A dynamic cell recruitment process drives growth of the Drosophila wing by overscaling the vestigial expression pattern

Organs mainly attain their size by cell growth and proliferation, but sometimes also grow through recruitment of undifferentiated cells. Here we investigate the participation of cell recruitment in establishing the pattern of Vestigial (Vg), the product of the wing selector gene in Drosophila. We find that the Vg pattern overscales along the dorsal-ventral (DV) axis of the wing imaginal disc, i.e., it expands faster than the DV length of the pouch. The overscaling of the Vg pattern cannot be explained by differential proliferation, apoptosis, or oriented-cell divisions, but can be recapitulated by a mathematical model that explicitly considers cell recruitment. When impairing cell recruitment genetically, we find that the Vg pattern almost perfectly scales and adult wings are approximately 20% smaller. Conversely, impairing cell proliferation results in very small wings, suggesting that cell recruitment and cell proliferation additively contribute to organ growth in this system. Furthermore, using fluorescent reporter tools, we provide direct evidence that cell recruitment is initiated between early and mid third-instar larval development. Altogether, our work quantitatively shows when, how, and by how much cell recruitment shapes the Vg pattern and drives growth of the Drosophila wing.Fil: Muñoz Nava, Luis Manuel. Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional; MéxicoFil: Alvarez, Hugo Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física de Líquidos y Sistemas Biológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física de Líquidos y Sistemas Biológicos; ArgentinaFil: Flores Flores, Marycruz. Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional; MéxicoFil: Chara, Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física de Líquidos y Sistemas Biológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física de Líquidos y Sistemas Biológicos; Argentina. Technische Universität Dresden; AlemaniaFil: Nahmad, Marcos. Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional; Méxic

FGF signaling and cell state transitions during organogenesis

Organogenesis is a complex choreography of morphogenetic processes, patterns and dynamic shape changes as well as the specification of cell fates. Although several molecular actors and context-specific mechanisms have already been identified, our general understanding of the fundamental principles that govern the formation of organs is far from comprehensive. The application of the concept of ‘rebuild it to understand it’ from synthetic biology represents a promising alternative to the classical approach of ‘break it to understand it’ in order to distill biological understanding from complex developmental processes. According to this ‘rebuilding’ concept, in this study we sought to develop an experimental approach to induce the formation of organs from progenitor cells ‘on demand’ and to investigate the minimum requirements for such a process. The zebrafish lateral line chain cells are a powerful in vivo model for our study because they are a group of naïve multipotent progenitor cells that display mesenchyme-like features. In order to bring these cells to form organs, we used the well-known role of the FGF signaling pathway as a driver of organogenesis in the lateral line and developed an inducible and constitutively active form of the fibroblast growth factor receptor 1a (chemoFGFR). The cell-autonomous induction of this chemoFGFR in chain cells effectively triggered the formation of fully mature organs and thus enabled spatial and temporal control of the organogenesis process. Next, we asked what it takes to form an organ de novo. We used a combination of real-time microscopy, single cell tracking, polarity quantification, and mosaic analysis to study the cell behaviors that result from chemoFGFR induction. The picture that emerges from these analyses is that de novo organs form through a genetically encoded self-assembly process that is based on the pattern of chemoFGFR induction. In this scenario, cells expressing chemoFGFR aggregate into clusters and epithelialize as they sort out of non-expressing cells. We found that this sorting process occurs through cell rearrangement and slithering, which involves an extensive remodeling of the cell-cell contacts. Chain cells that do not express chemoFGFR can envelop these chemoFGFR expressing cell clusters and form a rim at the cluster periphery. This multi-stage process leads to the establishment of the inside-outside pattern of de novo organs, which is used as a blueprint for cell differentiation. In summary, in this study we provide insights into the mechanisms involved in the self-assembly of organs from a naïve population of progenitor cells

Regulatory feedback on receptor and non-receptor synthesis for robust signaling.

Elaborate regulatory feedback processes are thought to make biological development robust, that is, resistant to changes induced by genetic or environmental perturbations. How this might be done is still not completely understood. Previous numerical simulations on reaction-diffusion models of Dpp gradients in Drosophila wing imaginal disc have showed that feedback (of the Hill function type) on (signaling) receptors and/or non-(signaling) receptors are of limited effectiveness in promoting robustness. Spatial nonuniformity of the feedback processes has also been shown theoretically to lead to serious shape distortion and a principal cause for ineffectiveness. Through mathematical modeling and analysis, the present article shows that spatially uniform nonlocal feedback mechanisms typically modify gradient shape through a shape parameter (that does not change with location). This in turn enables us to uncover new multi-feedback instrument for effective promotion of robust signaling gradients

Distinct mechanisms of planar polarization by the core and Fat-Dachsous planar polarity pathways in the Drosophila wing

Planar polarity describes the coordinated polarization of cells within a tissue plane, and in animals can be determined by the “core” or Fat-Dachsous pathways. Current models for planar polarity establishment involve two components: tissue-level “global” cues that determine the overall axis of polarity and cell-level feedback-mediated cellular polarity amplification. Here, we investigate the contributions of global cues versus cellular feedback amplification in the core and Fat-Dachsous pathways during Drosophila pupal wing development. We present evidence that these pathways generate planar polarity via distinct mechanisms. Core pathway function is consistent with strong feedback capable of self-organizing cell polarity, which can then be aligned with the tissue axis via weak or transient global cues. Conversely, generation of cell polarity by the Ft-Ds pathway depends on strong global cues in the form of graded patterns of gene expression, which can then be amplified by weak feedback mechanisms

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

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

An investigation into how the cell cycle and the Notch signalling pathway regulate pronephrogenesis in Xenopus laevis

The connections between cell cycle exit and terminal differentiation remain poorly understood. Cyclin dependent Kinase Inhibitors (CKIs) provide a possible link between entry into the quiescent state and differentiation. The initial aim of this project was to further investigate if the CKI p27Xic1 could promote differentiation in addition to, and independently of, its well characterised cell cycle exit function. p27Xic1 has been shown to be involved in cell fate determination during gliogenesis, neurogenesis, myogenesis and cardiogenesis and many mammalian Cip/ Kip CKI homologues of p27Xic1 have been described as important regulators of cellular processes beyond control of cell division. We aimed to investigate these roles during development of the embryonic kidney, the pronephros. We discovered that p27Xic1 does not affect differentiation during pronephrogenesis, but instead controls pronephric organ size through its cell cycle exit function. In addition we identified a previously unrecognised role for the cell cycle exit function of p27Xic1 in allocation of the somites during paraxial mesoderm segmentation. Preliminary results had suggested p27Xic1 expression in the pronephros was under the control of the Notch signalling pathway. Over-expressing a constitutively active form of Notch, Notch-ICD, and a dominant negative form of the Delta ligand, DeltaSTU, showed that both mis-activation and suppression of Notch signalling inhibited p27Xic1 expression. However, when investigating the effects these overexpressions had on pronephros development, we identified novel results indicating the Notch signalling pathway, which has previously been implicated in pronephros development, is essential for the separation of the proximal lateral and medial pronephric mesoderms. This process we propose is mediated by the Notch signalling pathway through the establishment of a boundary between these two distinct populations of cells, permitting both compartments to develop in isolation. The results in this thesis suggest novel mechanisms by which cell division controls X. laevis segmentation and organ size and how the Notch signalling pathway is able to pattern the pronephros anlagen such that the different compartments of the mature pronephros are able to develop, and thus function

An investigation into how the cell cycle and the Notch signalling pathway regulate pronephrogenesis in Xenopus laevis

The connections between cell cycle exit and terminal differentiation remain poorly understood. Cyclin dependent Kinase Inhibitors (CKIs) provide a possible link between entry into the quiescent state and differentiation. The initial aim of this project was to further investigate if the CKI p27Xic1 could promote differentiation in addition to, and independently of, its well characterised cell cycle exit function. p27Xic1 has been shown to be involved in cell fate determination during gliogenesis, neurogenesis, myogenesis and cardiogenesis and many mammalian Cip/ Kip CKI homologues of p27Xic1 have been described as important regulators of cellular processes beyond control of cell division. We aimed to investigate these roles during development of the embryonic kidney, the pronephros. We discovered that p27Xic1 does not affect differentiation during pronephrogenesis, but instead controls pronephric organ size through its cell cycle exit function. In addition we identified a previously unrecognised role for the cell cycle exit function of p27Xic1 in allocation of the somites during paraxial mesoderm segmentation. Preliminary results had suggested p27Xic1 expression in the pronephros was under the control of the Notch signalling pathway. Over-expressing a constitutively active form of Notch, Notch-ICD, and a dominant negative form of the Delta ligand, DeltaSTU, showed that both mis-activation and suppression of Notch signalling inhibited p27Xic1 expression. However, when investigating the effects these overexpressions had on pronephros development, we identified novel results indicating the Notch signalling pathway, which has previously been implicated in pronephros development, is essential for the separation of the proximal lateral and medial pronephric mesoderms. This process we propose is mediated by the Notch signalling pathway through the establishment of a boundary between these two distinct populations of cells, permitting both compartments to develop in isolation. The results in this thesis suggest novel mechanisms by which cell division controls X. laevis segmentation and organ size and how the Notch signalling pathway is able to pattern the pronephros anlagen such that the different compartments of the mature pronephros are able to develop, and thus function.EThOS - Electronic Theses Online ServiceBiotechnology and Biological Sciences Research Council (Great Britain) (BBSRC)GBUnited Kingdo

Different functions of Notch activation on formation and maintenance of rhombomere boundaries

Ph.DDOCTOR OF PHILOSOPH