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Non-invasive prenatal testing (NIPT) screens for common fetal chromosomal abnormalities through analysis of circulating cell-free DNA in maternal blood by massive parallel sequencing. NIPT reliability relies on both the estimation of the fetal fraction (ff) and on the sequencing depth (sd) but how these parameters are linked is unknown. Several bioinformatics tools have been developed to determine the ff but there is no universal ff threshold applicable across diagnostics laboratories. Thus, we developed two tools allowing the implementation of a strategy for NIPT results validation in clinical practice: GenomeMixer, a semi-supervised approach to create synthetic sequences and to estimate confidence intervals for NIPT validation and TRUST to estimate the reliability of NIPT results based on confidence intervals found in this study. We retrospectively validated these new tools on 2 cohorts for a total of 1439 samples with 31 confirmed aneuploidies. Through the analysis of the interrelationship between ff, sd and chromosomal aberration detection, we demonstrate that these parameters are profoundly connected and cannot be considered independently. Our tools take in account this critical relationship to improve NIPT reliability and facilitate cross laboratory standardization of this screening test., Competing Interests: The authors declare that they have no competing interests, (© 2022 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.)

Noninvasive prenatal testing (NIPT) consists of determining fetal aneuploidies by quantifying copy number alteration from the sequencing of cell-free DNA (cfDNA) from maternal blood. Due to the presence of cfDNA of fetal origin in maternal blood, in silico approaches have been developed to accurately predict fetal aneuploidies. Although NIPT is becoming a new standard in prenatal screening of chromosomal abnormalities, there are no integrated pipelines available to allow rapid, accurate and standardized data analysis in any clinical setting. Several tools have been developed, however often optimized only for research purposes or requiring enormous amount of retrospective data, making hard their implementation in a clinical context. Furthermore, no guidelines have been provided on how to accomplish each step of the data analysis to achieve reliable results. Finally, there is no integrated pipeline to perform all steps of NIPT analysis. To address these needs, we tested several tools for performing NIPT data analysis. We provide extensive benchmark of tools performances but also guidelines for running them. We selected the best performing tools that we benchmarked and gathered them in a computational pipeline. NiPTUNE is an open source python package that includes methods for fetal fraction estimation, a novel method for accurate gender prediction, a principal component analysis based strategy for quality control and fetal aneuploidies prediction. NiPTUNE is constituted by seven modules allowing the user to run the entire pipeline or each module independently. Using two cohorts composed by 1439 samples with 31 confirmed aneuploidies, we demonstrated that NiPTUNE is a valuable resource for NIPT analysis., (© The Author(s) 2021. Published by Oxford University Press.)

Left-right asymmetries in the zebrafish habenular nuclei are dependent upon the formation of the parapineal, a unilateral group of neurons that arise from the medially positioned pineal complex. In this study, we show that both the left and right habenula are competent to adopt left-type molecular character and efferent connectivity upon the presence of only a few parapineal cells. This ability to impart left-sided character is lost in parapineal cells lacking Sox1a function, despite the normal specification of the parapineal itself. Precisely timed laser ablation experiments demonstrate that the parapineal influences neurogenesis in the left habenula at early developmental stages as well as neurotransmitter phenotype and efferent connectivity during subsequent stages of habenular differentiation. These results reveal a tight coordination between the formation of the unilateral parapineal nucleus and emergence of asymmetric habenulae, ensuring that appropriate lateralised character is propagated within left and right-sided circuitry., Competing Interests: IL, VD, AF, SN, PB, SW No competing interests declared, (© 2019, Lekk et al.)

Although the left and right hemispheres of our brains develop with a high degree of symmetry at both the anatomical and functional levels, it has become clear that subtle structural differences exist between the two sides and that each is dominant in processing specific cognitive tasks. As the result of evolutionary conservation or convergence, lateralization of the brain is found in both vertebrates and invertebrates, suggesting that it provides significant fitness for animal life. This widespread feature of hemispheric specialization has allowed the emergence of model systems to study its development and, in some cases, to link anatomical asymmetries to brain function and behavior. Here, we present some of what is known about brain asymmetry in humans and model organisms as well as what is known about the impact of environmental and genetic factors on brain asymmetry development. We specifically highlight the progress made in understanding the development of epithalamic asymmetries in zebrafish and how this model provides an exciting opportunity to address brain asymmetry at different levels of complexity.

During echinoderm development, expression of nodal on the right side plays a crucial role in positioning of the rudiment on the left side, but the mechanisms that restrict nodal expression to the right side are not known. Here we show that establishment of left-right asymmetry in the sea urchin embryo relies on reciprocal signaling between the ectoderm and a left-right organizer located in the endomesoderm. FGF/ERK and BMP2/4 signaling are required to initiate nodal expression in this organizer, while Delta/Notch signaling is required to suppress formation of this organizer on the left side of the archenteron. Furthermore, we report that the H(+)/K(+)-ATPase is critically required in the Notch signaling pathway upstream of the S3 cleavage of Notch. Our results identify several novel players and key early steps responsible for initiation, restriction, and propagation of left-right asymmetry during embryogenesis of a non-chordate deuterostome and uncover a functional link between the H(+)/K(+)-ATPase and the Notch signaling pathway., Competing Interests: The authors have declared that no competing interests exist.

Echinoderms, which are phylogenetically related to vertebrates and produce large numbers of transparent embryos that can be experimentally manipulated, offer many advantages for the analysis of the gene regulatory networks (GRN) regulating germ layer formation. During development of the sea urchin embryo, the ectoderm is the source of signals that pattern all three germ layers along the dorsal-ventral axis. How this signaling center controls patterning and morphogenesis of the embryo is not understood. Here, we report a large-scale analysis of the GRN deployed in response to the activity of this signaling center in the embryos of the Mediterranean sea urchin Paracentrotus lividus, in which studies with high spatial resolution are possible. By using a combination of in situ hybridization screening, overexpression of mRNA, recombinant ligand treatments, and morpholino-based loss-of-function studies, we identified a cohort of transcription factors and signaling molecules expressed in the ventral ectoderm, dorsal ectoderm, and interposed neurogenic ("ciliary band") region in response to the known key signaling molecules Nodal and BMP2/4 and defined the epistatic relationships between the most important genes. The resultant GRN showed a number of striking features. First, Nodal was found to be essential for the expression of all ventral and dorsal marker genes, and BMP2/4 for all dorsal genes. Second, goosecoid was identified as a central player in a regulatory sub-circuit controlling mouth formation, while tbx2/3 emerged as a critical factor for differentiation of the dorsal ectoderm. Finally, and unexpectedly, a neurogenic ectoderm regulatory circuit characterized by expression of "ciliary band" genes was triggered in the absence of TGF beta signaling. We propose a novel model for ectoderm regionalization, in which neural ectoderm is the default fate in the absence of TGF beta signaling, and suggest that the stomodeal and neural subcircuits that we uncovered may represent ancient regulatory pathways controlling embryonic patterning., Competing Interests: The authors have declared that no competing interests exist.

Nodal factors play fundamental roles in induction and patterning of the mesoderm and endoderm in vertebrates, but whether this reflects an ancient role or one that evolved recently in vertebrates is not known. Here, we report that in addition to its primary role in patterning the ectoderm, sea urchin Nodal is crucial for patterning of the endoderm and skeletogenic mesoderm through the regulation of the expression of key transcription factors and signalling molecules, including BMP2/4 and FGFA. In addition, we uncovered an essential role for Nodal and BMP2/4 in the formation and patterning of the non-skeletogenic mesoderm. By comparing the effects of misexpressing Nodal or an activated Nodal receptor in clones of cells, we provide evidence that Nodal acts over a long range in the endomesoderm and that its effects on the blastocoelar cell precursors are likely to be direct. The activity of Nodal and BMP2/4 are antagonistic, and although bmp2/4 is transcribed in the ventral ectoderm downstream of Nodal, the BMP2/4 ligand is translocated to the dorsal side, where it activates signalling in the dorsal primary mesenchyme cells, the dorsal endoderm and in pigment cell precursors. Therefore, correct patterning of the endomesoderm depends on a balance between ventralising Nodal signals and dorsalising BMP2/4 signals. These experiments confirm that Nodal is a key regulator of dorsal-ventral polarity in the sea urchin and support the idea that the ventral ectoderm, like the Spemann organiser in vertebrates, is an organising centre that is required for patterning all three germ layers of the embryo.

Nodal is a key player in the process regulating oral-aboral axis formation in the sea urchin embryo. Expressed early within an oral organizing centre, it is required to specify both the oral and aboral ectoderm territories by driving an oral-aboral gene regulatory network. A model for oral-aboral axis specification has been proposed relying on the self activation of Nodal and the diffusion of the long-range antagonist Lefty resulting in a sharp restriction of Nodal activity within the oral field. Here, we describe the expression pattern of lefty and analyse its function in the process of secondary axis formation. lefty expression starts at the 128-cell stage immediately after that of nodal, is rapidly restricted to the presumptive oral ectoderm then shifted toward the right side after gastrulation. Consistently with previous work, neither the oral nor the aboral ectoderm are specified in embryos in which Lefty is overexpressed. Conversely, when Lefty's function is blocked, most of the ectoderm is converted into oral ectoderm through ectopic expression of nodal. Reintroducing lefty mRNA in a restricted territory of Lefty depleted embryos caused a dose-dependent effect on nodal expression. Remarkably, injection of lefty mRNA into one blastomere at the 8-cell stage in Lefty depleted embryos blocked nodal expression in the whole ectoderm consistent with the highly diffusible character of Lefty in other models. Taken together, these results demonstrate that Lefty is essential for oral-aboral axis formation and suggest that Lefty acts as a long-range inhibitor of Nodal signalling in the sea urchin embryo.

Nodal factors play crucial roles during embryogenesis of chordates. They have been implicated in a number of developmental processes, including mesoderm and endoderm formation and patterning of the embryo along the anterior-posterior and left-right axes. We have analyzed the function of the Nodal signaling pathway during the embryogenesis of the sea urchin, a non-chordate organism. We found that Nodal signaling plays a central role in axis specification in the sea urchin, but surprisingly, its first main role appears to be in ectoderm patterning and not in specification of the endoderm and mesoderm germ layers as in vertebrates. Starting at the early blastula stage, sea urchin nodal is expressed in the presumptive oral ectoderm where it controls the formation of the oral-aboral axis. A second conserved role for nodal signaling during vertebrate evolution is its involvement in the establishment of left-right asymmetries. Sea urchin larvae exhibit profound left-right asymmetry with the formation of the adult rudiment occurring only on the left side. We found that a nodal/lefty/pitx2 gene cassette regulates left-right asymmetry in the sea urchin but that intriguingly, the expression of these genes is reversed compared to vertebrates. We have shown that Nodal signals emitted from the right ectoderm of the larva regulate the asymmetrical morphogenesis of the coelomic pouches by inhibiting rudiment formation on the right side of the larva. This result shows that the mechanisms responsible for patterning the left-right axis are conserved in echinoderms and that this role for nodal is conserved among the deuterostomes. We will discuss the implications regarding the reference axes of the sea urchin and the ancestral function of the nodal gene in the last section of this review.

The sea urchin embryo is emerging as an attractive model to study morphogenetic processes such as directed migration of mesenchyme cells and cell sheet invagination, but surprisingly, few of the genes regulating these processes have yet been characterized. We present evidence that FGFA, the first FGF family member characterized in the sea urchin, regulates directed migration of mesenchyme cells, morphogenesis of the skeleton and gastrulation during early development. We found that at blastula stages, FGFA and a novel putative FGF receptor are expressed in a pattern that prefigures morphogenesis of the skeletogenic mesoderm and that suggests that FGFA is one of the elusive signals that guide migration of primary mesenchyme cells (PMCs). We first show that fgfA expression is correlated with abnormal migration and patterning of the PMCs following treatments that perturb specification of the ectoderm along the oral-aboral and animal-vegetal axes. Specification of the ectoderm initiated by Nodal is required to restrict fgfA to the lateral ectoderm, and in the absence of Nodal, fgfA is expressed ectopically throughout most of the ectoderm. Inhibition of either FGFA, FGFR1 or FGFR2 function severely affects morphogenesis of the skeleton. Furthermore, inhibition of FGFA and FGFR1 signaling dramatically delays invagination of the archenteron, prevents regionalization of the gut and abrogates formation of the stomodeum. We identified several genes acting downstream of fgfA in these processes, including the transcription factors pea3 and pax2/5/8 and the signaling molecule sprouty in the lateral ectoderm and SM30 and SM50 in the primary mesenchyme cells. This study identifies the FGF signaling pathway as an essential regulator of gastrulation and directed cell migration in the sea urchin embryo and as a key player in the gene regulatory network directing morphogenesis of the skeleton.

The Receptor Tyrosine kinase (RTK) and TGF-beta signaling pathways play essential roles during development in many organisms and regulate a plethora of cellular responses. From the genome sequence of Strongylocentrotus purpuratus, we have made an inventory of the genes encoding receptor tyrosine kinases and their ligands, and of the genes encoding cytokines of the TGF-beta superfamily and their downstream components. The sea urchin genome contains at least 20 genes coding for canonical receptor tyrosine kinases. Seventeen of the nineteen vertebrate RTK families are represented in the sea urchin. Fourteen of these RTK among which ALK, CCK4/PTK7, DDR, EGFR, EPH, LMR, MET/RON, MUSK, RET, ROR, ROS, RYK, TIE and TRK are present as single copy genes while pairs of related genes are present for VEGFR, FGFR and INSR. Similarly, nearly all the subfamilies of TGF-beta ligands identified in vertebrates are present in the sea urchin genome including the BMP, ADMP, GDF, Activin, Myostatin, Nodal and Lefty, as well as the TGF-beta sensu stricto that had not been characterized in invertebrates so far. Expression analysis indicates that the early expression of nodal, BMP2/4 and lefty is restricted to the oral ectoderm reflecting their role in providing positional information along the oral-aboral axis of the embryo. The coincidence between the emergence of TGF-beta-related factors such as Nodal and Lefty and the emergence of the deuterostome lineage strongly suggests that the ancestral function of Nodal could have been related to the secondary opening of the mouth which characterizes this clade, a hypothesis supported by functional data in the extant species. The sea urchin genome contains 6 genes encoding TGF-beta receptors and 4 genes encoding prototypical Smad proteins. Furthermore, most of the transcriptional activators and repressors shown to interact with Smads in vertebrates have orthologues in echinoderms. Finally, the sea urchin genome contains an almost complete repertoire of genes encoding extracellular modulators of BMP signaling including Chordin, Noggin, Sclerotin, SFRP, Gremlin, DAN and Twisted gastrulation. Taken together, these findings indicate that the sea urchin complement of genes of the RTK and TGF-beta signaling pathways is qualitatively very similar to the repertoire present in vertebrates, and that these genes are part of the common genetool kit for intercellular signaling of deuterostomes.

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes.

The asymmetric positioning of internal organs on the left or right side of the body is highly conserved in vertebrates and relies on a Nodal signaling pathway acting on the left side of the embryo. Whether the same pathway also regulates left-right asymmetry in invertebrates and what is the evolutionary origin of the mechanisms controlling left-right determination are not known. Here, we show that nodal regulates left-right asymmetry in the sea urchin but that, intriguingly, its expression is reversed compared to vertebrates. Nodal signals emitted from the right side of the larva prevent the right coelomic pouch from forming the imaginal rudiment. Inhibition of Nodal signaling after gastrulation causes formation of an ectopic rudiment on the right side, leading to twinned urchins after metamorphosis. In contrast, ectopic activation of the pathway prevents formation of the rudiment. Our results show that the mechanisms responsible for left-right determination are conserved within basal deuterostomes.

In the sea urchin embryo, the oral-aboral axis is specified after fertilization by mechanisms that are largely unknown. We report that early sea urchin embryos express Nodal and Antivin in the presumptive oral ectoderm and demonstrate that these genes control formation of the oral-aboral axis. Overexpression of nodal converted the whole ectoderm into oral ectoderm and induced ectopic expression of the orally expressed genes goosecoid, brachyury, BMP2/4, and antivin. Conversely, when the function of Nodal was blocked, by injection of an antisense Morpholino oligonucleotide or by injection of antivin mRNA, neither the oral nor the aboral ectoderm were specified. Injection of nodal mRNA into Nodal-deficient embryos induced an oral-aboral axis in a largely non-cell-autonomous manner. These observations suggest that the mechanisms responsible for patterning the oral-aboral axis of the sea urchin embryo may share similarities with mechanisms that pattern the dorsoventral axis of other deuterostomes.