Elise R. Pfaltzgraff

A dynamic code of dorsal neural tube genes regulates the segregation between neurogenic and melanogenic neural crest cells

Understanding when and how multipotent progenitors segregate into diverse fates is a key question during embryonic development. The neural crest (NC) is an exemplary model system with which to investigate the dynamics of progenitor cell specification, as it generates a multitude of derivatives. Based on ‘in ovo’ lineage analysis, we previously suggested an early fate restriction of premigratory trunk NC to generate neural versus melanogenic fates, yet the timing of fate segregation and the underlying mechanisms remained unknown. Analysis of progenitors expressing a Foxd3 reporter reveals that prospective melanoblasts downregulate Foxd3 and have already segregated from neural lineages before emigration. When this downregulation is prevented, late-emigrating avian precursors fail to upregulate the melanogenic markers Mitf and MC/1 and the guidance receptor Ednrb2, generating instead glial cells that express P0 and Fabp. In this context, Foxd3 lies downstream of Snail2 and Sox9, constituting a minimal network upstream of Mitf and Ednrb2 to link melanogenic specification with migration. Consistent with the gain-of-function data in avians, loss of Foxd3 function in mouse NC results in ectopic melanogenesis in the dorsal tube and sensory ganglia. Altogether, Foxd3 is part of a dynamically expressed gene network that is necessary and sufficient to regulate fate decisions in premigratory NC. Their timely downregulation in the dorsal neural tube is thus necessary for the switch between neural and melanocytic phases of NC development.

Neural crest and Schwann cell progenitor-derived melanocytes are two spatially segregated populations similarly regulated by Foxd3

Skin melanocytes arise from two sources: either directly from neural crest progenitors or indirectly from neural crest-derived Schwann cell precursors after colonization of peripheral nerves. The relationship between these two melanocyte populations and the factors controlling their specification remains poorly understood. Direct lineage tracing reveals that neural crest and Schwann cell progenitor-derived melanocytes are differentially restricted to the epaxial and hypaxial body domains, respectively. Furthermore, although both populations are initially part of the Foxd3 lineage, hypaxial melanocytes lose Foxd3 at late stages upon separation from the nerve, whereas we recently found that epaxial melanocytes segregate earlier from Foxd3-positive neural progenitors while still residing in the dorsal neural tube. Gain- and loss-of-function experiments in avians and mice, respectively, reveal that Foxd3 is both sufficient and necessary for regulating the balance between melanocyte and Schwann cell development. In addition, Foxd3 is also sufficient to regulate the switch between neuronal and glial fates in sensory ganglia. Together, we propose that differential fate acquisition of neural crest-derived cells depends on their progressive segregation from the Foxd3-positive lineage.

Bves and NDRG4 regulate directional epicardial cell migration through autocrine extracellular matrix deposition

The Bves and NDRG4 proteins interact to regulate directional cell movement by mediating cell surface fusion of internalized fibronectin for resecretion. This provides the first evidence of Bves/NDRG4 protein function within subcellular trafficking pathways and explains how the Bves complex diversely influences development, cancer, and repair.

Embryonic domains of the aorta derived from diverse origins exhibit distinct properties that converge into a common phenotype in the adult

Vascular smooth muscle cells (VSMCs) are derived from distinct embryonic origins. Vessels originating from differing smooth muscle cell populations have distinct vascular and pathological properties involving calcification, atherosclerosis, and structural defects such as aneurysm and coarctation. We hypothesized that domains within a single vessel, such as the aorta, vary in phenotype based on embryonic origin. Gene profiling and myographic analyses demonstrated that embryonic ascending and descending aortic domains exhibited distinct phenotypes. In vitro analyses demonstrated that VSMCs from each region were dissimilar in terms of cytoskeletal and migratory properties, and retention of different gene expression patterns. Using the same analysis, we found that these same two domains are indistinguishable in the adult vessel. Our data demonstrate that VSMCs from different embryonic origins are functionally distinct in the embryonic mouse, but converge to assume a common phenotype in the aorta of healthy adults. These findings have fundamental implications for aortic development, function and disease progression.

Functional Interaction Between Foxd3 and Pax3 in Cardiac Neural Crest Development

The transcription factors Foxd3 and Pax3 are important early regulators of neural crest (NC) progenitor cell properties. Homozygous mutations of Pax3 or a homozygous NC‐specific deletion of Foxd3 cause marked defects in most NC derivatives, but neither loss of both Foxd3 alleles nor loss of one Pax3 allele alone greatly affects overall development of cardiac NC derivatives. In contrast, compound mutant embryos homozygous for a NC‐specific Foxd3 mutation and heterozygous for Pax3 have fully penetrant persistent truncus arteriosus, severe thymus hypoplasia, and midgestation lethality. Foxd3; Pax3 compound mutant embryos have increased cell death in the neural folds and a drastic early reduction of NC cells, with an almost complete absence of NC caudal to the first pharyngeal arch. The genetic interaction between these genes implicates gene dosage‐sensitive roles for Foxd3 and Pax3 in cardiac NC progenitors. Foxd3 and Pax3 act together to affect survival and maintenance of cardiac NC progenitors, and loss of these progenitors catastrophically affects key aspects of later cardiovascular development. genesis, 2011.

Heterogeneity in vascular smooth muscle cell embryonic origin in relation to adult structure, physiology, and disease

Regional differences in vascular physiology and disease response exist throughout the vascular tree. While these differences in physiology and disease correspond to regional vascular environmental conditions, there is also compelling evidence that the embryonic origins of the smooth muscle inherent to the vessels may play a role. Here, we review what is known regarding the role of embryonic origin of vascular smooth muscle cells during vascular development. The focus of this review is to highlight the heterogeneity in the origins of vascular smooth muscle cells and the resulting regional physiologies of the vessels. Our goal is to stimulate future investigation into this area and provide a better understanding of vascular organogenesis and disease. Developmental Dynamics 244:410–416, 2015.

Transcriptional profiling reveals ductus arteriosus-specific genes that regulate vascular tone

Failure of the ductus arteriosus (DA) to close at birth can lead to serious complications. Conversely, certain profound congenital cardiac malformations require the DA to be patent until corrective surgery can be performed. In each instance, clinicians have a very limited repertoire of therapeutic options at their disposal - indomethacin or ibuprofen to close a patent DA (PDA) and prostaglandin E1 to maintain patency of the DA. Neither treatment is specific to the DA and both may have deleterious off-target effects. Therefore, more therapeutic options specifically targeted to the DA should be considered. We hypothesized the DA possesses a unique genetic signature that would set it apart from other vessels. A microarray was used to compare the genetic profiles of the murine DA and ascending aorta (AO). Over 4,000 genes were differentially expressed between these vessels including a subset of ion channel-related genes. Specifically, the alpha and beta subunits of large-conductance calcium-activated potassium (BKCa) channels are enriched in the DA. Gain- and loss-of-function studies showed inhibition of BKCa channels caused the DA to constrict, while activation caused DA relaxation even in the presence of O2. This study identifies subsets of genes that are enriched in the DA that may be used to develop DA-specific drugs. Ion channels that regulate DA tone, including BKCa channels, are promising targets. Specifically, BKCa channel agonists like NS1619 maintain DA patency even in the presence of O2 and may be clinically useful.

Autotaxin signaling governs phenotypic heterogeneity in visceral and parietal mesothelia.

Mesothelia, which cover all coelomic organs and body cavities in vertebrates, perform diverse functions in embryonic and adult life. Yet, mesothelia are traditionally viewed as simple, uniform epithelia. Here we demonstrate distinct differences between visceral and parietal mesothelia, the most basic subdivision of this tissue type, in terms of gene expression, adhesion, migration, and invasion. Gene profiling determined that autotaxin, a secreted lysophospholipase D originally discovered as a tumor cell-motility-stimulating factor, was expressed exclusively in the more motile and invasive visceral mesothelia and at abnormally high levels in mesotheliomas. Gain and loss of function studies demonstrate that autotaxin signaling is indeed a critical factor responsible for phenotypic differences within mesothelia. Furthermore, we demonstrate that known and novel small molecule inhibitors of the autotaxin signaling pathway dramatically blunt migratory and invasive behaviors of aggressive mesotheliomas. Taken together, this study reveals distinct phenotypes within the mesothelial cell lineage, demonstrates that differential autotaxin expression is the molecular underpinning for these differences, and provides a novel target and lead compounds to intervene in invasive mesotheliomas.

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types. NC also has the unique ability to influence the differentiation and maturation of target organs. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. NC multipotency was first described from explants of the avian neural tube. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter. The method presented here is adapted from protocols optimized for the culture of rat NC. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC.

Isolation and physiological analysis of mouse cardiomyocytes.

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.