Labib Rouhana

T/A cloning is a popular method for generating recombinant DNA plasmids. This method relies on single A:T nucleotide base pairs between PCR product ends and vector. Theoretically, the directionality of insert ligation with relation to the vector is random. However, we have continuously observed directionality bias using the pGEM-T Vector System for T/A cloning in a Course-based Undergraduate Research Experience (CURE). Cloning of over 400 inserts has shown directional bias higher than 74% ( p- value < 0.0005) “sense” to the T7 promoter of the vector. Awareness of biased insertion in our applications reduces time and cost in cloning and downstream analyses.

As the sister phylum to Bilateria, Cnidaria, holds a phylogenetic position that is crucial in evolutionary studies of animal development. However, the analysis of cnidarian species, as well as other emerging models, is limited by the scarcity of tools that are readily available and applied with ease. Commercial fluorescent dyes are an accessible option to increase visibility when transgenic approaches are out of reach. In this study, the commercial dye Acridine Orange (AO) was found to be suitable for long-term staining and live imaging of the cnidarian model Nematostella vectensis. Temporary incubation in media containing AO resulted in broad cellular labeling. Individuals stained with AO as zygotes retained the dye throughout embryogenesis, metamorphosis, and for longer than three months of growth, at which point the signal became restricted to subsets of cells. In contrast, staining of zebrafish zygotes with AO resulted in signal decay within the first couple of days of development and preferential accumulation in the yolk sac. AO is retained in N. vectensis adults after amputation and inherited by new tissue during regeneration. Surprisingly, AO was not found suitable for cell tracking via microinjection, as the dye dissipated within minutes of being injected into the cytoplasm of zygotes and blastomeres. Surprisingly, the subcellular distribution of AO signal did not match the pattern expected from it use in mammalian cell lines, where green fluorescence is observed when associated with cellular DNA and red fluorescence when bound to RNA. Instead, long-lived green fluorescence was observed throughout the cell surface and/or in the cytoplasm of N. vectensis stained with AO, whereas red emission was similarly distributed but short lived. Expanding the use of AO as a visualization tool will facilitate future studies of cnidarians and other underexplored animal models.

N⁶-methyladenosine (m⁶A) is one of the most prevalent post-transcriptional modifications of eukaryotic RNA molecules. This post-transcriptional modification is essential in biological contexts ranging from metabolism to cellular differentiation and neuronal function. While the role of m⁶A RNA regulation in the soma of planarian flatworms has been previously studied, the presence and biological relevance of this regulatory pathway in the germline of these or other lophotrochozoans remains unknown. Here, we characterize m⁶A RNA regulation factors in the planarian Schmidtea mediterranea and analyze their function in development of the germline. Enriched expression of orthologs of m⁶A methyltransferase complex components, namely Smed-METTL3, Smed-METTL14, and WTAP, was detected in ovaries and testes of S. mediterranea. Perturbation of these factors by RNA-interference (RNAi) disrupted intermediate steps in oogenesis and spermatogenesis, but the presence of germline stem cells was not affected. Expression of m⁶A “reader” homologs was also detected in the planarian gonads. Sperm development defects were observed upon Smed-YTHDF2-3 RNAi, and combined knockdown of Smed-YTHDF2-3, Smed-YTHDF2-2, or Smed-YTHDF1-2 with that of writers led to increased frequency of germline development defects. These results indicate that regulation of RNA bym⁶A methylation supports male and female germline development in planarian flatworms.

The Transient Receptor Potential superfamily of proteins (TRPs) form cation channels that are abundant in animal sensory systems. Amongst TRPs, the Melastatin-related family (TRPMs) is composed of members that respond to temperature, pH, sex hormones, and various other stimuli. Some TRPMs exhibit enriched expression in the gonads of vertebrate and invertebrate species, but their contributions to germline development remain to be determined. We identified twenty-one potential TRPMs in the planarian flatworm Schmidtea mediterranea and analyzed their anatomical distribution of expression by whole-mount in situ hybridization. Enriched expression of two TRPMs (Smed-TRPM-c and Smed-TRPM-l) was detected in testis, whereas eight TRPM genes had detectable expression in patterns representative of neuronal and/or sensory cell types. Functional analysis of TRPM homologs by RNA-interference (RNAi) revealed that disruption of normal levels of Smed-TRPM-c expression impaired sperm development, indicating a role for this receptor in supporting spermatogenesis. Smed-TRPM-l RNAi alone did not result in a detectable phenotype, but it did increase sperm development deficiencies when combined with Smed-TRPM-c RNAi. Fluorescence in situ hybridization revealed expression of Smed-TRPM-c in early spermatogenic cells within testes, suggesting cell-autonomous regulatory functions in germ cells for this gene. In addition, Smed-TRPM-c RNAi resulted in reduced numbers of presumptive germline stem cell clusters in asexual planarians, suggesting that Smed-TRPM-c supports the establishment, maintenance, and/or expansion of spermatogonial germline stem cells. While further research is needed to identify the factors that trigger Smed-TRPM-c activity, these findings reveal one of the few known examples for TRPM function in the direct regulation of sperm development.

The Transient Receptor Potential superfamily of proteins (TRPs) form cation channels that are abundant in animal sensory systems. Amongst TRPs, the Melastatin-related subfamily (TRPMs) is composed of members that respond to temperature, pH, sex hormones, and various other stimuli. Some TRPMs exhibit enriched expression in gonads of vertebrate and invertebrate species, but their contributions to germline development remain to be determined. We identified twenty-one potential TRPMs in the planarian flatworm Schmidtea mediterranea and analyzed their anatomical distribution of expression by whole-mount in situ hybridization. Enriched expression of two TRPMs ( Smed-TRPM-c and Smed-TRPM-l ) was detected in testis, whereas eight TRPM genes had detectable expression in patterns representative of neuronal and/or sensory cell types. Functional analysis of TRPM homologs by RNA-interference (RNAi) revealed that disruption of Smed-TRPM-c expression results in reduced sperm development, indicating a role for this receptor in supporting spermatogenesis. Smed-TRPM-l RNAi did not result in a detectable phenotype, but it increased sperm development deficiencies when combined with Smed-TRPM-c RNAi. Fluorescence in situ hybridization revealed expression of Smed-TRPM-c in early spermatogenic cells within testes, suggesting cell-autonomous regulatory functions in germ cells for this gene. In addition, Smed-TRPM-c RNAi resulted in reduced numbers of presumptive germline stem cell clusters in asexual planarians, suggesting that Smed-TRPM-c supports establishment, maintenance, and/or expansion of spermatogonial germline stem cells. While further research is needed to identify the factors that trigger Smed-TRPM-c activity, these findings reveal one of few known examples for TRPM function in direct regulation of sperm development.

Differential regulation of gene expression has produced the astonishing diversity of life on Earth. Understanding the origin and evolution of mechanistic innovations for control of gene expression is therefore integral to evolutionary and developmental biology. Cytoplasmic polyadenylation is the biochemical extension of polyadenosine at the 3′-end of cytoplasmic mRNAs. This process regulates the translation of specific maternal transcripts and is mediated by the Cytoplasmic Polyadenylation Element-Binding Protein family (CPEBs). Genes that code for CPEBs are amongst a very few that are present in animals but missing in nonanimal lineages. Whether cytoplasmic polyadenylation is present in non-bilaterian animals (i.e., sponges, ctenophores, placozoans, and cnidarians) remains unknown. We have conducted phylogenetic analyses of CPEBs, and our results show that CPEB1 and CPEB2 subfamilies originated in the animal stem lineage. Our assessment of expression in the sea anemone, Nematostella vectensis (Cnidaria), and the comb jelly, Mnemiopsis leidyi (Ctenophora), demonstrates that maternal expression of CPEB1 and the catalytic subunit of the cytoplasmic polyadenylation machinery (GLD2) is an ancient feature that is conserved across animals. Furthermore, our measurements of poly(A)-tail elongation reveal that key targets of cytoplasmic polyadenylation are shared between vertebrates, cnidarians, and ctenophores, indicating that this mechanism orchestrates a regulatory network that is conserved throughout animal evolution. We postulate that cytoplasmic polyadenylation through CPEBs was a fundamental innovation that contributed to animal evolution from unicellular life.

Differential regulation of gene expression has produced the astonishing diversity of life on Earth. Understanding the origin and evolution of mechanistic innovations for control of gene expression is therefore integral to evolutionary and developmental biology. Cytoplasmic polyadenylation is the biochemical extension of polyadenosine at the 3’-end of cytoplasmic mRNAs. This process regulates the translation of specific maternal transcripts and is mediated by the Cytoplasmic Polyadenylation Element Binding Protein family (CPEBs). Genes that code for CPEBs are amongst a very few that are present in animals but missing in non-animal lineages. Whether cytoplasmic polyadenylation is present in non bilaterian animals ( i.e. , sponges, ctenophores, placozoans, cnidarians) remains unknown. We have conducted phylogenetic analyses of CPEBs and our results show that CPEB1 and CPEB2 subfamilies originated in the animal stem lineage. Our assessment of expression in the sea anemone, Nematostella vectensis (Cnidaria), and the comb jelly, Mnemiopsis leidyi (Ctenophora), demonstrates that maternal expression of CPEB1 and the catalytic subunit of the cytoplasmic polyadenylation machinery (GLD2) is an ancient feature that is conserved across animals. Furthermore, our measurements of poly(A)-tail elongation reveal that key targets of cytoplasmic polyadenylation are shared between vertebrates, cnidarians, and ctenophores, indicating that this mechanism orchestrates a regulatory network that is conserved throughout animal evolution. We postulate that cytoplasmic polyadenylation through CPEBs was a fundamental innovation that contributed to animal evolution from unicellular life.

Detection of chemical stimuli is crucial for living systems and also contributes to quality of life in humans. Since loss of olfaction becomes more prevalent with aging, longer life expectancies have fueled interest in understanding the molecular mechanisms behind the development and maintenance of chemical sensing. Planarian flatworms possess an unsurpassed ability for stem cell-driven regeneration that allows them to restore any damaged or removed part of their bodies. This includes anteriorly-positioned lateral flaps known as auricles, which have long been thought to play a central role in chemotaxis. The contribution of auricles to the detection of positive chemical stimuli was tested in this study usingGirardia dorotocephala, a North American planarian species known for its morphologically prominent auricles. Behavioral experiments staged under laboratory conditions revealed that removal of auricles by amputation leads to a significant decrease in the ability of planarians to find food. However, full chemotactic capacity is observed as early as 2 days post-amputation, which is days prior from restoration of auricle morphology, but correlative with accumulation of ciliated cells in the position of auricle regeneration. Planarians subjected to x-ray irradiation prior to auricle amputation were unable to restore auricle morphology, but were still able to restore chemotactic capacity. These results indicate that although regeneration of auricle morphology requires stem cells, some restoration of chemotactic ability can still be achieved in the absence of normal auricle morphology, corroborating with the initial observation that chemotactic success is reestablished 2-days post-amputation in our assays. Transcriptome profiles of excised auricles were obtained to facilitate molecular characterization of these structures, as well as the identification of genes that contribute to chemotaxis and auricle development. A significant overlap was found between genes with preferential expression in auricles ofG. dorotocephalaand genes with reduced expression uponSoxB1knockdown inSchmidtea mediterranea, suggesting thatSoxB1has a conserved role in regulating auricle development and function. Models that distinguish between possible contributions to chemotactic behavior obtained from cellular composition, as compared to anatomical morphology of the auricles, are discussed.