Mechanisms of Early Brain Morphogenesis

In structures with obvious mechanical function, like the heart and bone, the relationship of mechanical forces to growth and development has been well studied. In contrast, other than the problem of neurulation: formation of the neural tube), developmental mechanisms in the nervous system have received relatively little attention. The central aim of this research is to characterize the biophysical mechanisms that shape the early embryonic brain. Experiments were performed primarily in the chicken brain, which is morphologically similar to humans during early stages of development. Proposed mechanisms were tested using computational models to ensure that hypotheses are consistent with physical law. The brain initially forms as a straight epithelial tube in the embryo: approximately 3 weeks gestation in humans). We first investigated a potential role for mechanical feedback in regulating the development of this structure. We find that the neuroepithelium actively stiffens under decreased loading and softens under increased loading. Nuclear shapes are elongated in stiffer brains and circular in softer brains, consistent with changes in cytoskeletal contractility and wall stress. These results suggest a role for stress-based mechanical feedback in regulating epithelial development. We next investigated the more specific role of cytoskeletal contraction in forming the primary brain vesicles and rhombomeres that subdivide the primitive brain tube. We show that a combination of circumferential contraction in the boundary regions and isotropic contraction between boundaries can generate realistic vesicle morphologies, whereas longitudinal contraction between boundaries likely causes rhombomere formation. Models are used to show how regional variations in contraction may be a function of brain geometry and morphogenetic plasticity. As an extension of the previous study, we show that enhancing contractility in the embryonic chicken brain induces morphologies reminiscent of more primitive species such as frog and fish. In particular, brain cross sections that are relatively circular transform into diamonds, triangles, and narrow slits, shapes that are present in normal zebrafish and Xenopus brains at comparable stages of development. Models show that these shapes are likely produced by locally elevated cytoskeletal contraction, indicating a potential role for differential contractility in early brain development and evolution. In summary, results from this thesis should improve our understanding of the biophysical mechanisms that establish and regulate phenotype in the developing brain. The research begins to establish the framework necessary to connect early-stage mechanisms to interspecies differences in brain morphogenesis that occur during later development

Biophysical Mechanisms of Early Heart Morphogenesis

The heart is the first functioning organ in the developing embryo. Initially, the heart is a relatively straight tube created by folding and fusion of the cardiogenic fields, which lie bilaterally within the blastoderm. Shortly after formation, the primitive heart tube (HT) undergoes the morphogenetic process of c-looping as it bends and twists into a c-shaped tube. All these transformations require physical forces, which remain poorly understood. The aim of this dissertation is to elucidate some of the biophysical mechanisms that create and shape the early HT. Our work involves a combination of ex ovo experiments and computational modeling. Experiments were performed on embryonic chicken hearts, which are morphologically similar to human hearts during development. First, we explored a somewhat puzzling aspect of early heart development. Previous studies have shown that myosin-II-based cytoskeletal contraction is required for fusion of the heart fields before looping begins, but not as these tissues continue to fuse and extend the length of the HT during subsequent c-looping. To investigate this fundamental change in behavior, we focused on the tissues around the anterior intestinal portal (AIP), where fusion takes place. Our results indicate that stiffness and tangential tension decreased bilaterally with distance from the embryonic midline along the AIP. The stiffness and tension gradients increased to peaks at Hamburger-Hamilton (HH) stage 9 and decreased immediately afterward. Along with experimental results of contraction inhibition, finite-element models indicate that the measured mechanical gradients are consistent with a relatively uniform contraction of the endoderm along the AIP. Taken together, these results suggest that, before looping begins at HH10, cytoskeletal contraction pulls the bilateral cardiogenic fields toward the midline where they begin to fuse to create the HT. By HH10, however, the fusion process is far enough along to enable apposing cardiac progenitor cells to subsequently undergo filopodia-mediated “zippering” without the continuing need for contraction. Next, in light of recently published data, we examined the possible role of differential hypertrophic growth in driving the bending component of c-looping. Using cultured isolated hearts, which bend without the complicating effects of external loads, we found that myocardial growth patterns correlate with bending. We also developed finite-element models that include previously measured regional changes in myocardial growth during c-looping. The simulations show that differential growth alone can produce results that agree reasonably well with trends in our experimental data, including changes in HT morphology and tissue strains and stresses. Incorporating other mechanisms into the model, such as active changes in myocardial cell shape, provides closer agreement. These results suggest that regional difference in hypertrophic myocardial growth is the primary cause of the bending component of c-looping, with other mechanisms playing lesser roles. Finally, we extended the model of the previous study to explore the physical plausibility of a hypothesis for the entire process of c-looping. According to our hypothesis, bending is driven primarily by differential hypertrophic growth in the myocardium, torsion is mainly caused by compressive loads exerted by the overlying splanchnopleuric membrane, and looping direction is determined by asymmetric regional growth in the omphalomesenteric veins at the caudal end of the HT. Our model includes both bending and torsion of the HT, realistic 3D geometry, and loads exerted by neighboring tissues. The behavior of the model is in reasonable agreement with available experimental data from control and mechanically perturbed embryos, offering support for our hypothesis. The results also suggest, however, that several other mechanisms contribute secondarily to normal looping, and we speculate that these mechanisms play backup roles when looping is perturbed. In summary, studies of this dissertation address several important questions during early cardiac development. The results should enrich our understanding of the underlying biophysical mechanisms

Using cilia mutants to study left-right asymmetry in zebrafish

A thesis submitted in fulfillment of the requirements for the degree of the Masters in Molecular Genetics and BiomedicineIn vertebrates, internal organs are positioned asymmetrically across the left-right (L-R) body axis. Events determining L-R asymmetry occur during embryogenesis, and are regulated by the coordinated action of genetic mechanisms. Embryonic motile cilia are essential in this process by generating a directional fluid flow inside the zebrafish organ of asymmetry, called Kupffer’s vesicle ﴾KV). A correct L-R formation is highly dependent on signaling pathways downstream of such flow, however detailed characterization of how its dynamics modulates these mechanisms is still lacking. In this project, fluid flow measurements were achieved by a non-invasive method, in four genetic backgrounds: Wild-type (WT), deltaD-/- mutants, Dnah7 morphants (MO) and control-MO embryos. Knockdown of Dnah7, a heavy chain inner axonemal dynein, renders cilia completely immotile and depletes the KV directional fluid flow, which we characterize here for the first time. By following the development of each embryo, we show that flow dynamics in the KV is already asymmetric and provides a very good prediction of organ laterality. Through novel experiments, we characterized a new population of motile cilia, an immotile population, a range of cilia beat frequencies and lengths, KV volumes and cilia numbers in live embryos. These data were crucial to perform fluid dynamics simulations, which suggested that the flow in embryos with 30 or more cilia reliably produces left situs; with fewer cilia, left situs is sometimes compromised through disruption of the dorsal anterior clustering of motile cilia. A rough estimate based upon the 30 cilium threshold and statistics of cilium number predicts 90% and 60% left situs in WT and deltaD-/- respectively, as observed experimentally. Cilia number and clustering are therefore critical to normal situs via robust asymmetric flow. Thus, our results support a model in which asymmetric flow forces registered in the KV pattern organ laterality in each embryo

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

Patterns of tooth replacement in osteichthyans: variations on a theme

Nonmammalian tooth-bearing vertebrates usually replace their teeth throughout life. Much about how a replacement pattern is generated has been learned from zebrafish. However, to understand general mechanisms of tooth replacement, advantage can be taken from studying other, “nonmodel” species. We have mapped the patterns of tooth replacement in widely divergent aquatic osteichthyans using 2D charts, in which one axis is time, the other linear spacing along the tooth row. New teeth that are generated simultaneously are considered part of the same odontogenic wave. Using this approach, it appears that a similar, general pattern underlies very distinctive dentitions in distantly related species. A simple shift in spacing of odontogenic waves, or in distance between subsequent tooth positions along a row (or both), can produce dramatically different dentitions between life stages within a species, or between closely related species. Examples will be presented from salmonids, cyprinids, and cichlids. Our observations suggest that lines linking subsequent positions may have more biological significance than replacement waves (usually linking alternate positions), often used to explain the generation of patterns. The presence of a general pattern raises questions about common control mechanisms. There is now increasing evidence, at least for the zebrafish, to support a role for stem cells in continuous tooth renewal and control of replacement patterns