Rusty Lansford - Academia.edu (original) (raw)

Papers by Rusty Lansford

The FASEB Journal, 2012

Vascular endothelial growth factor (VEGF) interacts with extracellular matrix (ECM) constituents;... more Vascular endothelial growth factor (VEGF) interacts with extracellular matrix (ECM) constituents; setting up morphogenetic gradients and regulating ligand presentation to cells. We are investigating the behavior of ECM-interacting VEGF165 and soluble VEGF121 during endocardial assembly in quail. We use fluorescent anti-VEGF antibodies and VEGF receptor bodies to visualize VEGF. ECM constituents are visualized by microinjection of fluorescent antibodies against fibronectin and fibrillin-2. All experiments are performed in Tie1::H2B-YFP quail embryos (nuclear labeling of endocardial cells). Endocardial cell, ECM, and VEGF tissue components are recorded using time lapse imaging, with their relative movements analyzed using tracing and image analysis techniques. During formation of the endocardium, ECM components move in a coordinated manner with endocardial precursors (tissue motion). However, neither VEGF over-activation (hypervascularization) or inactivation (blocking of endocardial differentiation) halts the tissue movement that results in formation (albeit abnormal) of a midline heart tube. Our results further the current understanding of the coordinated behavior of VEGF and ECM constituents of embryonic tissues and their collective role in cardiovascular morphogenesis. HL085694 (BJR); HL087136 (AC); Mathers Foundation (BJR, AC, CDL); AHA predoctoral award (AA).

The FASEB Journal, 2010

We studied vasculogenic sprouting, the multicellular patterning mechanism of vasculogenesis, with... more We studied vasculogenic sprouting, the multicellular patterning mechanism of vasculogenesis, with time-lapse microscopy in avian embryos, and in cultures of endothelial cells invading collagen I ge...

Frontiers in Physiology

The avian egg is a closed system that protects the growing embryo from external factors but preve... more The avian egg is a closed system that protects the growing embryo from external factors but prevents direct observation of embryo development. Various culture systems exist in the literature to study the development of the embryo for short periods of incubation (from 12 h up to a maximum of 60 h of egg incubation). A common flaw to these culture techniques is the inability to culture the unincubated avian blastoderm with intact tissue tensions on its native yolk. The goal of this work is to create a unique novel egg-in-cube system that can be used for long-term quail embryo culture initiated from its unincubated blastoderm stage. The egg-in-cube acts as an artificial transparent eggshell system that holds the growing embryo, making it amenable to microscopy. With the egg-in-cube system, quail embryos can be grown up to 9 days from the unincubated blastoderm (incubated in air, 20.9% O2), which improves to 15 days on switching to a hyperoxic environment of 60% O2. Using transgenic flu...

Leonardo, 2022

Scientists today can collect more data than they can perceive using traditional visualization met... more Scientists today can collect more data than they can perceive using traditional visualization methods. New technologies and sensors allow researchers to gather dynamic, complex multidimensional datasets—all of which must be carefully studied to reveal hidden patterns and narratives. The authors used an immersive platform to design a new visualization for real-time, intuitive spatial manipulations of time-based volumetric datasets via a wand-based gestural interface. The resulting work resolves microscopic tissue structures at a human scale in a room-based pixel space, facilitating research, discovery and in-person teaching and collaboration.

time lapse of a stage HH10 PGK1:H2B-chFP embryo ventral view 20x objective on a Zeiss 780 confoca... more time lapse of a stage HH10 PGK1:H2B-chFP embryo ventral view 20x objective on a Zeiss 780 confocal microscope and Zen 2011 black edition 64 bit software.Fig 5 and Fig

Frontiers in Cell and Developmental Biology, 2019

During early avian development, primordial germ cells (PGC) are highly migratory, moving from the... more During early avian development, primordial germ cells (PGC) are highly migratory, moving from the central area pellucida of the blastoderm to the anterior extra-embryonic germinal crescent. The PGCs soon move into the forming blood vessels by intravasation and travel in the circulatory system to the genital ridges where they participate in the organogenesis of the gonads. This complex cellular migration takes place in close association with a nascent extracellular matrix that matures in a precise spatio-temporal pattern. We first compiled a list of quail matrisome genes by bioinformatic screening of human matrisome orthologs. Next, we used single cell RNA-seq analysis (scRNAseq) to determine that PGCs express numerous ECM and ECM-associated genes in early embryos. The expression of select ECM transcripts and proteins in PGCs were verified by fluorescent in situ hybridization (FISH) and immunofluorescence (IF). Live imaging of transgenic quail embryos injected with fluorescent antibodies against fibronectin and laminin, showed that germinal crescent PGCs display rapid shape changes and morphological properties such as blebbing and filopodia while surrounded by, or in close contact with, an ECM fibril meshwork that is itself in constant motion. Injection of anti-β1 integrin CSAT antibodies resulted in a reduction of mature fibronectin and laminin fibril meshwork in the germinal crescent at HH4-5 but did not alter the active motility of the PGCs or their ability to populate the germinal crescent. These results suggest that integrin β1 receptors are important, but not required, for PGCs to successfully migrate during embryonic development, but instead play a vital role in ECM fibrillogenesis and assembly.

Development (Cambridge, England), Oct 1, 2016

In situ hybridization methods are used across the biological sciences to map mRNA expression with... more In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to ...

SummaryEmbryonic axis extension is a complex multi-tissue morphogenetic process responsible for t... more SummaryEmbryonic axis extension is a complex multi-tissue morphogenetic process responsible for the formation of the posterior part of the amniote body. Cells located in the caudal part of the embryo divide and rearrange to participate in the elongation of the different embryonic tissues (e.g. neural tube, axial and paraxial mesoderm, lateral plate, ectoderm, endoderm). We previously identified the paraxial mesoderm as a crucial player of axis elongation, but how movements and growth are coordinated between the different posterior tissues to drive morphogenesis remain largely unknown. Here we use the quail embryo as a model system to quantify cell behavior and movements in the various tissues of the elongating embryo. We first quantify the tissue-specific contribution to axis elongation by using 3D volumetric techniques, then quantify tissue-specific parameters such as cell density and proliferation at different embryonic stages. To be able to study cell behavior at a multi-tissue s...

Development, 2015

Embryogenesis is the coordinated assembly of tissues during morphogenesis by changes in individua... more Embryogenesis is the coordinated assembly of tissues during morphogenesis by changes in individual cell behaviors and collective cell movements. Dynamic imaging, combined with quantitative analysis, are ideal for investigating fundamental questions in developmental biology involving cellular differentiation, growth control, and morphogenesis. However, a reliable amniote model system amenable to the rigors of extended, high resolution imaging and cell tracking has been lacking. To address this shortcoming, we produced a novel transgenic quail that ubiquitously expresses nuclear localized monomer cherry fluorescent protein (chFP). We characterize the expression pattern of the chFP and provide concrete examples of how Tg(PGK1:H2B-chFP) quail can be used to dynamically image and analyze key morphogenetic events during embryonic stages X to 11.

Nature Communications, 2015

The diverse morphology of vertebrate skeletal system is genetically controlled, yet the means by ... more The diverse morphology of vertebrate skeletal system is genetically controlled, yet the means by which cells shape the skeleton remains to be fully illuminated. Here we perform quantitative analyses of cell behaviours in the growth plate cartilage, the template for long bone formation, to gain insights into this process. Using a robust avian embryonic organ culture, we employ time-lapse two-photon laser scanning microscopy to observe proliferative cells' behaviours during cartilage growth, resulting in cellular trajectories with a spreading displacement mainly along the tissue elongation axis. We build a novel software toolkit of quantitative methods to segregate the contributions of various cellular processes to the cellular trajectories. We find that convergent-extension, mitotic cell division, and daughter cell rearrangement do not contribute significantly to the observed growth process; instead, extracellular matrix deposition and cell volume enlargement are the key contributors to embryonic cartilage elongation.

Cold Spring Harbor protocols, 2014

This weeklong protocol for making and testing lentivirus has been used in the Advanced Topics in ... more This weeklong protocol for making and testing lentivirus has been used in the Advanced Topics in Molecular Neuroscience (ATMN) lecture and laboratory course at Cold Spring Harbor Laboratory (CSHL) for nearly a decade. Lentiviruses are derived from HIV-1 and are ideal vehicles for the delivery of multiple genes of interest into target cells. In this protocol, 2A peptide-linked sequences are used to create a bicistronic lentiviral construct containing a ubiquitous promoter (chick β actin with a cytomegalovirus [CMV] early enhancer) driving dual expression of two fluorescent proteins (FP): H2B-Cerulean (a nuclear-localized blue FP) and Dendra2 (a photoactivatable green FP that converts to red after exposure to UV light). Polymerase chain reaction (PCR) amplification of the bicistronic insert is followed by subcloning into a lentiviral vector and transfection into a packaging cell line. The resulting viral supernatants can be used to prepare concentrated stocks and infect cells for imag...

Cold Spring Harbor protocols, 2009

This protocol describes how to generate high-titer lentivirus for the production of transgenic Ja... more This protocol describes how to generate high-titer lentivirus for the production of transgenic Japanese quail. The virus is pseudotyped with vesicular stomatitis virus with the envelope G glycoprotein (VSV-g), which gives a broad infectious range and allows concentration of viral supernatants by ultracentrifugation. Using this method, we typically produce titers >1 x 10(8) transforming units (TU)/mL and recommend using a virus with a titer at least this high for in vivo work.

BMC Biology, 2014

Background: Parasympathetic signaling has been inferred to regulate epithelial branching as well ... more Background: Parasympathetic signaling has been inferred to regulate epithelial branching as well as organ regeneration and tumor development. However, the relative contribution of local nerve contact versus secreted signals remains unclear. Here, we show a conserved (vertebrates to invertebrates) requirement for intact local nerves in airway branching, persisting even when cholinergic neurotransmission is blocked. Results: In the vertebrate lung, deleting enhanced green fluorescent protein (eGFP)-labeled intrinsic neurons using a two-photon laser leaves adjacent cells intact, but abolishes branching. Branching is unaffected by similar laser power delivered to the immediately adjacent non-neural mesodermal tissue, by blocking cholinergic receptors or by blocking synaptic transmission with botulinum toxin A. Because adjacent vasculature and epithelial proliferation also contribute to branching in the vertebrate lung, the direct dependence on nerves for airway branching was tested by deleting neurons in Drosophila embryos. A specific deletion of neurons in the Drosophila embryo by driving cell-autonomous RicinA under the pan-neuronal elav enhancer perturbed Drosophila airway development. This system confirmed that even in the absence of a vasculature or epithelial proliferation, airway branching is still disrupted by neural lesioning. Conclusions: Together, this shows that airway morphogenesis requires local innervation in vertebrates and invertebrates, yet neurotransmission is dispensable. The need for innervation persists in the fly, wherein adjacent vasculature and epithelial proliferation are absent. Our novel, targeted laser ablation technique permitted the local function of parasympathetic innervation to be distinguished from neurotransmission.