Rajani M. George

Disruption of zebrafish (Danio rerio) reproduction upon chronic exposure to TiO2 nanoparticles

As common engineered nanomaterials, TiO(2) nanoparticles (nTiO(2)) are usually perceived as non-toxic, and have already been widely used in many products and applications. Such a perception might have been shaped by some short-term studies that revealed no/low toxicity of nTiO(2) to cells and eco-relevant organisms. However, given the ultimate release of nTiO(2) into the aquatic environment, which can act as a sink for engineered nanoparticles, their long-term impact on the environment and human health is still a concern and deserves more research efforts. Here, for the first time, we demonstrate that chronic exposure of zebrafish to 0.1 mg L(-1) nTiO(2), can significantly impair zebrafish reproduction. For instance, there was a 29.5% reduction in the cumulative number of zebrafish eggs after 13 weeks of nTiO(2) exposure. Thus, we provided timely information on indicating a serious risk of reproductive impairment of environments contaminated with low levels of nTiO(2) on aquatic organisms, leading to alterations in population dynamics and aquatic ecosystem balance, and thus warrants a careful scrutiny on toxicity assessment of nTiO(2), especially their long-term impact.

A Histological Comparison of the Original and Regenerated Tail in the Green Anole, Anolis carolinensis

This study provides a histological comparison of the mature regenerated and original tail of the lizard Anolis carolinensis. These data will provide a framework for future studies of this emerging model organism whose genome was recently published. This study demonstrated that the cartilage skeleton of the regenerated tail enclosed a spinal cord with an ependymal core, but there was no evidence that dorsal root ganglia or peripheral nerves are regenerated. The cartilage tube contained foramina that allowed the vasculature to cross, but was otherwise a rigid structure. The original tail has muscle groups arranged in quadrants in a regular pattern that attach to the vertebral column. The regenerated tail has irregular muscle bundles of variable number that form unusual attachments to each other and to the cartilage tube. Furthermore, the data show that there was increased connective tissue within the muscle bundles. Implications for functionality of the regenerated tail and for future biomechanical studies are discussed. Anat Rec, 2012.

Transcriptomic Analysis of Tail Regeneration in the Lizard Anolis carolinensis Reveals Activation of Conserved Vertebrate Developmental and Repair Mechanisms

Lizards, which are amniote vertebrates like humans, are able to lose and regenerate a functional tail. Understanding the molecular basis of this process would advance regenerative approaches in amniotes, including humans. We have carried out the first transcriptomic analysis of tail regeneration in a lizard, the green anole Anolis carolinensis, which revealed 326 differentially expressed genes activating multiple developmental and repair mechanisms. Specifically, genes involved in wound response, hormonal regulation, musculoskeletal development, and the Wnt and MAPK/FGF pathways were differentially expressed along the regenerating tail axis. Furthermore, we identified 2 microRNA precursor families, 22 unclassified non-coding RNAs, and 3 novel protein-coding genes significantly enriched in the regenerating tail. However, high levels of progenitor/stem cell markers were not observed in any region of the regenerating tail. Furthermore, we observed multiple tissue-type specific clusters of proliferating cells along the regenerating tail, not localized to the tail tip. These findings predict a different mechanism of regeneration in the lizard than the blastema model described in the salamander and the zebrafish, which are anamniote vertebrates. Thus, lizard tail regrowth involves the activation of conserved developmental and wound response pathways, which are potential targets for regenerative medical therapies.

Numb regulates Notch1, but not Notch3, during myogenesis

In the vertebrate embryo, skeletal muscle is derived from the myotome of the somites. Notch1-3 demonstrate overlapping and distinct expression patterns in mouse somites. Notch1 and Notch2 have been shown to be inhibitors of skeletal myogenesis. The current data demonstrate that Notch3 also is an effective inhibitor of MyoD induced myogenesis. Numb, an adaptor protein that promotes Notch degradation by recruiting the E3 ubiquitin ligase, Itch, is limited in expression to dividing cells of the dorsal medial lip of the dermomyotome and the myotome itself. Here the specificity of the four protein isoforms of Numb for the Notch receptors was examined. In transcription and myogenic differentiation assays, Notch1 was consistently negatively regulated by all four Numb isoforms, and Notch3 was not a target for Numb. Notch2 however was variably affected. Subsequent analyses showed that unlike Notch1, that Notch3 was not polyubiquitinated, nor degraded when co-expressed in cells with Numb. These data provide the first observations that Notch receptors are variably affected by Numb and will be important for the interpretation of the function of Notch and Numb interactions during the development of many different cells and tissues.

Numb-deficient satellite cells have regeneration and proliferation defects

Significance This study discloses a role for Numb in the activation and proliferation of adult muscle satellite cells and a unique function in the regulation of the muscle mass determinant Myostatin. Using two different genetic approaches to ablate Numb, one that ablated Numb in the myogenic lineage developmentally leading to reduced muscle mass. We determined that, in Numb-deficient muscle, regeneration was impaired, there was reduced stem cell proliferation, and there was an up-regulation of Myostatin. Overexpression of Numb suppressed Myostatin expression, and Myostatin-specific siRNA rescued the proliferation defect. These studies increase our knowledge of the signaling pathways involved in stem cell function and raise the possibility of regulating the Numb/Myostatin balance as a therapeutic approach to enhance muscle regeneration. The adaptor protein Numb has been implicated in the switch between cell proliferation and differentiation made by satellite cells during muscle repair. Using two genetic approaches to ablate Numb, we determined that, in its absence, muscle regeneration in response to injury was impaired. Single myofiber cultures demonstrated a lack of satellite cell proliferation in the absence of Numb, and the proliferation defect was confirmed in satellite cell cultures. Quantitative RT-PCR from Numb-deficient satellite cells demonstrated highly up-regulated expression of p21 and Myostatin, both inhibitors of myoblast proliferation. Transfection with Myostatin-specific siRNA rescued the proliferation defect of Numb-deficient satellite cells. Furthermore, overexpression of Numb in satellite cells inhibited Myostatin expression. These data indicate a unique function for Numb during the initial activation and proliferation of satellite cells in response to muscle injury.

Comparison of Intact Arabidopsis thaliana Leaf Transcript Profiles during Treatment with Inhibitors of Mitochondrial Electron Transport and TCA Cycle

Plant mitochondria signal to the nucleus leading to altered transcription of nuclear genes by a process called mitochondrial retrograde regulation (MRR). MRR is implicated in metabolic homeostasis and responses to stress conditions. Mitochondrial reactive oxygen species (mtROS) are a MRR signaling component, but whether all MRR requires ROS is not established. Inhibition of the cytochrome respiratory pathway by antimycin A (AA) or the TCA cycle by monofluoroacetate (MFA), each of which initiates MRR, can increase ROS production in some plant cells. We found that for AA and MFA applied to leaves of soil-grown Arabidopsis thaliana plants, ROS production increased with AA, but not with MFA, allowing comparison of transcript profiles under different ROS conditions during MRR. Variation in transcript accumulation over time for eight nuclear encoded mitochondrial protein genes suggested operation of both common and distinct signaling pathways between the two treatments. Consequences of mitochondrial perturbations for the whole transcriptome were examined by microarray analyses. Expression of 1316 and 606 genes was altered by AA and MFA, respectively. A subset of genes was similarly affected by both treatments, including genes encoding photosynthesis-related proteins. MFA treatment resulted in more down-regulation. Functional gene category (MapMan) and cluster analyses showed that genes with expression levels affected by perturbation from AA or MFA inhibition were most similarly affected by biotic stresses such as pathogens. Overall, the data provide further evidence for the presence of mtROS-independent MRR signaling, and support the proposed involvement of MRR and mitochondrial function in plant responses to biotic stress.

Characterization of the DNA-binding Properties of the Mohawk Homeobox Transcription Factor

Background: Mkx is a transcriptional repressor that regulates muscle and tendon differentiation. Results: MKX binds to nnACA recognition sites as a homodimer. Mkx regulates transcription through recognition sites in the Mkx and Sox6 loci. Conclusion: MKX has a novel DNA recognition mode and promotes slow muscle fiber type specification through Sox6. Significance: We provide insight into Mkx regulation of musculoskeletal-specific transcription. The homeobox transcription factor Mohawk (Mkx) is a potent transcriptional repressor expressed in the embryonic precursors of skeletal muscle, cartilage, and bone. MKX has recently been shown to be a critical regulator of musculoskeletal tissue differentiation and gene expression; however, the genetic pathways through which MKX functions and its DNA-binding properties are currently unknown. Using a modified bacterial one-hybrid site selection assay, we determined the core DNA-recognition motif of the mouse monomeric Mkx homeodomain to be A-C-A. Using cell-based assays, we have identified a minimal Mkx-responsive element (MRE) located within the Mkx promoter, which is composed of a highly conserved inverted repeat of the core Mkx recognition motif. Using the minimal MRE sequence, we have further identified conserved MREs within the locus of Sox6, a transcription factor that represses slow fiber gene expression during skeletal muscle differentiation. Real-time PCR and immunostaining of in vitro differentiated muscle satellite cells isolated from Mkx-null mice revealed an increase in the expression of Sox6 and down-regulation of slow fiber structural genes. Together, these data identify the unique DNA-recognition properties of MKX and reveal a novel role for Mkx in promoting slow fiber type specification during skeletal muscle differentiation.

Notch signaling represses GATA4-induced expression of genes involved in steroid biosynthesis

Notch2 and Notch3 and genes of the Notch signaling network are dynamically expressed in developing follicles, where they are essential for granulosa cell proliferation and meiotic maturation. Notch receptors, ligands, and downstream effector genes are also expressed in testicular Leydig cells, predicting a potential role in regulating steroidogenesis. In this study, we sought to determine if Notch signaling in small follicles regulates the proliferation response of granulosa cells to FSH and represses the up-regulation steroidogenic gene expression that occurs in response to FSH as the follicle grows. Inhibition of Notch signaling in small preantral follicles led to the up-regulation of the expression of genes in the steroid biosynthetic pathway. Similarly, progesterone secretion by MA-10 Leydig cells was significantly inhibited by constitutively active Notch. Together, these data indicated that Notch signaling inhibits steroidogenesis. GATA4 has been shown to be a positive regulator of steroidogenic genes, including STAR protein, P450 aromatase, and 3B-hydroxysteroid dehydrogenase. We observed that Notch downstream effectors HEY1, HEY2, and HEYL are able to differentially regulate these GATA4-dependent promoters. These data are supported by the presence of HEY/HES binding sites in these promoters. These studies indicate that Notch signaling has a role in the complex regulation of the steroidogenic pathway.

Identification of satellite cells from anole lizard skeletal muscle and demonstration of expanded musculoskeletal potential

The lizards are evolutionarily the closest vertebrates to humans that demonstrate the ability to regenerate entire appendages containing cartilage, muscle, skin, and nervous tissue. We previously isolated PAX7-positive cells from muscle of the green anole lizard, Anolis carolinensis, that can differentiate into multinucleated myotubes and express the muscle structural protein, myosin heavy chain. Studying gene expression in these satellite/progenitor cell populations from A. carolinensis can provide insight into the mechanisms regulating tissue regeneration. We generated a transcriptome from proliferating lizard myoprogenitor cells and compared them to transcriptomes from the mouse and human tissues from the ENCODE project using XGSA, a statistical method for cross-species gene set analysis. These analyses determined that the lizard progenitor cell transcriptome was most similar to mammalian satellite cells. Further examination of specific GO categories of genes demonstrated that among genes with the highest level of expression in lizard satellite cells were an increased number of genetic regulators of chondrogenesis, as compared to mouse satellite cells. In micromass culture, lizard PAX7-positive cells formed Alcian blue and collagen 2a1 positive nodules, without the addition of exogenous morphogens, unlike their mouse counterparts. Subsequent quantitative RT-PCR confirmed up-regulation of expression of chondrogenic regulatory genes in lizard cells, including bmp2, sox9, runx2, and cartilage specific structural genes, aggrecan and collagen 2a1. Taken together, these data suggest that tail regeneration in lizards involves significant alterations in gene regulation with expanded musculoskeletal potency.

Hand Factors in Cardiac Development: CARDIAC DEVELOPMENT

Congenital heart defects account for 1% of infant mortality and 10% of in utero deaths. As the vertebrate embryo develops, multiple tissue types develop in tandem to morphologically pattern the functional heart. Underlying cardiac development is a network of transcription factors known to tightly control these morphological events. Members of the Twist family of basic helix–loop–helix transcription factors, Hand1 and Hand2, are essential to this process. The expression patterns and functional role of Hand factors in neural crest cells, endocardium, myocardium, and epicardium is indicative of their importance during cardiogenesis; however, to date, an extensive understanding of the transcriptional targets of Hand proteins and their overall mechanism of action remain unclear. In this review, we summarize the recent findings that further outline the crucial functions of Hand factors during heart development and in post‐natal heart function. Anat Rec, 302:101–107, 2019.