigate the molecular mechanisms underlying exquisitely regulated cell fate decisions in developing embryos. The evolutionarily conserved miR-133 family of microRNAs is expressed in the myogenic lineage, but how it acts remains incompletely understood. Here, we performed genome-wide differential transcriptomics of miR-133 knockdown (KD) embryonic somites, the source of vertebrate skeletal muscle. These analyses, performed in chick embryos, revealed extensive downregulation of Sonic hedgehog (Shh) pathway components: patched receptors, Hedgehog interacting protein and the transcriptional activator Gli1. By contrast, Gli3, a transcriptional repressor, was de-repressed and confirmed as a direct miR-133 target. Phenotypically, miR-133 KD impaired myotome formation and growth by disrupting proliferation, extracellular matrix deposition and epithelialization. Together, these observations suggest that miR-133-mediated Gli3 silencing is crucial for embryonic myogenesis. Consistent with this idea, we found that activation of Shh signalling by either purmorphamine, or KD of Gli3 by antisense morpholino, rescued the miR-133 KD phenotype. Thus, we identify a novel Shh/myogenic regulatory factor/miR-133/Gli3 axis that connects epithelial morphogenesis with myogenic fate specification.