James D. Lauderdale

Axon Tracts Correlate with Netrin-1a Expression in the Zebrafish Embryo

Netrins are secreted molecules that can attract or repel growth cones from a variety of organisms. In order to clarify the extent and scope of the effects of netrins for guiding growth cones, we have analyzed netrin-1a within the relatively simple and well-characterized nervous system of zebrafish embryos. netrin-1a is expressed in dynamic patterns that suggest that it guides the growth cones of a wide variety of neurons. The spatiotemporal relationship of netrin-1a expression and extending growth cones further suggests that netrins may act to delineate specific pathways and stimulate axonal outgrowth in addition to attracting and repelling growth cones. Furthermore, aberrant outgrowth by commissural growth cones in the spinal cords of floating head mutants, in which netrin-1a expression is altered, is consistent with an in vivo, chemoattractive action of netrin-1a. These data suggest that netrins act on many growth cones and influence their behavior in a variety of ways.

Directed differentiation of human pluripotent cells to neural crest stem cells

Multipotent neural crest stem cells (NCSCs) have the potential to generate a wide range of cell types including melanocytes; peripheral neurons; and smooth muscle, bone, cartilage and fat cells. This protocol describes in detail how to perform a highly efficient, lineage-specific differentiation of human pluripotent cells to a NCSC fate. The approach uses chemically defined media under feeder-free conditions, and it uses two small-molecule compounds to achieve efficient conversion of human pluripotent cells to NCSCs in ∼15 d. After completion of this protocol, NCSCs can be used for numerous applications, including the generation of sufficient cell numbers to perform drug screens, for the development of cell therapeutics on an industrial scale and to provide a robust model for human disease. This protocol can be also be applied to patient-derived induced pluripotent stem cells and thus used to further the knowledge of human disease associated with neural crest development, for example, Treacher-Collins Syndrome.

PAX6 Regulates Melanogenesis in the Retinal Pigmented Epithelium through Feed-Forward Regulatory Interactions with MITF

During organogenesis, PAX6 is required for establishment of various progenitor subtypes within the central nervous system, eye and pancreas. PAX6 expression is maintained in a variety of cell types within each organ, although its role in each lineage and how it acquires cell-specific activity remain elusive. Herein, we aimed to determine the roles and the hierarchical organization of the PAX6-dependent gene regulatory network during the differentiation of the retinal pigmented epithelium (RPE). Somatic mutagenesis of Pax6 in the differentiating RPE revealed that PAX6 functions in a feed-forward regulatory loop with MITF during onset of melanogenesis. PAX6 both controls the expression of an RPE isoform of Mitf and synergizes with MITF to activate expression of genes involved in pigment biogenesis. This study exemplifies how one kernel gene pivotal in organ formation accomplishes a lineage-specific role during terminal differentiation of a single lineage.

Shared Enhancer Activity in the Limbs and Phallus and Functional Divergence of a Limb-Genital cis-Regulatory Element in Snakes.

The amniote phallus and limbs differ dramatically in their morphologies but share patterns of signaling and gene expression in early development. Thus far, the extent to which genital and limb transcriptional networks also share cis-regulatory elements has remained unexplored. We show that many limb enhancers are retained in snake genomes, suggesting that these elements may function in non-limb tissues. Consistent with this, our analysis of cis-regulatory activity in mice and Anolis lizards reveals that patterns of enhancer activity in embryonic limbs and genitalia overlap heavily. In mice, deletion of HLEB, an enhancer of Tbx4, produces defects in hindlimbs and genitalia, establishing the importance of this limb-genital enhancer for development of these different appendages. Further analyses demonstrate that the HLEB of snakes has lost hindlimb enhancer function while retaining genital activity. Our findings identify roles for Tbx4 in genital development and highlight deep similarities in cis-regulatory activity between limbs and genitalia.

REGULATION OF NETRIN-1A EXPRESSION BY HEDGEHOG PROTEINS

Netrins,a family of growth cone guidance molecules, are expressed both in the ventral neural tube and in subsets of mesodermal cells. In an effort to better understand the regulation ofnetrins,we examined the expression ofnetrin-1ain mutantcyclops, no tail,andfloating headzebrafish embryos, in which axial midline structures are perturbed.Netrin-1aexpression requires signals present in notochord and floor plate cells. In the myotome, but not the neural tube,netrin-1aexpression requiressonic hedgehog.In embryos lackingsonic hedgehog,thesonic-youlocus,netrin-1aexpression is reduced or absent in the myotomes but present in the neural tube. Embryos lackingsonic hedgehogexpresstiggy-winkle hedgehogin the floor plate, suggesting that, in the neural tube,tiggy-winkle hedgehogcan compensate for the lack ofsonic hedgehogin inducingnetrin-1aexpression. Ectopic expression ofsonic hedgehog, tiggy-winkle hedgehog,orechidna hedgehoginduces ectopicnetrin-1aexpression in the neural tube, and ectopic expression ofsonic hedgehogortiggy-winkle hedgehog,but notechidna hedgehog,induces ectopicnetrin-1aexpression in somites. These data demonstrate that in vertebratesnetrinexpression is regulated by Hedgehog signaling.

Characterization of cytokines associated with Th17 cells in the eyes of horses with recurrent uveitis

OBJECTIVE Equine recurrent uveitis (ERU) is a spontaneous disease that is the most common cause of blindness in horses, affecting up to 15% of the horse population. Th17 cells are a major cell population driving the pathogenesis in several mouse models of autoimmune inflammation, including experimental autoimmune uveitis. The purpose of this study is to investigate the role a Th17 cell-mediated response plays in the pathogenesis of ERU. PROCEDURE Banked, Davidsons-fixed equine globes histopathologically diagnosed with ERU (n = 7) were compared immunohistochemically with healthy control globes (n = 7). Immunohistochemical staining was performed using a pan-Leptospira antibody and antibodies against IL-6, IL-17, and IL-23. Additionally, immunostaining was performed for T-cell (CD3) and B-cell (CD79α) markers. Specificity of immunoreactivity was confirmed by western blot analysis. RESULTS Immunohistochemical staining was positive for IL-6, IL-17, and IL-23 within the cytoplasm of nonpigmented ciliary epithelial cells and mononuclear inflammatory cells infiltrating the iris, and ciliary body of ERU horses (n = 7) but negative in controls (n = 7). ERU-affected eyes were CD3 positive (n = 7) and CD79α negative (n = 7). Staining for Leptospira was negative in all ERU and control globes. CONCLUSIONS Strong immunoreactivity for IL-6, IL-17, and IL-23, in conjunction with the fact that T lymphocytes are the predominating inflammatory cells present in ERU, suggests that IL-17-secreting helper T-cells play a role in the pathogenesis of ERU. These findings suggest that horses with ERU may serve as a naturally occurring animal model for autoimmune uveitis.

Xenopus pax6 mutants affect eye development and other organ systems, and have phenotypic similarities to human aniridia patients.

Mutations in the Pax6 gene cause ocular defects in both vertebrate and invertebrate animal species, and the disease aniridia in humans. Despite extensive experimentation on this gene in multiple species, including humans, we still do not understand the earliest effects on development mediated by this gene. This prompted us to develop pax6 mutant lines in Xenopus tropicalis taking advantage of the utility of the Xenopus system for examining early development and in addition to establish a model for studying the human disease aniridia in an accessible lower vertebrate. We have generated mutants in pax6 by using Transcription Activator-Like Effector Nuclease (TALEN) constructs for gene editing in X. tropicalis. Embryos with putative null mutations show severe eye abnormalities and changes in brain development, as assessed by changes in morphology and gene expression. One gene that we found is downregulated very early in development in these pax6 mutants is myc, a gene involved in pluripotency and progenitor cell maintenance and likely a mediator of some key pax6 functions in the embryo. Changes in gene expression in the developing brain and pancreas reflect other important functions of pax6 during development. In mutations with partial loss of pax6 function eye development is initially relatively normal but froglets show an underdeveloped iris, similar to the classic phenotype (aniridia) seen in human patients with PAX6 mutations. Other eye abnormalities observed in these froglets, including cataracts and corneal defects, are also common in human aniridia. The frog model thus allows us to examine the earliest deficits in eye formation as a result of pax6 lesions, and provides a useful model for understanding the developmental basis for the aniridia phenotype seen in humans.

Estimating weak ratiometric signals in imaging data. I. Dual-channel data

Ratiometric fluorescent indicators are becoming increasingly prevalent in many areas of biology. They are used for making quantitative measurements of intracellular free calcium both in vitro and in vivo, as well as measuring membrane potentials, pH, and other important physiological variables of interest to researchers in many subfields. Often, functional changes in the fluorescent yield of ratiometric indicators are small, and the signal-to-noise ratio (SNR) is of order unity or less. In particular, variability in the denominator of the ratio can lead to very poor ratio estimates. We present a statistical optimization method for objectively detecting and estimating ratiometric signals in dual-wavelength measurements of fluorescent, ratiometric indicators that improves on standard methods. With the use of an appropriate statistical model for ratiometric signals and by taking the pixel-pixel covariance of an imaging dataset into account, we are able to extract user-independent spatiotemporal information that retains high resolution in both space and time.

New statistical methods enhance imaging of cameleon fluorescence resonance energy transfer in cultured zebrafish spinal neurons

Cameleons are genetically encoded fluorescence resonance energy transfer (FRET)-based Ca(2+) indicators. Attempts to use cameleons to detect neural activity in vertebrate systems have been largely frustrated by the small FRET signal, in contradistinction to the higher signals seen in Drosophila and Caenorhabditis elegans. We have developed a statistical optimization method capable of detecting small ratiometric signals in noisy imaging data, called statistical optimization for the analysis of ratiometric signals. Using this method, we can detect and estimate anticorrelated ratiometric signals with subcellular resolution in cultured, dissociated zebrafish spinal neurons expressing cameleon or loaded with fluo-4 and fura-red. This method may make it possible to use yellow cameleons for measuring neural activity at high resolution in transgenic animals.

Cellular Stiffness as a Novel Stemness Marker in the Corneal Limbus

Healthy eyes contain a population of limbal stem cells (LSCs) that continuously renew the corneal epithelium. However, each year, 1 million Americans are afflicted with severely reduced visual acuity caused by corneal damage or disease, including LSC deficiency (LSCD). Recent advances in corneal transplant technology promise to repair the cornea by implanting healthy LSCs to encourage regeneration; however, success is limited to transplanted tissues that contain a sufficiently high percentage of LSCs. Attempts to screen limbal tissues for suitable implants using molecular stemness markers are confounded by the poorly understood signature of the LSC phenotype. For cells derived from the corneal limbus, we show that the performance of cell stiffness as a stemness indicator is on par with the performance of ΔNP63α, a common molecular marker. In combination with recent methods for sorting cells on a biophysical basis, the biomechanical stemness markers presented here may enable the rapid purification of LSCs from a heterogeneous population of corneal cells, thus potentially enabling clinicians and researchers to generate corneal transplants with sufficiently high fractions of LSCs, regardless of the LSC percentage in the donor tissue.