A multiscale analysis of early flower development in Arabidopsis provides an integrated view of molecular regulation and growth control.

We have analyzed the link between the gene regulation and growth during the early stages of flower development in Arabidopsis. Starting from time-lapse images, we generated a 4D atlas of early flower development, including cell lineage, cellular growth rates, and the expression patterns of regulatory genes. This information was introduced in MorphoNet, a web-based platform. Using computational models, we found that the literature-based molecular network only explained a minority of the gene expression patterns. This was substantially improved by adding regulatory hypotheses for individual genes. Correlating growth with the combinatorial expression of multiple regulators led to a set of hypotheses for the action of individual genes in morphogenesis. This identified the central factor LEAFY as a potential regulator of heterogeneous growth, which was supported by quantifying growth patterns in a leafy mutant. By providing an integrated view, this atlas should represent a fundamental step toward mechanistic models of flower development

Single-cell approaches for understanding morphogenesis using Computational Morphodynamics

In multicellular organisms cells grow, divide and adopt different fates, resulting in tissues and organs with specific functions. In recent years, a number of studies have brought quantitative knowledge about how these processes are orchestrated, shed-ding new light on cells as active and central players in morphogenesis. We explore recent advances in understanding plant morphogenesis from a quantitative perspective, defining the re-search field of Computational Morphodynamics. The focus is on studies combining theoretical and experimental approaches integrating hypotheses of how molecular and mechanical regulation at the cellular level lead to tissue behaviour. Finally, we dis-cuss some of the main challenges for future work

Computational Morphodynamics: A Modeling Framework to Understand Plant Growth

Computational morphodynamics utilizes computer modeling to understand the development of living organisms over space and time. Results from biological experiments are used to construct accurate and predictive models of growth. These models are then used to make novel predictions that provide further insight into the processes involved, which can be tested experimentally to either confirm or rule out the validity of the computational models. This review highlights two fundamental challenges: (a) to understand the feedback between mechanics of growth and chemical or molecular signaling, and (b) to design models that span and integrate single cell behavior with tissue development. We review different approaches to model plant growth and discuss a variety of model types that can be implemented to demonstrate how the interplay between computational modeling and experimentation can be used to explore the morphodynamics of plant development

Role of histone H3 variant, HTR6, during stress response

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 18-10-2019Esta tesis tiene embargado el acceso al texto completo hasta el 18-04-2021Chromatin organization and function are essential for development and growth of all organisms as well as for their response to environmental challenges. Histones are key to channel intra- and extracellular signals and modulate chromatin dynamics, ultimately influencing genome functions. This is primarily achieved by post-translational modifications of histones and the exchange of canonical by variant histones. Here, we have sought to define the role of Arabidopsis thaliana HTR6, a histone H3 variant of unknown function. We found that HTR6 expression is induced in plants exposed to abscisic acid (ABA), a hormone that is highly involved in several types of abiotic stresses, such as to high salt. A spatiotemporal expression analysis in ABA-treated roots unveiled that HTR6 is expressed in the transition/elongation zone, where cells switch from the mitotic cell cycle to the endocycle and increase their nuclear ploidy. HTR6 expression is largely excluded from the root apical meristem as well as also from the differentiated zone. Moreover, HTR6 expression is confined to external cell layers, such as epidermis and cortex. At the cellular level, we found that HTR6 is mainly expressed during the S-phase in endocycling cells. Interestingly, its incorporation into chromatin relies on the HIRA and DEK3 chaperones and is independent of CAF-1. Consistent with this, HTR6 is a euchromatic histone deposited in a DNA replication-independent manner. This is a feature shared with the histone H3.3 variant. However, HTR6 possesses unique amino acid residues, with F41 being a striking one because it is shared with the canonical H3.1. We found that F41 residue is important to increase HTR6 turnover, as revealed by the increased amount of HTR6 carrying a F41Y mutation. This is similar to the role of the F41 of H3.1 in euchromatic regions. Other changes in the C-terminal region of HTR6 to the amino acids present in H3.3, such as N63I, V124I or A132G, are sufficient to increase HTR6 accumulation. In particular, the A132 residue of HTR6 seems important for the proteasome-mediated degradation of HTR6 beyond the transition zone. HTR6 plays a crucial role in restricting growth in response to ABA and salt, as demonstrated by the larger root length, root apical meristem and higher cortical cell number of the htr6-1 loss of function seedlings. Since the ploidy level of htr6-1 root nuclei remains unaffected, HTR6 might play a role in controlling the boundary between the root apical meristem and the transition/ elongation zone in response to stress. Furthermore, our transcriptomic analysis showed that the genes that failed to be activated in the htr6-1 mutant early after ABA treatment (10 minutes), are involved in cell wall biogenesis genes and lipid metabolism, and encode members of the TCP family of transcription factors. Furthermore, several LEA family genes failed to be downregulated in the htr6-1 mutant. Together, our studies have uncovered a key role of HTR6, a previously uncharacterized histone H3 variant, in the early response of Arabidopsis to the abiotic stressLa organización y la función de la cromatina son esenciales para el crecimiento y desarrollo de los organismos, y para su respuesta a cambios ambientales. Las histonas canalizan las señales intra y extracelulares para modular la dinámica de la cromatina y las funciones del genoma. Esto se consigue principalmente mediante modificaciones post-traduccionales de las histonas y la sustitución de las formas canónicas por sus variantes. En este trabajo hemos querido definir el papel de HTR6, una variante de la H3 de Arabidopsis thaliana de función desconocida. La expresión de HTR6 se induce tras exposición a ácido abscísico (ABA), una hormona involucrada en respuesta a estrés abiótico. El análisis espacio-temporal en raíces tratadas con ABA reveló que HTR6 se expresa entre las zonas de transición y elongación, donde las células inician los ciclos de endorreplicación e incrementan la ploidía. No se ha detectado expresión de HTR6 ni en el meristemo apical de la raíz ni en la zona de diferenciación. Asimismo, la expresión de HTR6 está restringida a las capas celulares externas (epidermis y córtex). A nivel celular, HTR6 se expresa durante la fase S de células en endorreplicación. Su incorporación a cromatina depende de las chaperonas HIRA y DEK3, siendo independiente de CAF-1. Así, HTR6 es una histona presente en eucromatina, depositada de manera independiente a la replicación del ADN. Esta es una característica compartida con la variante H3.3. Sin embargo, HTR6 posee aminoácidos específicos, siendo F41 de los más interesantes ya que se encuentra en H3.1. F41 es importante para su eliminación del nucleosoma, ya que la mutación F41Y aumenta su permanencia en cromatina, de manera similar a la función de F41 de H3.1 en eucromatina. Otros cambios en la región C-terminal de HTR6 a los presentes en H3.3, como N63I, V124I o A132G, son suficientes para incrementar la acumulación de HTR6. El residuo A132 de HTR6 parece tener un papel en la degradación de HTR6 por el proteasoma una vez pasada de la zona de transición. HTR6 es crucial para restringir el crecimiento de la planta en respuesta a ABA y sal, como demuestra la mayor longitud de la raíz y de su meristemo apical y por el mayor número de células corticales en plántulas mutantes con pérdida de función en htr6-1. Dado que la ploidía nuclear en raíces de htr6-1 no está afectada, HTR6 podría desempeñar un papel en el establecimiento de los límites entre el meristemo apical de la raíz y la zona de transición/elongación en situaciones de estrés. Además, nuestros análisis transcriptómicos mostraron que entre los genes que el mutante htr6-1 es incapaz de activar en respuesta a ABA (10 minutos), se encuentran genes implicados en la biogénesis de la pared celular y del metabolismo lipídico, y genes que codifican algunos factores de transcripción de la familia TCP. Igualmente, varios miembros de la familia LEA han perdido la regulación negativa en el mutante htr6-1. En conjunto, nuestro estudio ha puesto de manifiesto un papel importante de la histona HTR6, una variante de la histona H3 de función desconocida hasta ahora, en la respuesta temprana a estrés abiótico en Arabidopsis.This Thesis was supported by a grant from the Portuguese “Fundação para a Ciência e a Tecnologia” (FCT – SFRH/BD/105550/2014

UVR8 mediated spatial differences as a prerequisite for UV-B induced inflorescence phototropism

In Arabidopsis hypocotyls, phototropins are the dominant photoreceptors for the positive phototropism response towards unilateral ultraviolet-B (UV-B) radiation. We report a stark contrast of response mechanism with inflorescence stems with a central role for UV RESISTANCE LOCUS 8 (UVR8). The perception of UV-B occurs mainly in the epidermis and cortex with a lesser contribution of the endodermis. Unilateral UV-B exposure does not lead to a spatial difference in UVR8 protein levels but does cause differential UVR8 signal throughout the stem with at the irradiated side 1) increase of the transcription factor ELONGATED HYPOCOTYL 5 (HY5), 2) an associated strong activation of flavonoid biosynthesis genes and flavonoid accumulation, 3) increased GA2oxidase expression, diminished gibberellin1 levels and accumulation of DELLA protein REPRESSOR OF GA1 (RGA) and, 4) increased expression of the auxin transport regulator, PINOID, contributing to local diminished auxin signalling. Our molecular findings are in support of the Blaauw theory (1919), suggesting that differential growth occurs trough unilateral photomorphogenic growth inhibition. Together the data indicate phototropin independent inflorescence phototropism through multiple locally UVR8-regulated hormone pathways

Tree Peony Species Are a Novel Resource for Production of α-Linolenic Acid

Tree peony is known worldwide for its excellent ornamental and medical values, but recent reports that their seeds contain over 40% α-linolenic acid (ALA), an essential fatty acid for humans drew additional interest of biochemists. To understand the key factors that contribute to this rich accumulation of ALA, we carried out a comprehensive study of oil accumulation in developing seeds of nine wild tree peony species. The fatty acid content and composition was highly variable among the nine species; however, we selected a high- (P. rockii) and low-oil (P. lutea) accumulating species for a comparative transcriptome analysis. Similar to other oilseed transcriptomic studies, upregulation of select genes involved in plastidial fatty acid synthesis, and acyl editing, desaturation and triacylglycerol assembly in the endoplasmic reticulum was noted in seeds of P. rockii relative to P. lutea. Also, in association with the ALA content, transcript levels for fatty acid desaturases (SAD, FAD2 and FAD3), which encode for enzymes necessary for polyunsaturated fatty acid synthesis were higher in P. rockii compared to P. lutea. We further showed that the overexpression of PrFAD2 and PrFAD3 in Arabidopsis increased linoleic and α-linolenic acid content, respectively and modulated their final ratio in the seed oil. In conclusion, we identified the key steps that contribute to efficient ALA synthesis and validated the necessary desaturases in P. rockii that are responsible for not only increasing oil content but also modulating 18:2/18:3 ratio in seeds. Together, these results will aid to improve essential fatty acid content in seeds of tree peonies and other crops of agronomic interest

Analysis of craniofacial defects in Six1/Eya1-associated Branchio-Oto-Renal Syndrome

Poster Session I - Morphogenesis: 205/B10117th ISDB 2013 cum 72nd Annual Meeting of the Society for Developmental Biology, 7th Latin American Society of Developmental Biology Meeting and 11th Congreso de la Sociedad Mexicana de Biologia del Desarrollo.Branchio-Oto-Renal (BOR) syndrome patients exhibit craniofacial and renal anomalies as well as deafness. BOR syndrome is caused by mutations in Six1 or Eya1, both of which regulate cell proliferation and differentiation. The molecular mechanism underlying the craniofacial and branchial arch (BA) defects in BOR syndrome is unclear. We have found that Hoxb3 is up-regulated in the second branchial arch (BA2) of Six1-/- mutants. Moreover, Hoxb3 over-expression in transgenic mice leads to BA abnormalities which are similar to the BA defects in Six1-/- or Eya1-/- mutants, suggesting a regulatory relationship among Six1, Eya1 and Hoxb3 genes. The aim of this study is to investigate the molecular mechanism underlying abnormal BA development in BOR syndrome using Six1 and Eya1 mutant mice. Two potential Six1 binding sites were identified on the Hoxb3 gene. In vitro and in vivo Chromatin IP assays showed that Six1 could directly bind to one of the sites specifically. Furthermore, using a chick in ovo luciferase assay we showed that Six1 could suppress gene expression through one of the specific binding sites. On the other hand, in Six1-/- mutants, we found that the Notch ligand Jag1 was up-regulated in BA2. Similarly, in Hoxb3 transgenic mice, ectopic expression of Jag1 could be also detected in BA2. To investigate the activation of Notch signaling pathway, we found that Notch intracellular domain (NICD), a direct indicator of Notch pathway activation, was up-regulated in BAs of Six1-/-; Eya1-/- double mutants. Our results indicate that Hoxb3 and Notch signaling pathway are involved in mediating the craniofacial defects of Six1/Eya1-associated Branchio-Oto-Renal Syndrome.postprin

Sox10 regulates enteric neural crest cell migration in the developing gut

Concurrent Sessions 1: 1.3 - Organs to organisms: Models of Human Diseases: abstract no. 1417th ISDB 2013 cum 72nd Annual Meeting of the Society for Developmental Biology, VII Latin American Society of Developmental Biology Meeting and XI Congreso de la Sociedad Mexicana de Biologia del Desarrollo. The Conference's web site is located at http://www.inb.unam.mx/isdb/Sox10 is a HMG-domain containing transcription factor which plays important roles in neural crest cell survival and differentiation. Mutations of Sox10 have been identified in patients with Waardenburg-Hirschsprung syndrome, who suffer from deafness, pigmentation defects and intestinal aganglionosis. Enteric neural crest cells (ENCCs) with Sox10 mutation undergo premature differentiation and fail to colonize the distal hindgut. It is unclear, however, whether Sox10 plays a role in the migration of ENCCs. To visualize the migration behaviour of mutant ENCCs, we generated a Sox10NGFP mouse model where EGFP is fused to the N-terminal domain of Sox10. Using time-lapse imaging, we found that ENCCs in Sox10NGFP/+ mutants displays lower migration speed and altered trajectories compared to normal controls. This behaviour was cell-autonomous, as shown by organotypic grafting of Sox10NGFP/+ gut segments onto control guts and vice versa. ENCCs encounter different extracellular matrix (ECM) molecules along the developing gut. We performed gut explant culture on various ECM and found that Sox10NGFP/+ ENCCs tend to form aggregates, particularly on fibronectin. Time-lapse imaging of single cells in gut explant culture indicated that the tightly-packed Sox10 mutant cells failed to exhibit contact inhibition of locomotion. We determined the expression of adhesion molecule families by qPCR analysis, and found integrin expression unaffected while L1-cam and selected cadherins were altered, suggesting that Sox10 mutation affects cell adhesion properties of ENCCs. Our findings identify a de novo role of Sox10 in regulating the migration behaviour of ENCCs, which has important implications for the treatment of Hirschsprung disease.postprin