dataesr/scientific-paragraphs-categorization huggingface dataset:
These data are consistent with a massive accumulation of complexed iron in the absence of the MINPP1 enzyme, in the presence of high iron, and suggest the potential involvement of IP 6 -mediated abnormal cation homeostasis as the underlying disease mechanism. IP 6 accumulation causes cytosolic calcium depletion in mutant HEK293 cells and primary mouse neural progenitors. To further explore the involvement of the disruption of cellular cations homeostasis in PCH, we evaluated the intracellular free Ca 2+ levels in the absence of MINPP1, using the FLUO-4-AM calciumbinding indicator. Strikingly, we found a significant, close to 50% depletion of free basal Ca 2+ levels in MINPP1 -/-HEK293 cells, compared to control cell line (Fig. 5e ). To further validate the involvement of such calcium depletion in the neurological phenotype, we generated a CRISPR/Cas9-mediated Minpp1 -/- mouse model (Supplementary Fig.
Basal calcium levels influence neuronal physiology and cell survival  [47] [48] [49]  , and calcium signaling plays a role in neural induction and differentiation  [50] [51] [52] [53]. Consequently, a disruption in calcium homeostasis For (e), Ctrl vs. MINPP1 -/-: p = 0. 0021; for (f), Ctrl vs. Minpp1 -/-: p = 0. 007; for (g), Ctrl vs. MINPP1 -/-: p = 0. 0002; MINPP1 -/-vs. MINPP1 -/--MINPP1: p = 0. 0015; for h Ctrl vs. MINPP1 -/-: p = 0. 0018; MINPP1 -/-vs. MINPP1 -/--MINPP1: p = 0. 0402. For all panels, source data are provided as a source data file. could be involved in PCH disease pathogenesis. A link between MINPP1 and calcium regulation has been suggested previously but it was through the synthesis of I(1,4,5)P 3  54. Hypothetically, coupling the limitation of IP 6 -mediated chelation of calcium with the promotion of IP 3 synthesis could be an efficient way for MINPP1 to regulate calcium signaling dynamics and homeostasis.
05% following nucleofection and sorting. All iPSC lines were assessed for embryonic pluripotency markers OCT4, SOX2, SSEA-4, and TRA-1-81 (Supplementary Figs. 3a, b  and 6a,   b ). The absence of obvious chromosomal abnormalities was verified by CGH array 60 K (data not shown). The differentiation into mesoderm and endoderm lineage following embryoid body formation was assessed  64. In addition, no obvious differences in proliferation and apoptosis were observed among all the iPSC lines. iPSCs were maintained in vitronectin-coated (10 μg/ml; 07180, STEMCELL) 3. 5cm dishes (353001, Falcon) in complete mTeSR-1 medium (STEMCELL, 85850) supplemented with 1% Penicillin-Streptomycin (PS) (15140122, Gibco) in a humidified incubator (5% CO 2 , 37 °C). The media were replaced daily and iPSCs were mechanically passaged with ROCK inhibitor Y-27632 (10 μM; 72304, STEMCELL) every 7-8 days, with a 1:3 split ratio.
5 µg of DNA per well of a 6-well plate). Two days post transfection, single GFP + HEK293 cells were sorted into 96-well plates by Fluorescence-activated cell sorting (FACS) (BD FACSAria II SORP, BD Biosciences). Indel mutations of clones were detected by Sanger sequencing, and target editing efficiency was assessed by TIDE analysis (https://tide-calculator. nki. nl/; data not shown). For the generation of MINPP1 -/-iPSCs clones, transfection of pSpCas9 (sgRNA)-2A-GFP into iPSC line (Ctrl-I004) was performed using Amaxa 4D-Nucleofector X-Unit (AAF-1002X, Lonza) according to manufacturer's instructions. Briefly, iPSCs were pre-treated with Y-27632 (10 μM) for 1 h and further dissociated into single-cell suspension with Accutase (STEMCELL, 07920). 4 × 10 5 iPSCs were nucleofected per 20 μl nucleocuvette strip, using P3 Primary Cell 4D X Kit S (V4XP-3032, Lonza), 1 μg DNA per reaction, and the program CA-137.
After nucleofection, iPSCs were seeded on vitronectin pre-coated 12-well plates and cultured in complete mTeSR-1 medium supplemented with Y-27632 (10 μM). Two days post transfection, GFP + iPSCs were sorted by FACS and plated at low densities of 1-2 × 10 4 cells per 6 cm dish (Falcon, 353037) in mTeSR-1 medium supplemented with Y-27632 (10 μM). After 12-16 days, a subset of individual colonies was processed for DNA extraction using Quick-DNA/RNA Miniprep Plus Kit (D7003, Zymo) and Sanger sequenced. Target editing efficiency was assessed by TIDE analysis (https://tide-calculator. nki. nl/; data not shown). After clonal selection and expansion, no unusual chromosomal abnormalities were detected by CGH array (60 K, data not shown). Validation of MINPP1 -/-iPSC clones were further assessed by the absence of MINPP1 using western blot (Supplementary Fig.
SgRNAs were hybridized with Cas9 (wildtype) protein and injected into the pronucleus of the C57Bl/6J zygotes. Surviving zygotes were placed in KSOM medium (MR-106-D, Merck-Millipore) and cultured overnight to two-cell stage and then transferred into the oviduct of B6CBAF1 pseudo-pregnant females. The generated knockouts were validated by Sanger sequencing combined with tide TIDE analysis (https://tide-calculator. nki. nl/; data not shown). All Minpp1 -/-mice were backcrossed with C57BL/6J mice to remove potential off-targets. The Minpp1 -/-offspring were identified by polymerase chain reaction (PCR) genotyping (Supplementary Fig. 5a, b ). Brain histological characterization was performed with standard hematoxylin/eosin staining and analyzed with NDP. view. 2. Generation of stable HEK293 cell lines.
Next, the cell culture plate was left to shake for 15-30 min in the dark at room temperature, followed by transfer of the samples into flat-bottom 96-well microtiter plate (655101, Greiner). The optical density was read on a microplate reader at 560 nm (1681135, Biorad). Apoptosis assay. The apoptosis is measured using the Click-iT TUNEL Alexa Fluor 594 Imaging Assay (C10246, Thermofischer) according to manufacturer's instructions. For a positive control, cells are treated with tunicamycin (10 µg/ml, 3516/10, Biotechne) for 6 h to induce apoptosis (data not shown). For quantification analysis, a minimum of 5 fields per replicate were analyzed in ImageJ. The percentage of TUNEL + cells was counted relative to the total number of Hoechst + cells per field which was comparable (i. e. , Hoechst + ) between replicates and conditions.
The remaining cell lysate was mixed with equal volumes of 10 mM HCl and ironreleasing reagent (HCl 1. 4 M, KMnO 4 4. 5%) and incubated for 2 h at 60 °C. Once the samples reached room temperature, 30 μl of iron-detection reagent (6. 5 mM ferrozine, 6. 5 mM neocuproine, 2. 5 M ammonium acetate, and 1 M ascorbic acid) was added to each sample. After 30 min of incubation at room temperature, the samples were transferred to flat-bottom 96-well microtiter plate (Greiner, 655101). The optical density was read on a microplate reader at 560 nm (Biorad, 1681135). FAC was used as reference standard. The free iron levels (Fe 2+ and Fe 3+ ) were measured by using the Iron Assay Kit (Abcam, ab83366) according to manufacturer's instructions. In both iron-detection assays, the data are normalized against the protein levels, quantified via Pierce BCA Protein assay kit (23225, Thermo Scientific).
2% PBST for 1 h and further incubated overnight at 4 °C with the following primary antibodies diluted in either Odyssey-TM Blocking Buffer or 2. 5% dry milk diluted in 0. 2% PBST: anti-MINPP1 (1:2000, sc-10399, Santa-Cruz), anti-β Actin (1:5000, AM4302, Invitrogen). After three washes with 0. 2% PBST, the membranes were then incubated for 1 h at room temperature with HRP (1:10,000, sc-2314, sc-2020, Santa-Cruz) or IRDye-coupled (1:10,000, 925-68070, 925-32211, LICOR) secondary antibodies. After three washes with 0. 2% PBST, the membranes were developed either with HRP (ChemiDoc XRS, Biorad) or Odyssey CLx imaging system (LICOR) and analyzed with Image Studio Lite Ver 5. 2. Uncropped scan blots are available in the Source data file.
Acknowledgements The project is funded by the  French National Research Agency   ANR-16-CE12-0005-01  (V. C. ), the  Fondation pour la Recherche Médicale   FRM-DEQ20160334938  (L. C. and V. C. ), and  FRM-FDT201904007935  (E. U. ). This work was also supported by State funding from the  Agence Nationale de la Recherche  under " Investissements d'avenir" program  ( ANR-10-IAHU-01 ), the  Fondation Bettencourt Schueller , and the MSDAvenir fund ( DEVO-DECODE  project). A. S. and M. W. are supported by the  Medical Research Council (MRC)  award ( MR/T028904/1 ). We are grateful to families of patients for their participation and  Dr. B. Isidor  for the clinical-follow-up. We sincerely thank  S. Jaalouk ,  V. Meneghini ,  F. Petit , and  A. Rotig  for technical help and advice.
The growing interest in the lability of sex determination in non-model vertebrates such as amphibians and fishes has revealed high rates of sex chromosome turnovers among closely related species of the same clade. Can such lineages hybridize and admix with different sex-determining systems, or could the changes have precipitated their speciation? We addressed these questions in incipient species of toads (Bufonidae), where we identified a heterogametic transition and characterized their hybrid zone with genome-wide markers (RADseq). Adult and sibship data confirmed that the common toad B. bufo is female heterogametic (ZW), while its sister species the spined toad B. spinosus is male heterogametic (XY). Analysis of a fine scale transect across their parapatric ranges in southeastern France unveiled a narrow tension zone (∼10 km), with asymmetric mitochondrial and nuclear admixture over hundreds of kilometers southward and northward, respectively.
g. ,  Malone and Fontenot 2008). Nevertheless, sex chromosome turnovers and male-heterogamety do occur in this family  (Stöck et al. 2011). In Bufo, the experiments of  Ponse (1950)  and  Rostand (1952 Rostand ( ,1953) )  suggested contradictory patterns of heterogamety depending on the geographic origins of specimens. Recently,  Skorinov et al. (2018)  characterized a karyotypic dimorphism specific to males in B. spinosus, based on a few individuals. Hence, B. bufo and B. spinosus could differ in their heterogametic sex chromosomes (ZW versus XY), which in turn would allow testing whether such a recent turnover have affected their propensity to admix at range margins, and precipitated their incipient speciation. In this study, we revisit the B. bufo/spinosus hybrid zone with genome-wide data (RADseq), specifically to (i) confirm a putative heterogametic transition of sex-determining systems between the two species and (ii) illuminate their patterns of admixture along an extensive transect in southeastern France.
Sequences were manually aligned in Seaview  (Gouy et al. 2010 ) and matched against reference haplotypes from the two species (GenBank sequences from  Recuero et al. 2012). Finally, we further incorporated the average mitochondrial and nuclear allele frequencies of B. bufo/spinosus obtained for ∼200 populations of southeastern France and nearby Italy by  Arntzen et al. (2017, taken  from their tables S1 and S4, respectively), for complementation and comparison.
SCREENING FOR SEX-LINKED LOCI Sexed adults and siblings of B. bufo (libraries 1 and 3), as well as adults of B. spinosus (library 2), were screened for putative sexlinked markers, applying two independent approaches. The first approach, SLM finder, is an ad hoc pipeline developed to identify SNPs that show sex biases in allele frequencies (method I), heterozygosity (method II), or RADseq tags that are specific to only one sex (method III)  (Breslford et al. 2016b; Jeffries et al. 2018). For this, loci were constructed and SNPs were called using Stacks version 1. 48. Default parameters were used for the Ustacks and Cstacks modules, as these represented the best balance between stringency and data inclusion. For the Populations module, SNP genotypes were retained if the locus coverage was at least 8 reads (-m 8), if called for ≥80% of individuals of each sex (-r 0.
8, -p 2) and if heterozygosity across the whole dataset was not greater than 75% (-max_obs_het 0. 75), to remove over merged paralogous loci. In order to setup the optimal parameters with which to identify sex-linked markers, we ran SLM finder for 24 different combinations of the parameters underlying methods I and II (heterogametic and homogametic thresholds), and for six parameter values of method III (sex specificity threshold; Table  S2 ). For each set, we performed a permutation test whereby male and female sex assignments were randomly shuffled across the entire sample set 100 times, SLM finder was run, and the number of putative sex-linked markers obtained was recorded. This null distribution was then compared to the number of sex-linked markers flagged on the observed data (i. e. , real sex assignments).
If this number fell above the 95th percentile of the null distribution, we considered sex linkage to be significant (P < 0. 05). For a given method, we could further select the optimal parameter set as the one minimizing false positives, that is, showing the smallest proportion of sex-linked markers in the null distribution relative to the number of sex-linked markers in the observed data. The second approach, RadSex (https://github. com/ RomainFeron/RadSex), identifies sex specific RADseq tags or alleles directly from the raw read data (i. e. , without locus assembly or SNP calling), and compare their presence among samples of each sex. This approach has the advantage that it does not require the assembly of loci, which has two important consequences: (i) it is sensitive to only one parameter, namely the coverage of unique RAD tags (-min_cov), hence reducing the potential for parameter biases; (ii) complex loci that are usually discarded by assembling pipelines such as Stacks (e.
HYBRID ZONE ANALYSES The 200 hybrid zone samples (library 4) were processed with the denovo. pl pipeline of Stacks. Default stacking parameters were again used for Ustacks and Cstacks modules. In a first step, we filtered for SNPs genotyped in at least 90% of the samples (-r 0. 9) in all localities (-p 21), which yielded 8,560 SNPs (99. 3% of matrix completeness). The genetic structure of this initial dataset was explored by a Principal Component Analysis (PCA) on individual genotypes with the R package adegenet  (Jombart 2008). For specific hybrid zone analyses, we then called speciesdiagnostic SNPs, as those fixed between populations at the edges of our study area, specifically, between loc. 1-3 (B. spinosus) and loc. 20-21 (B. bufo), retaining only one SNP per RADseq tag, which yielded 950 SNPs.
We performed a PCA as above, and estimated the ancestry of individuals to either species with the Bayesian clustering algorithm of STRUCTURE  (Pritchard et al. 2000) , focusing on runs with K = 2. We applied the admixture model without pre-assignment of samples, and performed 10 replicate chains of 100,000 iterations, after a burn-in of 10,000. Replicates were combined with CLUMPP  (Jakobsson and Rosenberg 2007). For each population, we computed observed heterozygosity (H o ) with the R package hierfstat  (Goudet 2005) , linkage disequilibrium (correlation coefficient R 2 ) averaged over all pairs of loci with the R package genetics  (Warnes et al. 2013) , and the admixture linkage disequilibrium (D') as the variance in individual hybrid index  (Barton and Gale 1993) , here using the genome-wide individual ancestry obtained with STRUCTURE (individual Q scores) as a proxy.
SPECIES DISTRIBUTION MODELING To grasp whether environmental factors (adaptation to different ecological niches) contributed to mediate the transition between B. bufo and B. spinosus, we built species distribution models. We applied recent methodological recommendations to compute robust ecological niche models with MaxEnt 3. 4. 1  (Phillips et al. 2006) , such as occurrence filtering, using multiple combinations of model parameters (features and regularization multipliers), and multiple statistical criteria for model selection (partial ROC, omission rates, and AICc). A total of 14,504 localities known of B. bufo and B. spinosus were initially considered, combining our own records, museum collections, and previously published data. We first filtered this dataset to avoid spatial autocorrelation and duplication using NicheToolBox  (Osorio-Olvera et al.
2018). We then retained the localities at least 10 km (0. 093°) apart (see  Brown 2014) , and the final dataset comprised 3212 records for B. bufo and 886 records for B. spinosus (see Fig. S2 ). To compute the models, altitude and 19 bioclimatic layers representative of the climatic data over ∼1950-2000 were extracted from the WorldClim 1. 4 database (http://www. worldclim. org). Ten additional layers were considered: the aridity index (Global Aridity and Potential Evapo-Transpiration; http://www. cgiar-csi. org/data/global-aridity-and-pet-database), the global percent of tree coverage (https://github. com/globalmaps/gm_ ve_v1), and eight land cover variables (spatial homogeneity of global habitat, broadleaf forests, needleleaf forests, mixed forests, shrubs, barren, herbaceous, and cultivated vegetation; https://www.
earthenv. org/). To also consider topography in the model, four landscape layers were calculated with QGIS: aspect, exposition, slope, and terrain roughness index. These layers had a 30 arc seconds spatial resolution. All analyses were conducted under the WGS 84 projection with species-specific masks covering the areas of occurrence of B. bufo and B. spinosus. To eliminate predictor collinearity before generating these models, we calculated Pearsons's correlation coefficients for all pairs of bioclimatic variables using ENMTools  (Warren et al. 2010). For correlated pairs (│r│> 0. 75), the variable that appeared the most biologically important for the species was retained. The resulting dataset contained ten bioclimatic variables for B. bufo: Bio 1 (annual mean temperature; °C × 10), Bio 2 (mean diurnal range; °C × 10), Bio 3 (isothermality; Bio2/Bio7 × 100), Bio7 (temperature annual range; °C × 10), Bio 8 (mean temperature of wettest quarter; °C × 10), Bio 14 (precipitation of driest month; mm), Bio 15 (precipitation seasonality; CV), Bio 16 (precipitation of wettest quarter; mm), Bio 18 (precipitation of warmest quarter; mm), and Bio 19 (precipitation of coldest quarter; mm); and five bioclimatic variables for B.
SEX-LINKED MARKERS Testing different parameter combination using a permutation approach allowed to set up the optimal parameters set of SLM finder, which were used to identify putative sex-linked markers from the adult and sibling datasets of B. bufo, as well as the adult dataset of B. spinosus (Fig. S3  and Table  S2 ). For B. bufo, the adult dataset comprised 146,292,576, reads after demultiplexing, which contained 35,084 SNPs after the Stacks processing (average coverage = 32. 3 reads/locus). No support was found for either XY or ZW patterns of sex-linkage in this dataset, regardless of the parameter combination (Table  1 ). In contrast, the RadSex approach highlighted one significantly sexlinked marker consistent with a ZW system, that is, a sequence found among most of the females (19/20), but none of the males (0/21; Fig.
1 , left panel). This sequence contains an indel polymorphism and was therefore discarded by the Stacks pipeline, which is why it was not present (and flagged) among the loci screened by SLM finder. The B. bufo sibling dataset comprised 122,605,407 reads after demultiplexing, which contained 12,840 SNPs after the Stacks processing (average coverage = 39. 1 reads/locus). SLM finder tests also found evidence for a ZW system from this data. With the optimal thresholds, the frequency and heterozygosity methods of SLM finder reported 16 and 15 putative ZW loci, respectively (15 supported by both methods; Table  1 ). Only one putative XY marker was identified (by the heterozygosity method), which fell within the expected rate of false positive (Fig. S3 ). Note that for the heterozygosity method, more relaxed parameters on the homogametic threshold yielded a significant male-heterogametic signal (Fig.
S3 ). This could reflect Z-specific polymorphisms between the father's Zs (ZZ) and the mother's Z (ZW), hence mimicking an XY-like allele segregation in the full-sib clutch, as demonstrated for similar datasets (e. g. , Rana montezumae,  Jefrries et al. 2018). RadSex also suggested a ZW system among the sibling dataset: many tags were sequenced exclusively among the females (11-13/17), although the bias was not significant (Fig. 1 , central panel). The B. spinosus adult dataset comprised 452,840,434 reads after demultiplexing, which contained 28,090 SNPs (average coverage = 37. 2 reads/locus). SLM finder found strong evidence for an XY system (Table  1 ). Altogether, a total of 104 XY loci were flagged, 89 of them being detected by both the allele frequency and the heterozygosity methods, and in highly significant numbers (Fig.
HYBRID ZONE ANALYSES The distributions of the B. bufo and B. spinosus genomes in southeastern France are displayed in Fig. 2 , according to RADseq, introns and mitochondrial markers. Our initial RADseq dataset (8,560 SNPs) recovered the two gene pools as the main source of genetic structure, which built the first PCA component (PC1 in Fig. S4 ). It also highlighted some intraspecific structure: the southernmost B. spinosus samples (loc. 1-2), and the northernmost B. bufo samples (loc. 20-21) form the second and third PCA components, respectively (Fig. S4 ). As expected, the 950 SNPs fixed between the groups of populations at the edges of our transect (loc. 1-3 and loc. 20-21) mostly captured the genetic differentiation between the two species (63. 8% of variance explained by PC1 in Fig. S4 ).
The STRUCTURE ancestry coefficients obtained from this dataset closely matched the PC1 scores, indicative of robust inferences (Fig. S5 ). The clustering analysis supported a sharp nuclear transition between our loc. 14 and 15, located just 9 km apart in the hills north of Romans-sur-Isère (Fig. 2 ), associated with an increase of heterozygosity (H o ) and linkage disequilibrium (Fig. S6 ). Fainted traces of admixture were detected up to a hundred kilometers in either direction (loc. 7-14 and loc. 15-20, Fig. 2 ), but all ancestry coefficients reached above 0. 9, even close to the putative center (Fig. 2 , Fig. S7 ). Accordingly, the cline fitted to the average population ancestry (STRUCTURE's Q) along our transect was only 8. 6 km wide, with the best model involving mirrored introgression tails on both sides (Fig.
2018). Whether this heterogametic transition involves the same chromosome pair remains to be determined. No genome assembly is yet available for bufonids, and insufficient numbers of putatively sex-linked markers could be aligned to published references. The male-specific karyotype dimorphism found in B. spinosus by  Skorinov et al. (2018)  was on chromosome 6 (like in Strauchbufo radei,  Deng and Shang 1984) , but this needs clarification with additional samples. The amount of XY markers flagged here (Table  1 ) is comparable to that of homomorphic sex chromosomes identified in other amphibians with similar datasets and methodologies  (Brelsford et al. 2016b; Jeffries et al. 2018 ). We also found only two Y-specific tags (Table  1 ), which suggests little molecular differentiation between the Y and the X.
ACKNOWLEDGMENTS This study was only possible thanks to many fieldwork contributions by  B. Adam ,  L. Beligné ,  D. Combrisson ,  L. Courmont ,  D. Dufresnes ,  M. Dufresnes ,  R. Duguet ,  S. Gardien ,  J. M. Ferro ,  C. Jacquier ,  P. Joly ,  M Lathuillière ,  Q. Martinez ,  A. Movia ,  G. Maillet ,  L. Parrain ,  N. Parrain ,  M. Pezin ,  M. Pajković ,  P. Priol ,  A. Roux ,  C. Roy ,  A. Sprumont , V. Thary. We also thank  J. W. Arntzen  for useful discussions and  B. Wielstra  for comments on a previous version of the manuscript. This study was funded by the  Swiss National Science Foundation  (grant no. 31003A_166323  to N. P. and fellowship no. P2LAP3_171818  to C. D. ). S. N. L. was supported by the  RFBR  research project  20-04-00918.