Extracellular matrix dynamics in tubulogenesis.

Biological tubes form in a variety of shapes and sizes. Tubular topology of cells and tissues is a widely recognizable histological feature of multicellular life. Fluid secretion, storage, transport, absorption, exchange, and elimination-processes central to metazoans-hinge on the exquisite tubular architectures of cells, tissues, and organs.

Altered VEGF Signaling Leads to Defects in Heart Tube Elongation and Omphalomesenteric Vein Fusion in Quail Embryos.

Formation of the endocardial and myocardial heart tubes involves precise cardiac progenitor sorting and tissue displacements from the primary heart field to the embryonic midline-a process that is dependent on proper formation of conjoining great vessels, including the omphalomesenteric veins (OVs) and dorsal aortae. Using a combination of vascular endothelial growth factor (VEGF) over- and under-activation, fluorescence labeling of cardiac progenitors (endocardial and myocardial), and time-lapse imaging, we show that altering VEGF signaling results in previously unreported myocardial, in addition to vascular and endocardial phenotypes. Resultant data show: (1) exogenous VEGF leads to truncated endocardial and myocardial heart tubes and grossly dilated OVs; (2) decreased levels of VEGF receptor 2 tyrosine kinase signaling result in a severe abrogation of the endocardial tube, dorsal aortae, and OVs.

Live tissue antibody injection: A novel method for imaging ECM in limb buds and other tissues.

Understanding the morphogenesis and differentiation of tissues and organs from progenitor fields requires methods to visualize this process. Despite an ever-growing recognition that ECM plays an important role in tissue development, studies of ECM movement, and patterns in live tissue are scarce. Here, we describe a method in which a living limb bud is immunolabeled prior to fixation using fluorescent antibodies that recognize two ECM constituents, fibronectin and fibrillin 2.

Extracellular matrix motion and early morphogenesis.

For over a century, embryologists who studied cellular motion in early amniotes generally assumed that morphogenetic movement reflected migration relative to a static extracellular matrix (ECM) scaffold. However, as we discuss in this Review, recent investigations reveal that the ECM is also moving during morphogenesis. Time-lapse studies show how convective tissue displacement patterns, as visualized by ECM markers, contribute to morphogenesis and organogenesis.

Identification of emergent motion compartments in the amniote embryo.

The tissue scale deformations (≥ 1 mm) required to form an amniote embryo are poorly understood. Here, we studied ∼400 μm-sized explant units from gastrulating quail embryos. The explants deformed in a reproducible manner when grown using a novel vitelline membrane-based culture method.

Active cell and ECM movements during development.

Dynamic imaging of the extracellular matrix (ECM) and cells can reveal how tissues are formed. Displacement differences between cells and the adjacent ECM scaffold can be used to establish active movements of mesenchymal cells. Cells can also generate large-scale tissue movements in which cell and ECM displacements are shared.

Embryogenesis of the first circulating endothelial cells.

Prior to this study, the earliest appearance of circulating endothelial cells in warm-blooded animals was unknown. Time-lapse imaging of germ-line transformed Tie1-YFP reporter quail embryos combined with the endothelial marker antibody QH1 provides definitive evidence for the existence of circulating endothelial cells - from the very beginning of blood flow. Blood-smear counts of circulating cells from Tie1-YFP embryos showed that up to 30% of blood-borne cells are Tie1 positive; though cells expressing low levels of YFP were also positive for benzidine, a hemoglobin stain, suggesting that these cells were differentiating into erythroblasts.

Vascular Network Formation in Expanding versus Static Tissues: Embryos and Tumors.

In this perspectives article, we review scientific literature regarding de novo formation of vascular networks within tissues undergoing a significant degree of motion. Next, we contrast dynamic pattern formation in embryos to the vascularization of relatively static tissues, such as the retina. We argue that formation of primary polygonal vascular networks is an emergent process, which is regulated by biophysical mechanisms.

Spatial anisotropies and temporal fluctuations in extracellular matrix network texture during early embryogenesis.

Early stages of vertebrate embryogenesis are characterized by a remarkable series of shape changes. The resulting morphological complexity is driven by molecular, cellular, and tissue-scale biophysical alterations. Operating at the cellular level, extracellular matrix (ECM) networks facilitate cell motility.

Pattern formation during vasculogenesis.

Vasculogenesis, the assembly of the first vascular network, is an intriguing developmental process that yields the first functional organ system of the embryo. In addition to being a fundamental part of embryonic development, vasculogenic processes also have medical importance. To explain the organizational principles behind vascular patterning, we must understand how morphogenesis of tissue level structures can be controlled through cell behavior patterns that, in turn, are determined by biochemical signal transduction processes.

In vivo imaging of basement membrane movement: ECM patterning shapes Hydra polyps.

Growth and morphogenesis during embryonic development, asexual reproduction and regeneration require extensive remodeling of the extracellular matrix (ECM). We used the simple metazoan Hydra to examine the fate of ECM during tissue morphogenesis and asexual budding. In growing Hydra, epithelial cells constantly move towards the extremities of the animal and into outgrowing buds.

[A non directional cell migration gradient in the presomitic mesoderm contributes to axis elongation in chicken embryos].

Vertebrates are characterized by an elongated antero-posterior (AP) body axis. This particular shape arises during embryogenesis by mophogenetic events leading to elongation. Although elongation mechanisms that lead to the formation of the anterior part of the body are well described, the ones concerning the posterior part still remain poorly studied.

Dynamic analysis of vascular morphogenesis using transgenic quail embryos.

Background: One of the least understood and most central questions confronting biologists is how initially simple clusters or sheet-like cell collectives can assemble into highly complex three-dimensional functional tissues and organs. Due to the limits of oxygen diffusion, blood vessels are an essential and ubiquitous presence in all amniote tissues and organs. Vasculogenesis, the de novo self-assembly of endothelial cell (EC) precursors into endothelial tubes, is the first step in blood vessel formation.

A random cell motility gradient downstream of FGF controls elongation of an amniote embryo.

Vertebrate embryos are characterized by an elongated antero-posterior (AP) body axis, which forms by progressive cell deposition from a posterior growth zone in the embryo. Here, we used tissue ablation in the chicken embryo to demonstrate that the caudal presomitic mesoderm (PSM) has a key role in axis elongation. Using time-lapse microscopy, we analysed the movements of fluorescently labelled cells in the PSM during embryo elongation, which revealed a clear posterior-to-anterior gradient of cell motility and directionality in the PSM.

Rotation of organizer tissue contributes to left-right asymmetry.

Current hypotheses regarding vertebrate left-right asymmetry patterns are based on the presumption that genetic regulatory networks specify sidedness via extracellular morphogens and/or ciliary activity. We show empirical time-lapse evidence for an asymmetric rotation of epiblastic nodal tissue in avian embryos. This rotation spans the interval when initial symmetric expression of Shh and Fgf8 becomes asymmetrical with respect to the midline.

Chapter 5. Avian embryos a model for the study of primary vascular assembly in warm-blooded animals.

The formation of a primary vascular bed is a dynamic process, aspects of which are readily amenable to time-lapse imaging in avian embryos. At early developmental stages, the body plan of avian embryos is very similar to mammals and has many properties that make it ideal for imaging. We devised labeling, culturing, and imaging techniques that capture high-resolution images of intact avian embryos in four dimensions over large length scales (1 to 5000 microm).

Matrix metalloproteinase 2-integrin alpha(v)beta3 binding is required for mesenchymal cell invasive activity but not epithelial locomotion: a computational time-lapse study.

Cellular invasive behavior through three-dimensional collagen gels was analyzed using computational time-lapse imaging. A subpopulation of endocardial cells, derived from explanted quail cardiac cushions, undergoes an epithelial-to-mesenchymal transition and invades the substance of the collagen gels when placed in culture. In contrast, other endocardial cells remain epithelial and move over the gel surface.

The ECM moves during primitive streak formation--computation of ECM versus cellular motion.

Galileo described the concept of motion relativity--motion with respect to a reference frame--in 1632. He noted that a person below deck would be unable to discern whether the boat was moving. Embryologists, while recognizing that embryonic tissues undergo large-scale deformations, have failed to account for relative motion when analyzing cell motility data.

Vascular sprout formation entails tissue deformations and VE-cadherin-dependent cell-autonomous motility.

Embryonic and fetal vascular sprouts form within constantly expanding tissues. Nevertheless, most biological assays of vascular spouting are conducted in a static mechanical milieu. Here we study embryonic mouse allantoides, which normally give raise to an umbilical artery and vein.

Multicellular sprouting during vasculogenesis.

Living organisms, from bacteria to vertebrates, are well known to generate sophisticated multicellular patterns. Numerous recent interdisciplinary studies have focused on the formation and regulation of these structures. Advances in automatized microscopy allow the time-resolved tracking of embryonic development at cellular resolution over an extended area covering most of the embryo.

Mesodermal cell displacements during avian gastrulation are due to both individual cell-autonomous and convective tissue movements.

Gastrulation is a fundamental process in early development that results in the formation of three primary germ layers. During avian gastrulation, presumptive mesodermal cells in the dorsal epiblast ingress through a furrow called the primitive streak (PS), and subsequently move away from the PS and form adult tissues. The biophysical mechanisms driving mesodermal cell movements during gastrulation in amniotes, notably warm-blooded embryos, are not understood.

Electroporation and EGFP labeling of gastrulating quail embryos.

Labeling embryonic cells to trace their motion is a classical experimental approach with a host of techniques being used to mark live cells and tissues. Genetically engineered fluorescent protein vectors (DNA plasmids) are a recent technology well suited to time-resolved studies of cellular motion in live embryos. DNA plasmids encoding fluorescent proteins can be introduced into cells using several methods, including electroporation, a technique used widely for analysis of tissue culture and embryonic cells.

Extracellular matrix macroassembly dynamics in early vertebrate embryos.

This chapter focuses on the in vivo macroassembly dynamics of fibronectin and fibrillin-2--two prominent extracellular matrix (ECM) components, present in vertebrate embryos at the earliest stages of development. The ECM is an inherently dynamic structure with a well-defined position fate: ECM filaments are not only anchored to and move with established tissue boundaries, but are repositioned prior to the formation of new anatomical features. We distinguish two ECM filament relocation processes-each operating on different length scales.