Satish Srinivas Kitambi

Sox2 and Mitf cross-regulatory interactions consolidate progenitor and melanocyte lineages in the cranial neural crest

The cellular origin and molecular mechanisms regulating pigmentation of head and neck are largely unknown. Melanocyte specification is controlled by the transcriptional activity of Mitf, but no general logic has emerged to explain how Mitf and progenitor transcriptional activities consolidate melanocyte and progenitor cell fates. We show that cranial melanocytes arise from at least two different cellular sources: initially from nerve-associated Schwann cell precursors (SCPs) and later from a cellular source that is independent of nerves. Unlike the midbrain-hindbrain cluster from which melanoblasts arise independently of nerves, a large center of melanocytes in and around cranial nerves IX-X is derived from SCPs, as shown by genetic cell-lineage tracing and analysis of ErbB3-null mutant mice. Conditional gain- and loss-of-function experiments show genetically that cell fates in the neural crest involve both the SRY transcription factor Sox2 and Mitf, which consolidate an SCP progenitor or melanocyte fate by cross-regulatory interactions. A gradual downregulation of Sox2 in progenitors during development permits the differentiation of both neural crest- and SCP-derived progenitors into melanocytes, and an initial small pool of nerve-associated melanoblasts expands in number and disperses under the control of endothelin receptor B (Ednrb) and Wnt5a signaling.

RETRACTED: Vulnerability of Glioblastoma Cells to Catastrophic Vacuolization and Death Induced by a Small Molecule

Glioblastoma multiforme (GBM) is the most aggressive form of brain cancer with marginal life expectancy. Based on the assumption that GBM cells gain functions not necessarily involved in the cancerous process, patient-derived glioblastoma cells (GCs) were screened to identify cellular processes amenable for development of targeted treatments. The quinine-derivative NSC13316 reliably and selectively compromised viability. Synthetic chemical expansion reveals delicate structure-activity relationship and analogs with increased potency, termed Vacquinols. Vacquinols stimulate death by membrane ruffling, cell rounding, massive macropinocytic vacuole accumulation, ATP depletion, and cytoplasmic membrane rupture of GCs. The MAP kinase MKK4, identified by a shRNA screen, represents a critical signaling node. Vacquinol-1 displays excellent in vivo pharmacokinetics and brain exposure, attenuates disease progression, and prolongs survival in a GBM animal model. These results identify a vulnerability to massive vacuolization that can be targeted by small molecules and point to the possible exploitation of this process in the design of anticancer therapies.

Small molecule screen for compounds that affect vascular development in the zebrafish retina.

Blood vessel formation in the vertebrate eye is a precisely regulated process. In the human retina, both an excess and a deficiency of blood vessels may lead to a loss of vision. To gain insight into the molecular basis of vessel formation in the vertebrate retina and to develop pharmacological means of manipulating this process in a living organism, we further characterized the embryonic zebrafish eye vasculature, and performed a small molecule screen for compounds that affect blood vessel morphogenesis. The screening of approximately 2000 compounds revealed four small molecules that at specific concentrations affect retinal vessel morphology but do not produce obvious changes in trunk vessels, or in the neuronal architecture of the retina. Of these, two induce a pronounced widening of vessel diameter without a substantial loss of vessel number, one compound produces a loss of retinal blood vessels accompanied by a mild increase of their diameter, and finally one other generates a severe loss of retinal vessels. This work demonstrates the utility of zebrafish as a screening tool for small molecules that affect eye vasculature and presents several compounds of potential therapeutic importance.

Developmental toxicity of the environmental pollutant 4-nonylphenol in zebrafish

4-Nonylphenol (4-NP), an estrogen mimicking compound is produced by biodegradation of alkylethoxylates. It is well established that 4-NP can affect the development of aquatic animals by disrupting the endocrine signals. Here we show for the first time in zebrafish that 4-NP does not only target the neuroendocrine system but also the notochord and the muscle. The notochord malformation was first evident as distortions at 24hourspostfertilization (hpf) which within 24h appeared as kinks and herniations. The notochord phenotype was accompanied by reduced motility and impaired swimming behavior. Whole-mount in situ hybridization using chordamesoderm markers and electron microscopic analysis showed failure in the notochord differentiation and disruption of the perinotochordal basement membrane. Late larval stages of 4-NP treated embryos displayed abnormal mineralization, vertebral curvature, fusion of vertebral bodies and abnormal extension of haemal arches. The muscle structure and the maximal active force in isolated muscle preparations were similar between 4-NP exposed and of control embryos, suggesting that 4-NP did not induce major changes in striated muscle function. However, repeated electrical stimulation (>40Hz) of the 4-NP exposed larvae revealed an impaired relaxation between stimuli, possibly reflecting an alteration in the relaxant mechanisms (e.g. in cellular Ca(2+) removal) which could explain the abnormal swimming pattern exhibited by 4-NP exposed larvae. Additionally, we demonstrate that the expression levels of the stress hormone, corticotropin releasing hormonewere elevated in the brain following 4-NP treatment. We also observed a significant decrease in the transcript levels of luteinizing hormone b at early larval stages. Collectively, our results show that 4-NP is able to disrupt the notochord morphogenesis, muscle function and the neuroendocrine system. These data suggest that 4-NP enduringly affects the embryonic development in zebrafish and that this compound might exert these deleterious effects through diverse signaling pathways.

Spatiotemporal features of neurogenesis in the retina of medaka, Oryzias latipes.

The vertebrate retina is very well conserved in evolution. Its structure and functional features are very similar in phyla as different as primates and teleost fish. Here, we describe the spatiotemporal characteristics of neurogenesis in the retina of a teleost, medaka, and compare them with other species, primarily the zebrafish. Several intriguing differences are observed between medaka and zebrafish. For example, photoreceptor differentiation in the medaka retina starts independently in two different areas, and at more advanced stages of differentiation, medaka and zebrafish retinae display obviously different patterns of the photoreceptor cell mosaic. Medaka and zebrafish evolutionary lineages are thought to have separated from each other 110 million years ago, and so the differences between these species are not unexpected, and may be exploited to gain insight into the architecture of developmental pathways. Importantly, this work highlights the benefits of using multiple teleost models in parallel to understand a developmental process. Developmental Dynamics 237:3870–3881, 2008.

Small molecule screening platform for assessment of cardiovascular toxicity on adult zebrafish heart

BackgroundCardiovascular toxicity is a major limiting factor in drug development and requires multiple cost-effective models to perform toxicological evaluation. Zebrafish is an excellent model for many developmental, toxicological and regenerative studies. Using approaches like morpholino knockdown and electrocardiogram, researchers have demonstrated physiological and functional similarities between zebrafish heart and human heart. The close resemblance of the genetic cascade governing heart development in zebrafish to that of humans has propelled the zebrafish system as a cost-effective model to conduct various genetic and pharmacological screens on developing embryos and larvae. The current report describes a methodology for rapid isolation of adult zebrafish heart, maintenance ex vivo, and a setup to perform quick small molecule throughput screening, including an in-house implemented analysis script.ResultsAdult zebrafish were anesthetized and after rapid decapitation the hearts were isolated. The short time required for isolation of hearts allows dissection of multiple fishes, thereby obtaining a large sample size. The simple protocol for ex vivo culture allowed maintaining the beating heart for several days. The in-house developed script and spectral analyses allowed the readouts to be presented either in time domain or in frequency domain. Taken together, the current report offers an efficient platform for performing cardiac drug testing and pharmacological screens.ConclusionThe new methodology presents a fast, cost-effective, sensitive and reliable method for performing small molecule screening. The variety of readouts that can be obtained along with the in-house developed analyses script offers a powerful setup for performing cardiac toxicity evaluation by researchers from both academics and industry.

14-Membered Macrocyclic Ring-Derived Toolbox: The Identification of Small Molecule Inhibitors of Angiogenesis and Early Embryo Development in Zebrafish Assay

A highly practical and modular synthesis to obtain a diverse 14-membered ring-based macrocyclic toolbox is achieved. These compounds were further tested in zebrafish assays related to early embryonic development, angiogenesis, and neurogenesis, respectively. 1.4c was identified as an antiangiogenesis agent.

Stem cells: a model for screening, discovery and development of drugs

The identification of normal and cancerous stem cells and the recent advances made in isolation and culture of stem cells have rapidly gained attention in the field of drug discovery and regenerative medicine. The prospect of performing screens aimed at proliferation, directed differentiation, and toxicity and efficacy studies using stem cells offers a reliable platform for the drug discovery process. Advances made in the generation of induced pluripotent stem cells from normal or diseased tissue serves as a platform to perform drug screens aimed at developing cell-based therapies against conditions like Parkinson’s disease and diabetes. This review discusses the application of stem cells and cancer stem cells in drug screening and their role in complementing, reducing, and replacing animal testing. In addition to this, target identification and major advances in the field of personalized medicine using induced pluripotent cells are also discussed.

Cell and small animal models for phenotypic drug discovery

The phenotype-based drug discovery (PDD) approach is re-emerging as an alternative platform for drug discovery. This review provides an overview of the various model systems and technical advances in imaging and image analyses that strengthen the PDD platform. In PDD screens, compounds of therapeutic value are identified based on the phenotypic perturbations produced irrespective of target(s) or mechanism of action. In this article, examples of phenotypic changes that can be detected and quantified with relative ease in a cell-based setup are discussed. In addition, a higher order of PDD screening setup using small animal models is also explored. As PDD screens integrate physiology and multiple signaling mechanisms during the screening process, the identified hits have higher biomedical applicability. Taken together, this review highlights the advantages gained by adopting a PDD approach in drug discovery. Such a PDD platform can complement target-based systems that are currently in practice to accelerate drug discovery.

Erg Channel Is Critical in Controlling Cell Volume during Cell Cycle in Embryonic Stem Cells

Abstract The cell cycle progression in mouse embryonic stem cells (mESCs) is controlled by ion fluxes that alter cell volume [1]. This suggests that ion fluxes might control dynamic changes in morphology over the cell cycle, such as rounding up of the cell at mitosis. However, specific channels regulating such dynamic changes and the possible interactions with actomyosin complex have not been clearly identified. Following RNAseq transcriptome analysis of cell cycle sorted mESCs, we found that expression of the K+ ion channel Erg1 peaked in G1 cell cycle phase, which was confirmed by immunostaining. Inhibition of Erg channel activity caused loss of G1 phase cells via non-apoptotic cell death. Cells first lost the ability of membrane blebbing, a typical feature of cultured embryonic stem cells. Continued Erg inhibition further increased cell volume and the cell eventually ruptured. In addition, atomic force measurements on live cells revealed a decreased cortical stiffness after treatment, suggesting alterations in actomyosin organization. When the intracellular osmotic pressure was experimentally decreased by hypertonic solution or block of K+ ion import via the Na, K-ATPase, cell viability was restored and cells acquired normal volume and blebbing activity. Our results suggest that Erg channels have a critical function in K+ ion homeostasis of mESCs over the cell cycle, and that cell death following Erg inhibition is a consequence of the inability to regulate cell volume.