Joan M. Lemire

Induction of Vertebrate Regeneration by a Transient Sodium Current

Amphibians such as frogs can restore lost organs during development, including the lens and tail. To design biomedical therapies for organ repair, it is necessary to develop a detailed understanding of natural regeneration. Recently, ion transport has been implicated as a functional regulator of regeneration. Whereas voltage-gated sodium channels play a well known and important role in propagating action potentials in excitable cells, we have identified a novel role in regeneration for the ion transport function mediated by the voltage-gated sodium channel, NaV1.2. A local, early increase in intracellular sodium is required for initiating regeneration following Xenopus laevis tail amputation, and molecular and pharmacological inhibition of sodium transport causes regenerative failure. NaV1.2 is absent under nonregenerative conditions, but misexpression of human NaV1.5 can rescue regeneration during these states. Remarkably, pharmacological induction of a transient sodium current is capable of restoring regeneration even after the formation of a nonregenerative wound epithelium, confirming that it is the regulation of sodium transport that is critical for regeneration. Our studies reveal a previously undetected competency window in which cells retain their intrinsic regenerative program, identify a novel endogenous role for NaV in regeneration, and show that modulation of sodium transport represents an exciting new approach to organ repair.

Transmembrane voltage potential controls embryonic eye patterning in Xenopus laevis

Uncovering the molecular mechanisms of eye development is crucial for understanding the embryonic morphogenesis of complex structures, as well as for the establishment of novel biomedical approaches to address birth defects and injuries of the visual system. Here, we characterize change in transmembrane voltage potential (Vmem) as a novel biophysical signal for eye induction in Xenopus laevis. During normal embryogenesis, a striking hyperpolarization demarcates a specific cluster of cells in the anterior neural field. Depolarizing the dorsal lineages in which these cells reside results in malformed eyes. Manipulating Vmem of non-eye cells induces well-formed ectopic eyes that are morphologically and histologically similar to endogenous eyes. Remarkably, such ectopic eyes can be induced far outside the anterior neural field. A Ca2+ channel-dependent pathway transduces the Vmem signal and regulates patterning of eye field transcription factors. These data reveal a new, instructive role for membrane voltage during embryogenesis and demonstrate that Vmem is a crucial upstream signal in eye development. Learning to control bioelectric initiators of organogenesis offers significant insight into birth defects that affect the eye and might have significant implications for regenerative approaches to ocular diseases.

Overexpression of the V3 variant of versican alters arterial smooth muscle cell adhesion, migration, and proliferation in vitro

Versican is an extracellular matrix proteoglycan produced by many cells. Although versican is generally known as a large chondroitin sulfate proteoglycan (CSPG), the smallest splice variant, V3, consists only of the amino‐ and carboxy‐terminal globular domains and is therefore predicted to be a small glycoprotein, lacking CS chains. The large size, negative charge, and ability of versican variants to form pericellular coats with hyaluronan are responsible for many of its effects. V3, lacking the large size and high charge density, but retaining the hyaluronan‐binding domain of the larger isoforms, may have different effects on cell phenotype. To determine whether V3 alters cell phenotype, Fisher rat arterial smooth muscle cells (ASMCs), which express the larger CSPG versican splice forms (V0 and V1) were retrovirally transduced with the rat V3 cDNA. Northern analysis for versican RNAs confirmed that cells transduced with V3 retrovirus, but not cells tranduced with the empty vector, expressed RNA of the size expected for V3/neor bicistronic RNA. V3 overexpressing cells were more spread on tissue culture plastic, had a smaller length‐to‐breadth ratio and were more resistant to release from the culture dish by trypsin. Interference reflection microscopy of sparsely plated cells showed larger areas of close contact between the V3 expressing cells and the coverslip, in comparison to control cells. Focal contacts in the periphery of V3 expressing cells were larger. Growth and migration studies revealed that V3 transduced cells grow slower and migrate a shorter distance in a scratch wound assay. The increased adhesion and the inhibition of migration and proliferation resulting from V3 overexpression are the opposites of the known and predicted effects of the other variants of versican. V3 may exert these effects through changes in pericellular coat formation, either by competing with larger isoforms for hyaluronan‐binding, or by altering other components of the pericellular matrix. J. Cell. Physiol. 190: 38–45, 2002.

Bioelectric signaling regulates head and organ size during planarian regeneration.

A main goal of regenerative medicine is to replace lost or damaged tissues and organs with functional parts of the correct size and shape. But the proliferation of new cells is not sufficient; we will also need to understand how the scale and ultimate form of newly produced tissues are determined. Using the planarian model system, we report that membrane voltage-dependent bioelectric signaling determines both head size and organ scaling during regeneration. RNA interference of the H+,K+-ATPase ion pump results in membrane hyperpolarization, which has no effect on the amount of new tissue (blastema) that is regenerated yet produces regenerates with tiny ‘shrunken’ heads and proportionally oversized pharynges. Our data show that this disproportionality results from a lack of the apoptosis required to adjust head and organ size and placement, highlighting apoptotic remodeling as the link between bioelectric signaling and the establishment of organ size during regeneration.

Versican/PG-M Isoforms in Vascular Smooth Muscle Cells

The expression of increased amounts of proteoglycans in the extracellular matrix may play a role in vascular stenosis and lipid retention. The large chondroitin sulfate proteoglycan versican is synthesized by vascular smooth muscle cells (SMCs), accumulates during human atherosclerosis and restenosis, and has been shown to bind LDLs. We recently demonstrated that adult rat aortic SMCs express several versican mRNAs. Four versican splice variants, V0, V1, V2, and V3, have recently been described, which differ dramatically in length. These variants differ in the extent of modification by glycosaminoglycan chains, and V3 may lack glycosaminoglycan chains. In this study, we characterized versican RNAs from rat SMCs by cloning, sequencing, and hybridization with domain-specific probes. DNA sequence was obtained for the V3 isoform, and for a truncated V0 isoform. By hybridization of polyadenylated RNA with domain-specific probes, we determined that the V0, V1, and V3 isoforms are present in vascular SMCs. We confirmed the presence of the V3 isoform in polyadenylated RNA and in RT-PCR products by hybridization with an oligonucleotide that spans the splice junction between the hyaluronan-binding domain and the epidermal growth factor-like domain. In addition, a novel splice variant was cloned by PCR amplification from both rat and human SMC RNA. This appears to be an incompletely spliced variant, retaining the final intron. PCR analysis shows that this intron can be retained in both V1 and V3 isoforms. The predicted translation product of this variant would have a different carboxy-terminus than previously described versican isoforms.

Transmembrane potential of GlyCl-expressing instructor cells induces a neoplastic-like conversion of melanocytes via a serotonergic pathway

SUMMARY Understanding the mechanisms that coordinate stem cell behavior within the host is a high priority for developmental biology, regenerative medicine and oncology. Endogenous ion currents and voltage gradients function alongside biochemical cues during pattern formation and tumor suppression, but it is not known whether bioelectrical signals are involved in the control of stem cell progeny in vivo. We studied Xenopus laevis neural crest, an embryonic stem cell population that gives rise to many cell types, including melanocytes, and contributes to the morphogenesis of the face, heart and other complex structures. To investigate how depolarization of transmembrane potential of cells in the neural crest’s environment influences its function in vivo, we manipulated the activity of the native glycine receptor chloride channel (GlyCl). Molecular-genetic depolarization of a sparse, widely distributed set of GlyCl-expressing cells non-cell-autonomously induces a neoplastic-like phenotype in melanocytes: they overproliferate, acquire an arborized cell shape and migrate inappropriately, colonizing numerous tissues in a metalloprotease-dependent fashion. A similar effect was observed in human melanocytes in culture. Depolarization of GlyCl-expressing cells induces these drastic changes in melanocyte behavior via a serotonin-transporter-dependent increase of extracellular serotonin (5-HT). These data reveal GlyCl as a molecular marker of a sparse and heretofore unknown cell population with the ability to specifically instruct neural crest derivatives, suggest transmembrane potential as a tractable signaling modality by which somatic cells can control stem cell behavior at considerable distance, identify a new biophysical aspect of the environment that confers a neoplastic-like phenotype upon stem cell progeny, reveal a pre-neural role for serotonin and its transporter, and suggest a novel strategy for manipulating stem cell behavior.

Inhibition of Versican Synthesis by Antisense Alters Smooth Muscle Cell Phenotype and Induces Elastic Fiber Formation In Vitro and in Neointima After Vessel Injury

The proteoglycan versican is implicated in several atherogenic events, including stimulation of vascular smooth muscle cell (VSMC) growth and migration, retention of lipoproteins, and promotion of thrombogenesis. A high content of intimal versican also correlates with a low content of elastin, suggesting an inhibitory role for versican in elastogenesis. To determine whether reduced production of versican can be used to enhance elastogenesis, we transduced Fischer rat VSMC (FRSMC) with a versican antisense sequence using the retroviral vector LXSN. Stable expression of versican antisense (LVaSN) significantly reduced versican production, induced a flattened morphology, reduced cell proliferation and migration, increased tropoelastin synthesis, increased elastin binding protein (S-Gal/EBP), and increased deposition of elastic fibers in long-term cultures. Add-back of chondroitin sulfate chains, or versican, decreased S-Gal/EBP and elastic fiber formation. LVaSN cells seeded into balloon catheter-injured rat carotid arteries formed neointimae containing low levels versican, increased amounts of S-Gal/EBP, and increased elastin deposits 7 days postinjury. At 4 weeks, neointimae formed from LVaSN cells were highly structured and contained multiple layers of elastic fibers and lamellae. These results indicate a central role for versican and its constituent chondroitin sulfate chains in controlling cell phenotype, elastogenesis, and intimal structure.

Histone Deacetylase activity is necessary for left-right patterning during vertebrate development

BackgroundConsistent asymmetry of the left-right (LR) axis is a crucial aspect of vertebrate embryogenesis. Asymmetric gene expression of the TGFβ superfamily member Nodal related 1 (Nr1) in the left lateral mesoderm plate is a highly conserved step regulating the situs of the heart and viscera. In Xenopus, movement of maternal serotonin (5HT) through gap-junctional paths at cleavage stages dictates asymmetry upstream of Nr1. However, the mechanisms linking earlier biophysical asymmetries with this transcriptional control point are not known.ResultsTo understand how an early physiological gradient is transduced into a late, stable pattern of Nr1 expression we investigated epigenetic regulation during LR patterning. Embryos injected with mRNA encoding a dominant-negative of Histone Deacetylase (HDAC) lacked Nr1 expression and exhibited randomized sidedness of the heart and viscera (heterotaxia) at stage 45. Timing analysis using pharmacological blockade of HDACs implicated cleavage stages as the active period. Inhibition during these early stages was correlated with an absence of Nr1 expression at stage 21, high levels of heterotaxia at stage 45, and the deposition of the epigenetic marker H3K4me2 on the Nr1 gene. To link the epigenetic machinery to the 5HT signaling pathway, we performed a high-throughput proteomic screen for novel cytoplasmic 5HT partners associated with the epigenetic machinery. The data identified the known HDAC partner protein Mad3 as a 5HT-binding regulator. While Mad3 overexpression led to an absence of Nr1 transcription and randomized the LR axis, a mutant form of Mad3 lacking 5HT binding sites was not able to induce heterotaxia, showing that Mad3s biological activity is dependent on 5HT binding.ConclusionHDAC activity is a new LR determinant controlling the epigenetic state of Nr1 from early developmental stages. The HDAC binding partner Mad3 may be a new serotonin-dependent regulator of asymmetry linking early physiological asymmetries to stable changes in gene expression during organogenesis.

Distinct Rat Aortic Smooth Muscle Cells Differ in Versican/PG-M Expression

Smooth muscle cells (SMCs) with distinct phenotypes are present in blood vessels, and distinct culture types appear when SMCs are maintained in vitro. For example, cultured SMCs from rat adult media grow as bipolar cells, which differ in gene expression from the predominantly cobblestone-shaped SMCs from rat pup aortas and rat neointimas that we call pi SMCs. Since proteoglycans are present at different concentrations in the normal intima and media and are elevated in atherosclerotic plaque, we sought to determine whether pi and adult medial SMC types synthesize different or unique proteoglycans that are characteristic of each phenotype. [35S]sulfate-labeled proteoglycans were purified by ion-exchange chromatography. An adult medial SMC line synthesized a large proteoglycan (0.2 Kav on Sepharose CL-2B) that was not detectable in a pi SMC line. Digestion of this proteoglycan with chondroitin ABC lyase revealed three core glycoproteins of 330, 370, and 450 kD. By Western blot analysis, the two smallest of these reacted with two antibodies to the human fibroblast proteoglycan versican. RNAs hybridizing to versican probes were found only in adult medial-type SMCs, including an adult medial type clone from pup aorta, by Northern blot analysis. Both SMC types synthesize RNAs that hybridize to probes for other proteoglycans, such as perlecan, biglycan, and decorin. We conclude that rat pi SMC cultures, unlike monkey, human, and rat adult medial SMC cultures, express little or no versican. This difference in expression may be responsible for the different morphologies and growth properties of the two cell types.

Early, nonciliary role for microtubule proteins in left–right patterning is conserved across kingdoms

Many types of embryos’ bodyplans exhibit consistently oriented laterality of the heart, viscera, and brain. Errors of left–right patterning present an important class of human birth defects, and considerable controversy exists about the nature and evolutionary conservation of the molecular mechanisms that allow embryos to reliably orient the left–right axis. Here we show that the same mutations in the cytoskeletal protein tubulin that alter asymmetry in plants also affect very early steps of left–right patterning in nematode and frog embryos, as well as chirality of human cells in culture. In the frog embryo, tubulin α and tubulin γ-associated proteins are required for the differential distribution of maternal proteins to the left or right blastomere at the first cell division. Our data reveal a remarkable molecular conservation of mechanisms initiating left–right asymmetry. The origin of laterality is cytoplasmic, ancient, and highly conserved across kingdoms, a fundamental feature of the cytoskeleton that underlies chirality in cells and multicellular organisms.