LIM-HD transcription factors control axial patterning and specify distinct neuronal and intestinal cell identities in planarians.

Adult planarians can regenerate the gut, eyes and even a functional brain. Proper identity and patterning of the newly formed structures require signals that guide and commit their adult stem cells. During embryogenesis, LIM-homeodomain (LIM-HD) transcription factors act in a combinatorial 'LIM code' to control cell fate determination and differentiation. However, our understanding about the role these genes play during regeneration and homeostasis is limited. Here, we report the full repertoire of LIM-HD genes in Schmidtea mediterranea. We found that lim homeobox (lhx) genes appear expressed in complementary patterns along the cephalic ganglia and digestive system of the planarian, with some of them being co-expressed in the same cell types. We have identified that Smed-islet1, -lhx1/5-1, -lhx2/9-3, -lhx6/8, -lmx1a/b-2 and -lmx1a/b-3 are essential to pattern and size the planarian brain as well as for correct regeneration of specific subpopulations of dopaminergic, serotonergic, GABAergic and cholinergic neurons, while Smed-lhx1/5.2 and -lhx2/9.2 are required for the proper expression of intestinal cell type markers, specifically the goblet subtype. LIM-HD are also involved in controlling axonal pathfinding (lhx6/8), axial patterning (islet1, lhx1/5-1, lmx1a/b-3), head/body proportions (islet2) and stem cell proliferation (lhx3/4, lhx2/9-3, lmx1a/b-2, lmx1a/b-3). Altogether, our results suggest that planarians might present a combinatorial LIM code that controls axial patterning and axonal growing and specifies distinct neuronal and intestinal cell identities.

Analysis of the P. lividus sea urchin genome highlights contrasting trends of genomic and regulatory evolution in deuterostomes.

Sea urchins are emblematic models in developmental biology and display several characteristics that set them apart from other deuterostomes. To uncover the genomic cues that may underlie these specificities, we generated a chromosome-scale genome assembly for the sea urchin Paracentrotus lividus and an extensive gene expression and epigenetic profiles of its embryonic development. We found that, unlike vertebrates, sea urchins retained ancestral chromosomal linkages but underwent very fast intrachromosomal gene order mixing. We identified a burst of gene duplication in the echinoid lineage and showed that some of these expanded genes have been recruited in novel structures (water vascular system, Aristotle's lantern, and skeletogenic micromere lineage). Finally, we identified gene-regulatory modules conserved between sea urchins and chordates. Our results suggest that gene-regulatory networks controlling development can be conserved despite extensive gene order rearrangement.

FoxK1 is Required for Ectodermal Cell Differentiation During Planarian Regeneration.

Forkhead box (Fox) genes belong to the "winged helix" transcription factor superfamily. The function of some Fox genes is well known, such as the role of foxO in controlling metabolism and longevity and foxA in controlling differentiation of endodermal tissues. However, the role of some Fox factors is not yet well characterized. Such is the case of FoxK genes, which are mainly studied in mammals and have been implicated in diverse processes including cell proliferation, tissue differentiation and carcinogenesis. Planarians are free-living flatworms, whose importance in biomedical research lies in their regeneration capacity. Planarians possess a wide population of pluripotent adult stem cells, called neoblasts, which allow them to regenerate any body part after injury. In a recent study, we identified three foxK paralogs in the genome of Schmidtea mediterranea. In this study, we demonstrate that foxK1 inhibition prevents regeneration of the ectodermal tissues, including the nervous system and the epidermis. These results correlate with foxK1 expression in neoblasts and in neural progenitors. Although the triggering of wound genes expression, polarity reestablishment and proliferation was not affected after foxK1 silencing, the apoptotic response was decreased. Altogether, these results suggest that foxK1 would be required for differentiation and maintenance of ectodermal tissues.

Decoding Stem Cells: An Overview on Planarian Stem Cell Heterogeneity and Lineage Progression.

Planarians are flatworms capable of whole-body regeneration, able to regrow any missing body part after injury or amputation. The extraordinary regenerative capacity of planarians is based upon the presence in the adult of a large population of somatic pluripotent stem cells. These cells, called neoblasts, offer a unique system to study the process of stem cell specification and differentiation in vivo. In recent years, FACS-based isolation of neoblasts, RNAi functional analyses as well as high-throughput approaches such as single-cell sequencing have allowed a rapid progress in our understanding of many different aspects of neoblast biology. Here, we summarize our current knowledge on the molecular signatures that define planarian neoblasts heterogeneity, which includes a percentage of truly pluripotent stem cells, and guide the commitment of pluripotent neoblasts into lineage-specific progenitor cells, as well as their differentiation into specific planarian cell types.

MAPK and GSK3/ß-TRCP-mediated degradation of the maternal Ets domain transcriptional repressor Yan/Tel controls the spatial expression of nodal in the sea urchin embryo.

In the sea urchin embryo, specification of the dorsal-ventral axis critically relies on the spatially restricted expression of nodal in the presumptive ventral ectoderm. The ventral restriction of nodal expression requires the activity of the maternal TGF-ß ligand Panda but the mechanism by which Panda restricts nodal expression is unknown. Similarly, what initiates expression of nodal in the ectoderm and what are the mechanisms that link patterning along the primary and secondary axes is not well understood. We report that in Paracentrotus lividus, the activity of the maternally expressed ETS-domain transcription factor Yan/Tel is essential for the spatial restriction of nodal. Inhibiting translation of maternal yan/tel mRNA disrupted dorsal-ventral patterning in all germ layers by causing a massive ectopic expression of nodal starting from cleavage stages, mimicking the phenotype caused by inactivation of the maternal Nodal antagonist Panda. We show that like in the fly or in vertebrates, the activity of sea urchin Yan/Tel is regulated by phosphorylation by MAP kinases. However, unlike in the fly or in vertebrates, phosphorylation by GSK3 plays a central role in the regulation Yan/Tel stability in the sea urchin. We show that GSK3 phosphorylates Yan/Tel in vitro at two different sites including a ß-TRCP ubiquitin ligase degradation motif and a C-terminal Ser/Thr rich cluster and that phosphorylation of Yan/Tel by GSK3 triggers its degradation by a ß-TRCP/proteasome pathway. Finally, we show that, Yan is epistatic to Panda and that the activity of Yan/Tel is required downstream of Panda to restrict nodal expression. Our results identify Yan/Tel as a central regulator of the spatial expression of nodal in Paracentrotus lividus and uncover a key interaction between the gene regulatory networks responsible for patterning the embryo along the dorsal-ventral and animal-vegetal axes.

Characterization of celiac disease related oat proteins: bases for the development of high quality oat varieties suitable for celiac patients.

Some studies have suggested that the immunogenicity of oats depends on the cultivar. RP-HPLC has been proposed as a useful technique to select varieties of oats with reduced immunogenicity. The aim of this study was to identify both the avenin protein patterns associated with low gluten content and the available variability for the development of new non-toxic oat cultivars. The peaks of alcohol-soluble avenins of a collection of landraces and cultivars of oats have been characterized based on the RP-HPLC elution times. The immunotoxicity of oat varieties for patients with celiac disease (CD) has been tested using a competitive ELISA based on G12 monoclonal antibody. The oat lines show, on average, seven avenin peaks giving profiles with certain similarities. Based on this similarity, most of the accessions have been grouped into avenin patterns. The variability of RP-HPLC profiles of the collection is great, but not sufficient to uniquely identify the different varieties of the set. Overall, the immunogenicity of the collection is less than 20 ppm. However, there is a different distribution of toxicity ranges between the different peak patterns. We conclude that the RP-HPLC technique is useful to establish groups of varieties differing in degree of toxicity for CD patients.

Compliance with objectives based on different guidelines (KDIGO/S.E.N.) and analysis of the individual variability of mineral metabolism in haemodialysis patients in the medium term.

OBJECTIVE: To assess the level of compliance and variability of mineral metabolism parameters over time in a sample of haemodialysis patients for the different ranges proposed (KDIGO guidelines/S.E.N recommendations) in both groups and individuals continuously. MATERIAL AND METHOD: Every four months, we collected data on calcium, phosphorus, PTH and treatment in a sample of 44 patients followed up continuously for 32 months. We established the percentages of patients who complied with the objectives set for each parameter in both ranges: optimal (KDIGO) and acceptable (S.E.N.) in each control and the percentage that individually complied with the objectives in at least 75% of the determinations. RESULTS: Compliance with the objective using the optimal range improved, although PTH did not exceed 50%. Using the acceptable range, the objective was achieved in the three parameters in over 70% and over 50% of patients achieved the three simultaneously while using the optimal range, 30% was never achieved. Individually, compliance with the optimal range was continuously achieved in 52.3% (calcium), 45.5% (phosphorus) and in only one patient in PTH, while when using the acceptable range, compliance was achieved in 84.1% (calcium) and 70.5% (phosphorus and PTH). CONCLUSIONS: The use of less stringent criteria than the KDIGO guidelines in calcium, phosphorus and PTH objectives allows patients to remain continuously within appropriate ranges with less intervention and less individual variability.

Cumplimiento de objetivos en función de diferentes guías (KDIGO/S. E. N. ) y análisis de la variabilidad individual del metabolismo mineral en pacientes en hemodiálisis a medio plazo / Compliance with objectives based on different guidelines (KDIGO/S. E. N. ) and analysis of the individual variability of mineral metabolism in haemodialysis patients in the medium term

Objetivos: Valorar el grado de cumplimiento y la variabilidad de los parámetros del metabolismo mineral a lo largo del tiempo en una muestra de pacientes en hemodiálisis según los distintos rangos propuestos (Guía KDIGO/Recomendaciones S.E.N.) tanto a nivel de grupo como individual de forma continuada. Material y métodos: Recogimos cuatrimestralmente los datos de calcio, fósforo, hormona paratiroidea (PTH) y de tratamiento en una muestra de 44 pacientes seguidos de forma continuada durante 32 meses. Establecimos los porcentajes de pacientes que cumplían los objetivos propuestos para cada parámetro en ambos rangos: óptimo (KDIGO) y aceptable (S.E.N.) en cada control y el porcentaje que cumplían individualmente los objetivos en al menos un 75 % de las determinaciones. Resultados: El cumplimiento del objetivo utilizando el rango óptimo mejoró, aunque en la PTH no superó el 50 %. Utilizando el rango aceptable, el objetivo se alcanzaba en los tres parámetros por encima del 70 % y más del 50 % de los pacientes cumplían los tres simultáneamente, mientras que utilizando el rango óptimo nunca se alcanzó el 30 %. A nivel individual el cumplimiento del rango óptimo se alcanzó de forma continuada en el 52,3 % (calcio), 45,5% (fósforo) y solo un paciente en la PTH, mientras que utilizando el rango aceptable el cumplimiento se alcanzó en el 84,1 % (calcio) y el 70,5 % (fósforo y PTH). Conclusiones: El uso de criterios menos exigentes que los KDIGO en los objetivos de calcio, fósforo y PTH permite mantener de forma continuada a los pacientes en rangos adecuados con menor intervencionismo y menor variabilidad individual

Objective: To assess the level of compliance and variability of mineral metabolism parameters over time in a sample of haemodialysis patients for the different ranges proposed (KDIGO guidelines/S.E.N recommendations) in both groups and individuals continuously. Material and method: Every four months, we collected data on calcium, phosphorus, PTH and treatment in a sample of 44 patients followed up continuously for 32 months. We established the percentages of patients who complied with the objectives set for each parameter in both ranges: optimal (KDIGO) and acceptable (S.E.N.) in each control and the percentage that individually complied with the objectives in at least 75% of the determinations. Results: Compliance with the objective using the optimal range improved, although PTH did not exceed 50%. Using the acceptable range, the objective was achieved in the three parameters in over 70% and over 50% of patients achieved the three simultaneously while using the optimal range, 30% was never achieved. Individually, compliance with the optimal range was continuously achieved in 52.3% (calcium), 45.5% (phosphorus) and in only one patient in PTH, while when using the acceptable range, compliance was achieved in 84.1% (calcium) and 70.5% (phosphorus and PTH). Conclusions: The use of less stringent criteria than the KDIGO guidelines in calcium, phosphorus and PTH objectives allows patients to remain continuously within appropriate ranges with less intervention and less individual variability (AU)

Nodal: master and commander of the dorsal-ventral and left-right axes in the sea urchin embryo.

Recent studies suggest that specification of the dorsal-ventral and left-right axes of the sea urchin embryo relies on Nodal-expressing signalling centres located in the ventral ectoderm and in the archenteron that share striking similarities with vertebrate organising centres. Nodal and its downstream target BMP2/4 pattern all three germ layers along the dorsal-ventral axis, repress neural fates and control morphogenesis of the larva. Moreover, Nodal establishes left-right asymmetry by repressing formation of the adult rudiment and inhibiting germline cells differentiation on the right side, while BMP2/4 promotes expression of mesodermal genes on the left side. These findings provide a framework for future studies and raise new questions regarding the events upstream and downstream of Nodal and BMP signalling during axis formation.

Noggin and noggin-like genes control dorsoventral axis regeneration in planarians.

Planarians regenerate a whole animal from a small body piece within a few days. Recent studies have shown that the bone morphogenetic protein (BMP) pathway is required to reestablish the dorsoventral (DV) axis. In vertebrates, the specification of the DV axis depends on the coordinated action of a dual organizer defined by BMP and antidorsalizing morphogenetic protein (ADMP) under the control of several factors, including the inhibitors chordin and noggin. Planarians have an expanded noggin family (up to ten members), which have been classified as canonical noggin (nog) and noggin-like (nlg) genes, the latter carrying an insertion within the noggin domain. Here we show that a BMP/ADMP organizer governs DV axis reestablishment during planarian regeneration, highlighting a greater-than-thought conservation of the mechanisms that establish this axis in protostomes and deuterostomes. Also, we report that whereas noggin genes function as canonical BMP inhibitors, the silencing of planarian nlg8 induces ectopic neurogenesis and enhances ventralizing bmp(RNAi) phenotypes. Finally, we show that noggin-like genes are conserved from cnidarian to vertebrates and that both planarian nlg8 and Xenopus nlg ventralize Xenopus embryos when overexpressed. Remarkably, this ventralization is not associated with an increase in SMAD1/5/8 phosphorylation.

Expression pattern of the expanded noggin gene family in the planarian Schmidtea mediterranea.

Noggin genes are mainly known as inhibitors of the Bone Morphogenetic Protein (BMP) signalling pathway. Noggin genes play an important role in various developmental processes such as axis formation and neural differentiation. In vertebrates, inhibition of the BMP pathway is usually carried out together with other inhibitory molecules: chordin and follistatin. Recently, it has been shown in planarians that the BMP pathway has a conserved function in the maintenance and re-establishment of the dorsoventral axis during homeostasis and regeneration. In an attempt to further characterize the BMP pathway in this model we have undertaken an in silico search of noggin genes in the genome of Schmidtea mediterranea. In contrast to other systems in which between one and four noggin genes have been reported, ten genes containing a noggin domain are present in S. mediterranea. These genes have been classified into two groups: noggin genes (two genes) and noggin-like genes (eight genes). Noggin-like genes are characterized by the presence of an insertion of 50-60 amino acids in the middle of the noggin domain. Here, we report the characterization of this expanded family of noggin genes in planarians as well as their expression patterns in both intact and regenerating animals. In situ hybridizations show that planarian noggin genes are expressed in a variety of cell types located in different regions of the planarian body.

The BMP pathway is essential for re-specification and maintenance of the dorsoventral axis in regenerating and intact planarians.

The bone morphogenetic protein (BMP) pathway has been shown to play an important role in the establishment of the dorsoventral axis during development in both vertebrate and invertebrate species. In an attempt to unravel the role of BMPs in pattern formation during planarian regeneration, we studied this signaling pathway in Schmidtea mediterranea. Here, we functionally characterize planarian homologues of two key elements of the pathway: Smed-BMP and Smed-Smad1. Whole-mount in situ hybridization showed that Smed-BMP is expressed at the planarian dorsal midline, suggesting a role in dorsoventral patterning, while Smed-Smad1 is widely expressed throughout the mesenchyme and in the central nervous system. RNA interference (RNAi) knockdowns of Smed-BMP or Smed-Smad1 led to the disappearance of dorsal markers along with the ectopic expression of ventral markers on the dorsal side of the treated animals. In almost all cases, a duplicated central nervous system differentiated dorsally after Smed-BMP or Smed-Smad1 RNAi. These defects were observed not only during regeneration but also in intact non-regenerating animals. Our results suggest that the BMP signaling pathway is conserved in planarians and that it plays a key role in the regeneration and maintenance of the dorsoventral axis.