Cell-Based Multi-Parametric Model of Cleft Progression during Submandibular Salivary Gland Branching Morphogenesis

Cleft formation during submandibular salivary gland branching morphogenesis is the critical step initiating the growth and development of the complex adult organ. Previous experimental studies indicated requirements for several epithelial cellular processes, such as proliferation, migration, cell-cell adhesion, cell-extracellular matrix (matrix) adhesion, and cellular contraction in cleft formation; however, the relative contribution of each of these processes is not fully understood since it is not possible to experimentally manipulate each factor independently. We present here a comprehensive analysis of several cellular parameters regulating cleft progression during branching morphogenesis in the epithelial tissue of an early embryonic salivary gland at a local scale using an on lattice Monte-Carlo simulation model, the Glazier-Graner-Hogeweg model. We utilized measurements from time-lapse images of mouse submandibular gland organ explants to construct a temporally and spatially relevant cell-based 2D model. Our model simulates the effect of cellular proliferation, actomyosin contractility, cell-cell and cell-matrix adhesions on cleft progression, and it was used to test specific hypotheses regarding the function of these parameters in branching morphogenesis. We use innovative features capturing several aspects of cleft morphology and quantitatively analyze clefts formed during functional modification of the cellular parameters. Our simulations predict that a low epithelial mitosis rate and moderate level of actomyosin contractility in the cleft cells promote cleft progression. Raising or lowering levels of contractility and mitosis rate resulted in non-progressive clefts. We also show that lowered cell-cell adhesion in the cleft region and increased cleft cell-matrix adhesions are required for cleft progression. Using a classifier-based analysis, the relative importance of these four contributing cellular factors for effective cleft progression was determined as follows: cleft cell contractility, cleft region cell-cell adhesion strength, epithelial cell mitosis rate, and cell-matrix adhesion strength

LIM Kinase Regulation of Cytoskeletal Dynamics is Required for Salivary Gland Branching Morphogenesis

Coordinated actin microfilament and microtubule dynamics is required for salivary gland development, although the mechanisms by which they contribute to branching morphogenesis are not defined. Because LIM kinase (LIMK) regulates both actin and microtubule organization, we investigated the role of LIMK signaling in mouse embryonic submandibular salivary glands using ex vivo organ cultures. Both LIMK 1 and 2 were necessary for branching morphogenesis and functioned to promote epithelial early- and late-stage cleft progression through regulation of both microfilaments and microtubules. LIMK-dependent regulation of these cytoskeletal systems was required to control focal adhesion protein– dependent fibronectin assembly and integrin β1 activation, involving the LIMK effectors cofilin and TPPP/p25, for assembly of the actin- and tubulin-based cytoskeletal systems, respectively. We demonstrate that LIMK regulates the early stages of cleft formation—cleft initiation, stabilization, and progression—via establishment of actin stability. Further, we reveal a novel role for the microtubule assembly factor p25 in regulating stabilization and elongation of late-stage progressing clefts. This study demonstrates the existence of multiple actin- and microtubule-dependent stabilization steps that are controlled by LIMK and are required in cleft progression during branching morphogenesis

Reiterative FGF signaling determines the identity and morphology of the lacrimal gland

Indiana University-Purdue University Indianapolis (IUPUI)The lacrimal gland plays an essential role in protection of the ocular surface by secreting the aqueous component of the tear film. Deficiency in the lacrimal gland is the main cause of dry eye disease, but existing treatments only alleviate the symptoms without curing the underlying disease. To develop curative measures, a thorough understanding of lacrimal gland development is needed. Lacrimal gland is formed as a result of interaction between the neural crest-derived mesenchyme and the conjunctival epithelium. The mesenchyme secretes the chemo-attractive signal of Fgf10, which binds to epithelial Fgfr2b and co-receptor heparan sulphate proteoglycans, to promote budding and branching morphogenesis of the lacrimal gland. However, the mechanism by which Fgf10 expression is regulated within the neural crest and the direct downstream targets of Fgf signaling in the epithelium are currently unknown. In this study, we show that FGF signaling mediated by protein phosphatase Shp2 is required for the proper patterning and differentiation of the neural crest-derived mesenchyme to produce Fgf10. Genetic evidence further demonstrates that Shp2 is recruited by Frs2α to activate Ras-MAPK signaling downstream to Fgfr1 and Fgfr2 but not to Pdgfrα in the neural crest. By differential gene expression analysis, we identified homeodomain transcription factor Alx4 as the key effector of Shp2 signaling to control expression of Fgf10 in the periocular mesenchyme. Loss of function ALX4/Alx4 mutation disrupted lacrimal gland development in both human and mouse. Our results reveal a FGF-Shp2-Alx4-Fgf10 axis in regulating neural crests during lacrimal gland development. In addition, we also show that Fgf signaling cascade mediated by Pea3 family of transcription factors are critical for lacrimal gland duct elongation and branching. High-throughput gene expression analysis revealed that Pea3 genes were important for establishing the tissue identity of the lacrimal gland. Loss of Pea3 resulted in upregulation of Notch signaling with the concomitant loss in the expression of the members of Six family of transcription factors and a switch of cell fate to the epidermal skin-like cells. These findings show that Fgf signaling is used reiteratively to establish the identity of both the epithelium and mesenchyme of the lacrimal gland.2 year

PRELIMINARY FINDINGS OF A POTENZIATED PIEZOSURGERGICAL DEVICE AT THE RABBIT SKULL

The number of available ultrasonic osteotomes has remarkably increased. In vitro and in vivo studies have revealed differences between conventional osteotomes, such as rotating or sawing devices, and ultrasound-supported osteotomes (Piezosurgery®) regarding the micromorphology and roughness values of osteotomized bone surfaces. Objective: the present study compares the micro-morphologies and roughness values of osteotomized bone surfaces after the application of rotating and sawing devices, Piezosurgery Medical® and Piezosurgery Medical New Generation Powerful Handpiece. Methods: Fresh, standard-sized bony samples were taken from a rabbit skull using the following osteotomes: rotating and sawing devices, Piezosurgery Medical® and a Piezosurgery Medical New Generation Powerful Handpiece. The required duration of time for each osteotomy was recorded. Micromorphologies and roughness values to characterize the bone surfaces following the different osteotomy methods were described. The prepared surfaces were examined via light microscopy, environmental surface electron microscopy (ESEM), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM) and atomic force microscopy. The selective cutting of mineralized tissues while preserving adjacent soft tissue (dura mater and nervous tissue) was studied. Bone necrosis of the osteotomy sites and the vitality of the osteocytes near the sectional plane were investigated, as well as the proportion of apoptosis or cell degeneration. Results and Conclusions: The potential positive effects on bone healing and reossification associated with different devices were evaluated and the comparative analysis among the different devices used was performed, in order to determine the best osteotomes to be employed during cranio-facial surgery

The expansion and diversification of the claudin gene family: insight from the lamprey

Dissertation submitted to the Faculty of Science, University of the Witswatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. May 2015 in JohannesburgClaudins are a large gene family found in all vertebrates. Claudins encode tetraspan membrane proteins, involved in the structure and function of the tight junctions. This association of cells leads to the formation of the epithelial sheet which is involved in many functions such as embryo morphogenesis. The NCBI database shows 27 claudins identified in humans; 23 in mice and 17 in Xenopus. This suggests that an increase in gene family size may correlate with the evolution of more complex vertebrates. In this study claudins from the most basal extant vertebrate, the sea lamprey, were investigated. RNA used to build up the lamprey genome by Jeramiah Smith (Smith et al., 2012), was used for lamprey claudin sequences. Additionally this study identified 2 more claudins (Cldn B & Cldn F). The phylogenetic tree constructed using claudins from higher vertebrate model organisms and the invertebrates Ciona intestinalis and Drosophila melanogaster; showed that lamprey claudins are evolutionarily more distantly related to their orthologs in higher vertebrates. Furthermore some claudins in lamprey did not show any homologs in higher vertebrates and vice versa, indicating the emergence of novel members in higher vertebrates. However lamprey Cldn A was found to be homologous to CLDN 3 in higher vertebrates. This is interesting since CLDN 3 is involved in the development of two vertebrate specific traits; one of which is the ear placode. Thus Cldn A (renamed Cldn 3B), was made a focus of this study. RNA in situ hybridization using probes designed from individual UTRs showed localised expression of Cldn 3B in the ear placode, pharyngeal pouch, pericardial cavity and the fin fold whereas Cldn B (renamed Cldn 8B) was mostly expressed in the pharyngeal pouch and ear placode much like its orthologs in higher vertebrates. Knockout experiments showed that Cldn 3B is involved in sealing and expansion of the ear placode and pharyngeal arches during development whereas Cldn 8B is involved in determining ear placode development. Thus claudins are seen to be heavily involved in the morphology of vertebrate specific traits therefore an expansion in this gene family would affect the complexity of vertebrates during evolution

Current Frontiers and Perspectives in Cell Biology

A numerous internationally renowned authors in the pages of this book present the views of the fields of cell biology and their own research results or review of current knowledge. Chapters are divided into five sections that are dedicated to cell structures and functions, genetic material, regulatory mechanisms, cellular biomedicine and new methods in cell biology. Multidisciplinary and often quite versatile approach by many authors have imposed restrictions of this classification, so it is certain that many chapters could belong to the other sections of this book. The current frontiers, on the manner in which they described in the book, can be a good inspiration to many readers for further improving, and perspectives which are highlighted can be seen in many areas of fundamental biology, biomedicine, biotechnology and other applications of knowledge of cell biology. The book will be very useful for beginners to gain insight into new area, as well as experts to find new facts and expanding horizons

Analysis of junctional and F-actin dynamics during blood vessel morphogenesis

Organ morphogenesis relies on dynamic cell behaviors, which are highly coordinated to ensure a functional cellular organ architecture. During vascular morphogenesis, the process of angiogenesis is driven by cell migration, cell shape changes and cell rearrangements. Here, a dynamic balance between inter-endothelial cell adhesion and plasticity allows angiogenic sprouting while maintaining the endothelial seal. Previous analyses on blood vessel formation and anastomosis in zebrafish have shown that junctional remodeling is central to many aspects of morphogenetic endothelial cell-cell interactions. In particular, the adhesion molecule VE-cadherin (Cdh5) is essential for coordinated cell shape changes during multicellular tube formation, as loss of VE-cadherin was shown to inhibit cell rearrangements (Sauteur et al., 2014). This study also proposed an active, force generating function for VE-cadherin in this process. This hypothesis is supported by our study showing that cell elongation is mediated by junction-based lamellipodia (JBL), which are thought to provide a tractile force for junction elongation (Paatero et al., 2018). In my thesis, my goal was to further analyze the molecular mechanisms which underly JBL function. In particular I focused on molecular players that influence F-actin dynamics or contractility (Arp2/3, Rac1 and ROCK) in order to identify critical players in the process of the cell elongation movement. Furthermore, I elucidated, how JBL might generate motile forces and how these forces are transmitted onto endothelial cell junctions (e.g. VE-cadherin) In the course of my experiments I identified the actomyosin contractility as an important basis for junctional ring elongation. Inhibition of ROCK did interfere with the correct localization of junctional protein ZO1 as it is abrogated formation of double junctions- a hallmark during JBL oscillations and an indispensable step to since it leads to the formation of a new attachment site. Furthermore, I found that the establishment of differential VE-cadherin tension is also ROCK-dependent, which might provide the basis for junctional remodeling. Junctional rearrangements were not only impaired after inhibition of ROCK. Also, interference with the F-actin dynamics significantly altered junctional ring elongation. Arp2/3 (and concomitant formation of branched F-actin networks) is necessary for maintaining junctional stability and responsible for the correct localization of F-actin during the process, whereas Rac1 mostly seem to play a role in the induction phase of the JBL. Last but not least I generated two new transgenic fish lines (fli:iRFP-UCHD and kdrl:mCherry-PA-Rac1), which will open up a whole lot of new possibilities for future experiments. Making use of the photoactivatable Rac1 will give manifold new insights into processes during vascular morphogenesis, which underly Rac1 activity (sprouting, anastomosis, etc.). In summary JBL function and subsequent endothelial cell rearrangements rely on a tight interplay between generation and maintenance of a dynamic F-actin cytoskeleton and regulation of junctional proteins. The F-actin cytoskeleton furthermore provide a basis for local force generation, which is reflected in differential VE-cadherin tension and thus exert mechanical forces, which in turn are a major driver of the process of junctional ring elongation. Last but not least my experiments suggest, that JBL formation and local protrusion formation might be a general mechanism of endothelial cells to induce cell movements and cell shape changes (e.g. in the dorsa aorta)

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

The regulation of Fibronectin matrix assembly by Tenascin-C

Fibronectin (FN) is a ubiquitous component of the extracellular matrix (ECM). Its assembly into 3D fibrillar matrices is essential during development and tissue repair to maintain tissue architecture and provide environmental signals to cells. However, FN deposition must be tightly regulated as excessive assembly is a major hallmark of fibrotic diseases and cancer. Tenascin-C (TN-C) is a large, multi-domain ECM glycoprotein that co-localizes with newly synthesized FN fibrils in vivo during development, wound repair and tumorigenesis. However, it is not known precisely how FN and TN-C interact within the ECM, or whether their interaction has any functional relevance. I have demonstrated that distinct domains of TN-C inhibit FN matrix assembly by fibroblasts, whereas full length TN-C has no effect. I have identified regions within TN-C domains that are essential for binding to fibrillar FN, but not to soluble FN, and mapped where they interact within the FN molecule. I have found that domains containing these regions interfere with inter-molecular FN-FN interactions during fibrillogenesis. I also identified other TN-C domains that interfered with FN matrix assembly by FNindependent mechanisms. I demonstrated that one of these TN-C domains was internalized by fibroblasts causing morphological changes that may interfere with cytoskeleton organization or cell surface receptor availability to prevent the maintenance of a fibrillar FN matrix at the cell surface. Recently emerging evidence indicates that deposition of FN by epithelial and endothelial cells is absolutely vital for tubulogenesis and the formation of new blood vessels. Without this scaffold, the cells fail to generate sufficient tensional force required for the morphogenesis and migration essential for tubule formation. I have shown that specific domains of TN-C can inhibit tubulogenesis by epithelial cells within a 3D collagen matrix and may also affect angiogenesis of endothelial cells within a 3D collagen matrix environment. These data suggest that proteolysis of TN-C during tissue remodelling may create fragments that act to limit FN matrix assembly. Persistent TN-C expression during fibrosis and tumour growth may contribute to uncontrolled FN deposition and angiogenesis during disease progression