Identification of a heterogeneous and dynamic ciliome during embryonic development and cell differentiation.

Primary cilia are nearly ubiquitous organelles that transduce molecular and mechanical signals. Although the basic structure of the cilium and the cadre of genes that contribute to ciliary formation and function (the ciliome) are believed to be evolutionarily conserved, the presentation of ciliopathies with narrow, tissue-specific phenotypes and distinct molecular readouts suggests that an unappreciated heterogeneity exists within this organelle. Here, we provide a searchable transcriptomic resource for a curated primary ciliome, detailing various subgroups of differentially expressed genes within the ciliome that display tissue and temporal specificity. Genes within the differentially expressed ciliome exhibited a lower level of functional constraint across species, suggesting organism and cell-specific function adaptation. The biological relevance of ciliary heterogeneity was functionally validated by using Cas9 gene-editing to disrupt ciliary genes that displayed dynamic gene expression profiles during osteogenic differentiation of multipotent neural crest cells. Collectively, this novel primary cilia-focused resource will allow researchers to explore longstanding questions related to how tissue and cell-type specific functions and ciliary heterogeneity may contribute to the range of phenotypes associated with ciliopathies.

Molecular compartmentalization in a syncytium: restricted mobility of proteins within the sea urchin skeletogenic mesenchyme.

Multinucleated cells, or syncytia, are found in diverse taxa. Their biological function is often associated with the compartmentalization of biochemical or cellular activities within the syncytium. How such compartments are generated and maintained is poorly understood. The sea urchin embryonic skeleton is secreted by a syncytium, and local patterns of skeletal growth are associated with distinct sub-domains of gene expression within the syncytium. For such molecular compartments to be maintained and to control local patterns of skeletal growth: (1) the mobility of TFs must be restricted to produce stable differences in the transcriptional states of nuclei within the syncytium; and (2) the mobility of biomineralization proteins must also be restricted to produce regional differences in skeletal growth. To test these predictions, we expressed fluorescently tagged forms of transcription factors and biomineralization proteins in sub-domains of the skeletogenic syncytium. We found that both classes of proteins have restricted mobility within the syncytium and identified motifs that limit their mobility. Our findings have general implications for understanding the functional and molecular compartmentalization of syncytia.

An optimized Tet-On system for conditional control of gene expression in sea urchins.

Sea urchins and other echinoderms are important experimental models for studying developmental processes. The lack of approaches for conditional gene perturbation, however, has made it challenging to investigate the late developmental functions of genes that have essential roles during early embryogenesis and genes that have diverse functions in multiple tissues. The doxycycline-controlled Tet-On system is a widely used molecular tool for temporally and spatially regulated transgene expression. Here, we optimized the Tet-On system to conditionally induce gene expression in sea urchin embryos. Using this approach, we explored the roles the MAPK signaling plays in skeletogenesis by expressing genes that perturb the pathway specifically in primary mesenchyme cells during later stages of development. We demonstrated the wide utility of the Tet-On system by applying it to a second sea urchin species and in cell types other than the primary mesenchyme cells. Our work provides a robust and flexible platform for the spatiotemporal regulation of gene expression in sea urchins, which will considerably enhance the utility of this prominent model system.

microRNA-1 regulates sea urchin skeletogenesis by directly targeting skeletogenic genes and modulating components of signaling pathways.

microRNAs are evolutionarily conserved non-coding RNAs that direct post-transcriptional regulation of target transcripts. In vertebrates, microRNA-1 (miR-1) is expressed in muscle and has been found to play critical regulatory roles in vertebrate angiogenesis, a process that has been proposed to be analogous to sea urchin skeletogenesis. Results indicate that both miR-1 inhibitor and miR-1 mimic-injected larvae have significantly less F-actin enriched circumpharyngeal muscle fibers and fewer gut contractions. In addition, miR-1 regulates the positioning of skeletogenic primary mesenchyme cells (PMCs) and skeletogenesis of the sea urchin embryo. Interestingly, the gain-of-function of miR-1 leads to more severe PMC patterning and skeletal branching defects than its loss-of-function. The results suggest that miR-1 directly suppresses Ets1/2, Tbr, and VegfR7 of the skeletogenic gene regulatory network, and Nodal, and Wnt1 signaling components. This study identifies potential targets of miR-1 that impacts skeletogenesis and muscle formation and contributes to a deeper understanding of miR-1's function during development.

Expression and Transcriptional Targets of TGFß-RII in Paracentrotus lividus Larval Skeletogenesis.

Organisms from the five kingdoms of life use minerals to harden their tissues and make teeth, shells and skeletons, in the process of biomineralization. The sea urchin larval skeleton is an excellent system to study the biological regulation of biomineralization and its evolution. The gene regulatory network (GRN) that controls sea urchin skeletogenesis is known in great details and shows similarity to the GRN that controls vertebrates' vascularization while it is quite distinct from the GRN that drives vertebrates' bone formation. Yet, transforming growth factor beta (TGF-ß) signaling regulates both sea urchin and vertebrates' skeletogenesis. Here, we study the upstream regulation and identify transcriptional targets of TGF-ß in the Mediterranean Sea urchin species, Paracentrotus lividus. TGF-ßRII is transiently active in the skeletogenic cells downstream of vascular endothelial growth factor (VEGF) signaling, in P. lividus. Continuous perturbation of TGF-ßRII activity significantly impairs skeletal elongation and the expression of key skeletogenic genes. Perturbation of TGF-ßRII after skeletal initiation leads to a delay in skeletal elongation and minor changes in gene expression. TGF-ß targets are distinct from its transcriptional targets during vertebrates' bone formation, suggesting that the role of TGF-ß in biomineralization in these two phyla results from convergent evolution.

The tolerance to hypoxia is defined by a time-sensitive response of the gene regulatory network in sea urchin embryos.

Deoxygenation, the reduction of oxygen level in the oceans induced by global warming and anthropogenic disturbances, is a major threat to marine life. This change in oxygen level could be especially harmful to marine embryos that use endogenous hypoxia and redox gradients as morphogens during normal development. Here, we show that the tolerance to hypoxic conditions changes between different developmental stages of the sea urchin embryo, possibly due to the structure of the gene regulatory networks (GRNs). We demonstrate that during normal development, the bone morphogenetic protein (BMP) pathway restricts the activity of the vascular endothelial growth factor (VEGF) pathway to two lateral domains and this restriction controls proper skeletal patterning. Hypoxia applied during early development strongly perturbs the activity of Nodal and BMP pathways that affect the VEGF pathway, dorsal-ventral (DV) and skeletogenic patterning. These pathways are largely unaffected by hypoxia applied after DV-axis formation. We propose that the use of redox and hypoxia as morphogens makes the sea urchin embryo highly sensitive to environmental hypoxia during early development, but the GRN structure provides higher tolerance to hypoxia at later stages.

Is African non-annual killifish Fundulopanchax gardneri (Teleostei; Cyprinodontiformes; Nothobranchiidae) true non-annual?

BACKGROUND: Annual or seasonal killifishes (Cyprinodontiformes: Nothobranchiidae) are unique among fish in their ability to enter into developmental arrests (diapauses: DI, DII, and DIII). They have a short lifespan and their embryos are exceptionally tolerant to a variety of environmental stresses. These traits make them a popular model for studying vertebrate diapause, aging, stress tolerance, genome adaptation, and evolution. In such issues, in a comparative evolutionary framework, Fundulopanchax gardneri, a popular aquarium fish from Africa, is commonly used as a representative non-annual model though its development is not studied in detail and whether it includes diapauses remains uncertain. RESULTS: We described in detail for the first time embryonic development of F. gardneri and revealed it to resemble that in the undoubtedly annual Austrofundulus limnaeus killifish in displaying two developmental depressions. However, if compared with A. limnaeus, these developmental states look like "less intense" versions of DII and DIII rather than true diapauses. CONCLUSIONS: To determine whether developmental depressions in F. gardneri represent "true" diapauses or only their functional equivalents, detailed studies of embryonic development of different killifish both annual and non-annual are needed. Before that, acceptance of F. gardneri as a representative non-annual fish seems premature.

Soluble adenylyl cyclase coordinates intracellular pH homeostasis and biomineralization in calcifying cells of a marine animal.

Biomineralizing cells concentrate dissolved inorganic carbon (DIC) and remove protons from the site of mineral precipitation. However, the molecular regulatory mechanisms that orchestrate pH homeostasis and biomineralization of calcifying cells are poorly understood. Here, we report that the acid-base sensing enzyme soluble adenylyl cyclase (sAC) coordinates intracellular pH (pHi) regulation in the calcifying primary mesenchyme cells (PMCs) of sea urchin larvae. Single-cell transcriptomics, in situ hybridization, and immunocytochemistry elucidated the spatiotemporal expression of sAC during skeletogenesis. Live pHi imaging of PMCs revealed that the downregulation of sAC activity with two structurally unrelated small molecules inhibited pHi regulation of PMCs, an effect that was rescued by the addition of cell-permeable cAMP. Pharmacological sAC inhibition also significantly reduced normal spicule growth and spicule regeneration, establishing a link between PMC pHi regulation and biomineralization. Finally, increased expression of sAC mRNA was detected during skeleton remineralization and exposure to CO2-induced acidification. These findings suggest that transcriptional regulation of sAC is required to promote remineralization and to compensate for acidic stress. This work highlights the central role of sAC in coordinating acid-base regulation and biomineralization in calcifying cells of a marine animal.

Cilia kinases in skeletal development and homeostasis.

Primary cilia are dynamic compartments that regulate multiple aspects of cellular signaling. The production, maintenance, and function of cilia involve more than 1000 genes in mammals, and their mutations disrupt the ciliary signaling which manifests in a plethora of pathological conditions-the ciliopathies. Skeletal ciliopathies are genetic disorders affecting the development and homeostasis of the skeleton, and encompass a broad spectrum of pathologies ranging from isolated polydactyly to lethal syndromic dysplasias. The recent advances in forward genetics allowed for the identification of novel regulators of skeletogenesis, and revealed a growing list of ciliary proteins that are critical for signaling pathways implicated in bone physiology. Among these, a group of protein kinases involved in cilia assembly, maintenance, signaling, and disassembly has emerged. In this review, we summarize the functions of cilia kinases in skeletal development and disease, and discuss the available and upcoming treatment options.

Distinct regulatory states control the elongation of individual skeletal rods in the sea urchin embryo.

BACKGROUND: Understanding how gene regulatory networks (GRNs) control developmental progression is a key to the mechanistic understanding of morphogenesis. The sea urchin larval skeletogenesis provides an excellent platform to tackle this question. In the early stages of sea urchin skeletogenesis, skeletogenic genes are uniformly expressed in the skeletogenic lineage. Yet, during skeletal elongation, skeletogenic genes are expressed in distinct spatial sub-domains. The regulation of differential gene expression during late skeletogenesis is not well understood. RESULTS: Here we reveal the dynamic expression of the skeletogenic regulatory genes that define a specific regulatory state for each pair of skeletal rods, in the sea urchin Paracentrotus lividus. The vascular endothelial growth factor (VEGF) signaling, essential for skeleton formation, specifically controls the migration of cells that form the postoral and distal anterolateral skeletogenic rods. VEGF signaling also controls the expression of regulatory genes in cells at the tips of the postoral rods, including the transcription factors Pitx1 and MyoD1. Pitx1 activity is required for normal skeletal elongation and for the expression of some of VEGF target genes. CONCLUSIONS: Our study illuminates the fine-tuning of the regulatory system during the transition from early to late skeletogenesis that gives rise to rod-specific regulatory states.

Analysis of the DNA-binding properties of Alx1, an evolutionarily conserved regulator of skeletogenesis in echinoderms.

Alx1, a homeodomain-containing transcription factor, is a highly conserved regulator of skeletogenesis in echinoderms. In sea urchins, Alx1 plays a central role in the differentiation of embryonic primary mesenchyme cells (PMCs) and positively regulates the transcription of most biomineralization genes expressed by these cells. The alx1 gene arose via duplication and acquired a skeletogenic function distinct from its paralog (alx4) through the exonization of a 41-amino acid motif (the D2 domain). Alx1 and Alx4 contain glutamine-50 paired-type homeodomains, which interact preferentially with palindromic binding sites in vitro. Chromatin immunoprecipitation sequencing (ChIP-seq) studies have shown, however, that Alx1 binds both to palindromic and half sites in vivo. To address this apparent discrepancy and explore the function of the D2 domain, we used an endogenous cis-regulatory module associated with Sp-mtmmpb, a gene that encodes a PMC-specific metalloprotease, to analyze the DNA-binding properties of Alx1. We find that Alx1 forms dimeric complexes on TAAT-containing half sites by a mechanism distinct from the well-known mechanism of dimerization on palindromic sites. We used transgenic reporter assays to analyze the functional roles of half sites in vivo and demonstrate that two sites with partially redundant functions are essential for the PMC-specific activity of the Sp-mtmmpb cis-regulatory module. Finally, we show that the D2 domain influences the DNA-binding properties of Alx1 in vitro, suggesting that the exonization of this motif may have facilitated the acquisition of new transcriptional targets and consequently a novel developmental function.

Genome-wide identification of binding sites and gene targets of Alx1, a pivotal regulator of echinoderm skeletogenesis.

Alx1 is a conserved regulator of skeletogenesis in echinoderms and evolutionary changes in Alx1 sequence and expression have played a pivotal role in modifying programs of skeletogenesis within the phylum. Alx1 regulates a large suite of effector genes that control the morphogenetic behaviors and biomineral-forming activities of skeletogenic cells. To better understand the gene regulatory control of skeletogenesis by Alx1, we used genome-wide ChIP-seq to identify Alx1-binding sites and direct gene targets. Our analysis revealed that many terminal differentiation genes receive direct transcriptional inputs from Alx1. In addition, we found that intermediate transcription factors previously shown to be downstream of Alx1 all receive direct inputs from Alx1. Thus, Alx1 appears to regulate effector genes by indirect, as well as direct, mechanisms. We tested 23 high-confidence ChIP-seq peaks using GFP reporters and identified 18 active cis-regulatory modules (CRMs); this represents a high success rate for CRM discovery. Detailed analysis of a representative CRM confirmed that a conserved, palindromic Alx1-binding site was essential for expression. Our work significantly advances our understanding of the gene regulatory circuitry that controls skeletogenesis in sea urchins and provides a framework for evolutionary studies.

Gene Expression Detects the Factors Influencing the Reproductive Success and the Survival Rates of Paracentrotus lividus Offspring.

The increase in the demand for Paracentrotus lividus roe, a food delicacy, causes increased pressure on its wild stocks. In this scenario, aquaculture facilities will mitigate the effects of anthropogenic pressures on the wild stocks of P. lividus. Consequently, experimental studies should be conducted to enhance techniques to improve efficient aquaculture practices for these animals. Here, we for the first time performed molecular investigations on cultured sea urchins. We aimed at understanding if maternal influences may significantly impact the life of future offspring, and how the culture conditions may impact the development and growth of cultured specimens. Our findings demonstrate that the outcomes of in vitro fertilization of P. lividus are influenced by maternal influences, but these effects are largely determined by culture conditions. In fact, twenty-three genes involved in the response to stress and skeletogenesis, whose expressions were measured by Real Time qPCR, were differently expressed in sea urchins cultured in two experimental conditions, and the results were largely modified in offspring deriving from two groups of females. The findings herein reported will be critical to develop protocols for the larval culture of the most common sea urchin, both for research and industrial production purposes for mass production.

Regulation of FGF-2, FGF-18 and Transcription Factor Activity by Perlecan in the Maturational Development of Transitional Rudiment and Growth Plate Cartilages and in the Maintenance of Permanent Cartilage Homeostasis.

The aim of this study was to highlight the roles of perlecan in the regulation of the development of the rudiment developmental cartilages and growth plate cartilages, and also to show how perlecan maintains permanent articular cartilage homeostasis. Cartilage rudiments are transient developmental templates containing chondroprogenitor cells that undergo proliferation, matrix deposition, and hypertrophic differentiation. Growth plate cartilage also undergoes similar changes leading to endochondral bone formation, whereas permanent cartilage is maintained as an articular structure and does not undergo maturational changes. Pericellular and extracellular perlecan-HS chains interact with growth factors, morphogens, structural matrix glycoproteins, proteases, and inhibitors to promote matrix stabilization and cellular proliferation, ECM remodelling, and tissue expansion. Perlecan has mechanotransductive roles in cartilage that modulate chondrocyte responses in weight-bearing environments. Nuclear perlecan may modulate chromatin structure and transcription factor access to DNA and gene regulation. Snail-1, a mesenchymal marker and transcription factor, signals through FGFR-3 to promote chondrogenesis and maintain Acan and type II collagen levels in articular cartilage, but prevents further tissue expansion. Pre-hypertrophic growth plate chondrocytes also express high Snail-1 levels, leading to cessation of Acan and CoI2A1 synthesis and appearance of type X collagen. Perlecan differentially regulates FGF-2 and FGF-18 to maintain articular cartilage homeostasis, rudiment and growth plate cartilage growth, and maturational changes including mineralization, contributing to skeletal growth.

Generation of a new mouse line with conditionally activated signaling through the BMP receptor, ACVR1: A tool to characterize pleiotropic roles of BMP functions.

BMP signaling plays pleiotropic roles in various tissues during embryogenesis and after birth. We have previously generated a constitutively activated Acvr1(ca-Acvr1) transgenic mouse line (line L35) through pronuclei injection to investigate impacts of enhanced BMP signaling in a tissue specific manner. However, line L35 shows a restricted expression pattern of the transgene. Here, we generated another ca-Acvr1 transgenic line, line A11, using embryonic stem (ES) transgenesis. The generated line A11 shows distinctive phenotypes from line L35, along with very limited expression levels of the transgene. When the transgene is activated in the neural crest cells in a Cre-dependent manner, line A11 exhibits cleft palate and shorter jaws, while line L35 develops ectopic cartilages and highly hypomorphic facial structures. When activated in limb buds, line A11 develops organized but smaller limb skeletal structures, while line L35 forms disorganized limbs with little mineralization. Additionally, no heterotopic ossification (HO) is identified in line A11 when bred with NFATc1-Cre mice even after induction of tissue injury, which is an established protocol for HO for line L35. Therefore, the newly generated conditional ca-Acvr1 mouse line A11 provides an additional resource to dissect highly context dependent functions of BMP signaling in development and disease.

Podoplanin is dispensable for mineralized tissue formation and maintenance in the Swiss outbred mouse background.

Podoplanin, PDPN, is a mucin-type transmembrane glycoprotein widely expressed in many tissues, including lung, kidney, lymph nodes, and mineralized tissues. Its function is critical for lymphatic formation, differentiation of type I alveolar epithelial lung cells, and for bone response to biomechanical loading. It has previously been shown that Pdpn null mice die at birth due to respiratory failure emphasizing the importance of Pdpn in alveolar lung development. During the course of generation of Pdpn mutant mice, we found that most Pdpn null mice in the 129S6 and C57BL6/J mixed genetic background die at the perinatal stage, similar to previously published studies with Pdpn null mice, while all Pdpn null mice bred with Swiss outbred mice survived. Surviving mutant mice in the 129S6 and C57BL6/J mixed genetic background showed alterations in the osteocyte lacunocanalicular network, especially reduced osteocyte canaliculi in the tibial cortex with increased tibial trabecular bone. However, adult Pdpn null mice in the Swiss outbred background showed no overt differences in their osteocyte lacunocnalicular network, bone density, and no overt differences when challenged with exercise. Together, these data suggest that genetic variations present in the Swiss outbred mice compensate for the loss of function of PDPN in lung, kidney, and bone.

The biological regulation of sea urchin larval skeletogenesis - From genes to biomineralized tissue.

Biomineralization is the process in which soft organic tissues use minerals to produce shells, skeletons and teeth for various functions such as protection and physical support. The ability of the cells to control the time and place of crystal nucleation as well as crystal orientation and stiffness is far beyond the state-of-the art of human technologies. Thus, understanding the biological control of biomineralization will promote our understanding of embryo development as well as provide novel approaches for material engineering. Sea urchin larval skeletogenesis offers an excellent platform for functional analyses of both the molecular control system and mineral uptake and deposition. Here we describe the current understanding of the genetic, molecular and cellular processes that underlie sea urchin larval skeletogenesis. We portray the regulatory genes that define the specification of the skeletogenic cells and drive the various morphogenetic processes that occur in the skeletogenic lineage, including: epithelial to mesenchymal transition, cell migration, spicule cavity formation and mineral deposition into the spicule cavity. We describe recent characterizations of the size, motion and mineral concentration of the calcium-bearing vesicles in the skeletogenic cells. We review the distinct specification states within the skeletogenic lineage that drive localized skeletal growth at the tips of the spicules. Finally, we discuss the surprising similarity between the regulatory network and cellular processes that drive sea urchin skeletogenesis and those that control vertebrate vascularization. Overall, we illustrate the novel insights on the biological regulation and evolution of biomineralization, gained from studies of the sea urchin larval skeletogenesis.

Devonian agglutinated polychaete tubes: all in all it's just another grain in the wall.

Biomineralized and organic metazoan tubular skeletons are by far the most common in the fossil record. However, several groups of organisms are also able to agglutinate particles to construct more rigid structures. Here we present a novel type of agglutinated tube from the austral and endemic palaeobiota of the Malvinokaffric realm (Devonian, Brazil). This fossil is characterized by an agglutinated tube made of silt-sized particles forming an unusual flanged morphology that is not known from the fossil record. Besides being able to select specific particles, these organisms probably lived partially buried and were detritus/suspension feeders. Comparisons across different modern groups show that these fossils are strongly similar to tubes made by polychaetes, specifically from the family Maldanidae. If this interpretation is correct, then an early divergence of the Sedentaria clade may have occurred before the Devonian.

Postnatal ossification sequences in Acrocephalus scirpaceus and Chroicocephalus ridibundus (Aves: Neognathae): The precocial-altricial spectrum and evolution of compound bones in birds.

Although the development of the avian skeleton has attracted considerable attention, most of the studies have been concentrated on the embryonic period, while studies on the postnatal period are rare. We studied the postnatal development of the skeleton in two phylogenetically distant birds, an altricial passerine Acrocephalus scirpaceus and a semiprecocial charadriiform Chroicocephalus ridibundus. The neonates of the former, despite being altricial, have well-ossified skeleton-the degree of development approaches that of the semiprecocial gull. However, after hatching the limb bones (particularly those of the hind limb) ossify earlier in the gull which is probably related to faster acquisition of locomotor abilities. We have observed that, in contrast to previous reports from neognathous birds, in the ankle of the gull, the ascending process fuses with the astragalus rather than with the calcaneum. This type of development is present in palaeognaths and nonavian dinosaurs but has not yet been reported in neognaths. This indicates a greater diversity within Neognathae and suggests a more complex scenario for the evolution of the avian ankle. However, data from a greater number of species are needed to establish the developmental sequence ancestral for neognathous birds. Furthermore, the sequence of bone fusions in the wrist of Acrocephalus is similar to the fossil-documented evolutionary sequence observed in the phylogeny of early birds, with the semilunate carpal and major metacarpal fusing first, followed by the alular metacarpal fusing with the major metacarpal and then the major and minor metacarpal fusing proximally. These data underscore the importance of developmental studies for reconstructing the evolutionary history.

Regulation and Role of Transcription Factors in Osteogenesis.

Bone is a dynamic tissue constantly responding to environmental changes such as nutritional and mechanical stress. Bone homeostasis in adult life is maintained through bone remodeling, a controlled and balanced process between bone-resorbing osteoclasts and bone-forming osteoblasts. Osteoblasts secrete matrix, with some being buried within the newly formed bone, and differentiate to osteocytes. During embryogenesis, bones are formed through intramembraneous or endochondral ossification. The former involves a direct differentiation of mesenchymal progenitor to osteoblasts, and the latter is through a cartilage template that is subsequently converted to bone. Advances in lineage tracing, cell sorting, and single-cell transcriptome studies have enabled new discoveries of gene regulation, and new populations of skeletal stem cells in multiple niches, including the cartilage growth plate, chondro-osseous junction, bone, and bone marrow, in embryonic development and postnatal life. Osteoblast differentiation is regulated by a master transcription factor RUNX2 and other factors such as OSX/SP7 and ATF4. Developmental and environmental cues affect the transcriptional activities of osteoblasts from lineage commitment to differentiation at multiple levels, fine-tuned with the involvement of co-factors, microRNAs, epigenetics, systemic factors, circadian rhythm, and the microenvironments. In this review, we will discuss these topics in relation to transcriptional controls in osteogenesis.