Regulated peristalsis into the acidic region of the _Drosophila_ larval midgut is controlled by a novel component of the Autonomic Nervous System

The underlying cellular and molecular mechanisms that regulate and coordinate critical physiological processes such as peristalsis are complex, often cryptic, and involve the integration of multiple tissues and organ systems within the organism. We have identified a completely novel component of the larval autonomic nervous system in the _Drosophila_ larval midgut that is essential for the peristaltic movement of food from the anterior midgut into the acidic region of the midgut. We have named this region the Superior Cupric Autonomic Nervous System or SCANS. Located at the junction of the anterior and the acidic portions of the midgut, the SCANS is characterized by a cluster of a novel neuro-enteroendocrine cells that we call Lettuce Head Cells, a valve, and two anterior muscular tethers to the dorsal gastric caeca. Using cell ablation and ectopic activation via expression of the _Chlamydomonas reinhardtii_ blue-light activated channelrhodopsin, we demonstrate that the SCANS and in particular the Lettuce Head Cells are both necessary and sufficient for peristalsis and perhaps serve a larger role by coordinating digestion throughout the anterior midgut with development and growth

Developmental characteristics of a novel cell type in the larval midgut of Drosophila melanogaster

In the Drosophila larval midgut, the development pathways associated with the specialized cell types found in the middle midgut region have been well characterized. In this region determination between the cell types is dependent on differential signaling of two signaling molecules Wg and Dpp. This differential signaling from the mesoderm controls the specification of the underlying endoderm in the developing embryonic midgut. The homeotic gene lab is expressed and required for the formation of the copper cells in the middle midgut. In a recent study, a group of cells was discovered at the anterior and middle midgut junction region in 3rd instar larvae. These cells (LHCs) expressed GFP in UASCD8GFP;DJ752Gal4 larvae and also expressed the hormone DH31. In my study, I performed an overexpression enhancer-promoter screen for genes that are involved with the development of the LHCs. I also carried out immunohistochemistry assays in mutant larvae to determine the extent known genes play in the development of the LHCs and neighboring MIP-expressing cells. In mutant larvae for wg, dpp, and lab, the morphology of the MIP-expressing cells was disrupted. The screen yielded 80 lines that produced a positive phenotype in the LHCs. I discuss three lines in further detail (sax, kis, fusl) and evaluate the possibilities of not just LHC development, but overall endocrine cell specification in the Drosophila larval midgut

Reduction of Paraoxonase Expression Followed by Inactivation across Independent Semiaquatic Mammals Suggests Stepwise Path to Pseudogenization.

Convergent adaptation to the same environment by multiple lineages frequently involves rapid evolutionary change at the same genes, implicating these genes as important for environmental adaptation. Such adaptive molecular changes may yield either change or loss of protein function; loss of function can eliminate newly deleterious proteins or reduce energy necessary for protein production. We previously found a striking case of recurrent pseudogenization of the Paraoxonase 1 (Pon1) gene among aquatic mammal lineages-Pon1 became a pseudogene with genetic lesions, such as stop codons and frameshifts, at least four times independently in aquatic and semiaquatic mammals. Here, we assess the landscape and pace of pseudogenization by studying Pon1 sequences, expression levels, and enzymatic activity across four aquatic and semiaquatic mammal lineages: pinnipeds, cetaceans, otters, and beavers. We observe in beavers and pinnipeds an unexpected reduction in expression of Pon3, a paralog with similar expression patterns but different substrate preferences. Ultimately, in all lineages with aquatic/semiaquatic members, we find that preceding any coding-level pseudogenization events in Pon1, there is a drastic decrease in expression, followed by relaxed selection, thus allowing accumulation of disrupting mutations. The recurrent loss of Pon1 function in aquatic/semiaquatic lineages is consistent with a benefit to Pon1 functional loss in aquatic environments. Accordingly, we examine diving and dietary traits across pinniped species as potential driving forces of Pon1 functional loss. We find that loss is best associated with diving activity and likely results from changes in selective pressures associated with hypoxia and hypoxia-induced inflammation

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

The Evolution of the Krüppel-Like Factor Gene Family and Their Function During Embryonic Development in the Ctenophore Mnemiopsis leidyi

How modifications during development influence large-scale evolutionary changes is a major emphasis in the field of evolutionary developmental biology (evo-devo). Comparing the underlying cellular and molecular basis of development in diverse organisms can provide insight into how alterations of these processes contribute to morphological diversity. Ctenophores, or comb jellies, are a phylum of marine invertebrates and, due to their unique phylogenetic position as one of the earliest branching lineages of extant animals, are an ideal system for investigating the origin and evolution of metazoan character traits. For example, the animal through-gut is thought to have originated in the metazoan stem lineage prior to diversification of Bilateria. However, as part of my dissertation, I showed that ctenophores possess a functional through-gut. These results suggest that the origin of the through-gut may have been much earlier during metazoan diversification than previously thought. Additionally, my studies of gene function in ctenophores provide insight into the molecular mechanisms that, in some part, may be driving early metazoan diversification. Members of the Krüppel-like factor (Klf) gene family are known to play a role in many aspects of development, including maintenance of stem cell renewal and pluripotency, and regulating the balance between cellular proliferation and differentiation. Functional studies of these transcription factors have been restricted to only a handful of bilaterian animals, with little investigation into the evolutionary history of this gene family. To evaluate how the Klf gene family could influence evolutionary changes driving metazoan diversification, I examined the origins and evolutionary history of the Klf gene family, and characterized their function in the ctenophore, Mnemiopsis leidyi. Ultimately, I showed that Klf genes were present in the stem lineage leading to Metazoa, and they regulate cellular proliferation in putative stem cell niches during development in M. leidyi. My findings suggest that regulation of stem cell proliferation was an ancestral function of Klfs in metazoans.</p

Krüppel-like factor gene function in the ctenophore Mnemiopsis suggests an ancient role in promoting cell proliferation in metazoan stem cell niches

Abstract All animals possess pluripotent stem cells that during an organism’s lifespan give rise to differentiated cell types and are capable of self-renewal. To date studies assessing the function of explicit genes driving the regulation of stem cell renewal and pluripotency have been characterized in bilaterians and a small number of cnidarians. Transcription factor genes critical for regulation of stem cell self-renewal and pluripotency include O ct4 , S ox2 , K lf4 , and c- m yc (OSKM) (1–3). In particular, the role of Klfs in stem cell renewal and pluripotency has been thoroughly investigated (1, 2, 4–9). In mouse embryonic stem cells the three Krüppel-like factor ( Klf) genes Klf2 , Klf4 , and Klf5 participate in an auto-regulatory activation loop and maintain a transcriptional circuit with Oct4 , Sox2 , c-myc , and Nanog regulating stem cell self-renewal (4). While OSKM gene homologs have been found in the genomes of many metazoans (3, 10, 11), their function is unknown in non-bilaterians. Ctenophora, an ancient non-bilaterian lineage (12–14), possess putative stem cells (11, 15, 16) and have prodigious regenerative capacity (17, 18), making them informative models for investigating the evolution and genetic underpinnings of stem cell maintenance in Metazoa. Ctenophores possess homologs of the OSKM genes (11, 19–21), and while the expression patterns of ctenophore Sox paralogs are associated with high rates of cell proliferation in putative stem cell niches (11, 19), little is known about Pou , Klf , and c-myc expression or function. Here we examine the expression of Klf homologs in the ctenophore Mnemiopsis leidyi and disrupt zygotic Klf gene function during embryogenesis using both morpholino oligonucleotides and CRISPR-Cas9. We find that zygotic MleKlf5a and MleKlf5b play a role in patterning the gastrovascular cavity, pharynx, apical organ, and tentacle bulbs in M. leidyi . Simultaneous zygotic knockdown of both MleKlf5a and MleKlf5b results in the diminution or loss of cell proliferation in endodermal stem cell niches and aberrant endodermal patterning. Our results highlight an ancestral functional role for Klf transcription factors in the regulation of rapid cell proliferation in metazoan stem cell niches. Significance All animals possess pluripotent stem cells that are capable of self-renewal and give rise to differentiated cell types. However studies assessing the function of genes regulating stem cells have been characterized only in bilaterians and cnidarians. Mnemiopsis leidyi , a member of the early diverging non-bilaterian phylum Ctenophora, has emerged as an important model system for understanding early animal evolution. M. leidyi possess putative stem cell niches in which the expression of ‘stemness’ genes have been described. Here we characterize the zygotic function of Krüppel-like factor ( Klf) genes during M. leidyi embryogenesis. Our results highlight an ancestral role for Klf genes in regulating cell proliferation in metazoan endodermal stem cell niches, suggesting very deep conservation of the core transcriptional circuitry critical for stem cell maintenance among extant metazoans

Krüppel-like factor gene function in the ctenophore Mnemiopsis leidyi assessed by CRISPR/Cas9-mediated genome editing

ABSTRACT The Krüppel-like factor (Klf) gene family encodes transcription factors that play an important role in the regulation of stem cell proliferation, cell differentiation and development in bilaterians. Although Klf genes have been shown to specify functionally various cell types in non-bilaterian animals, their role in early-diverging animal lineages has not been assessed. Thus, the ancestral activity of these transcription factors in animal development is not well understood. The ctenophore Mnemiopsis leidyi has emerged as an important non-bilaterian model system for understanding early animal evolution. Here, we characterize the expression and functional role of Klf genes during M. leidyi embryogenesis. Zygotic Klf gene function was assessed with both CRISPR/Cas9-mediated genome editing and splice-blocking morpholino oligonucleotide knockdown approaches. Abrogation of zygotic Klf expression during M. leidyi embryogenesis resulted in abnormal development of several organs, including the pharynx, tentacle bulbs and apical organ. Our data suggest an ancient role for Klf genes in regulating endodermal patterning, possibly through regulation of cell proliferation

Hypoxia Inducible Factor (HIF) transcription factor family expansion, diversification, divergence and selection in eukaryotes

<div><p>Hypoxia inducible factor (HIF) transcription factors are crucial for regulating a variety of cellular activities in response to oxygen stress (hypoxia). In this study, we determine the evolutionary history of HIF genes and their associated transactivation domains, as well as perform selection and functional divergence analyses across their four characteristic domains. Here we show that the HIF genes are restricted to metazoans: At least one HIF-α homolog is found within the genomes of non-bilaterians and bilaterian invertebrates, while most vertebrate genomes contain between two and six HIF-α genes. We also find widespread purifying selection across all four characteristic domain types, bHLH, PAS, NTAD, CTAD, in HIF-α genes, and evidence for Type I functional divergence between HIF-1α, HIF-2α /EPAS, and invertebrate HIF genes. Overall, we describe the evolutionary histories of the HIF transcription factor gene family and its associated transactivation domains in eukaryotes. We show that the NTAD and CTAD domains appear <i>de novo</i>, without any appearance outside of the HIF-α subunits. Although they both appear in invertebrates as well as vertebrate HIF- α sequences, there seems to have been a substantial loss across invertebrates or were convergently acquired in these few lineages. We reaffirm that HIF-1α is phylogenetically conserved among most metazoans, whereas HIF-2α appeared later. Overall, our findings can be attributed to the substantial integration of this transcription factor family into the critical tasks associated with maintenance of oxygen homeostasis and vascularization, particularly in the vertebrate lineage.</p></div

KLF/SP Transcription Factor Family Evolution: Expansion, Diversification, and Innovation in Eukaryotes

The Krüppel-like factor and specificity protein (KLF/SP) genes play key roles in critical biological processes including stem cell maintenance, cell proliferation, embryonic development, tissue differentiation, and metabolism and their dysregulation has been implicated in a number of human diseases and cancers. Although many KLF/SP genes have been characterized in a handful of bilaterian lineages, little is known about the KLF/SP gene family in nonbilaterians and virtually nothing is known outside the metazoans. Here, we analyze and discuss the origins and evolutionary history of the KLF/SP transcription factor family and associated transactivation/repression domains. We have identified and characterized the complete KLF/SP gene complement from the genomes of 48 species spanning the Eukarya. We have also examined the phylogenetic distribution of transactivation/repression domains associated with this gene family. We report that the origin of the KLF/SP gene family predates the divergence of the Metazoa. Furthermore, the expansion of the KLF/SP gene family is paralleled by diversification of transactivation domains via both acquisitions of pre-existing ancient domains as well as by the appearance of novel domains exclusive to this gene family and is strongly associated with the expansion of cell type complexity