Summer Undergraduate Research Program 2021

Genetic analysis of soma-germline communication during spermatid differentiation
Sperm are perhaps the most highly specialized and modified cells in the animal world. They are specifically designed to be efficient couriers of the male’s genetic information. Animal sperm vary in shapes and sizes to carry out this task. Nonetheless, the general process of sperm production is largely similar from insects to mammals. Construction of a sperm is accomplished with the assistance of other cells in the testis. While many studies have uncovered genes that are active in sperm during their development, little is known about the genes and functions of the support cells in the testis. In the fruit fly, Drosophila melanogaster, each 64-spermatid cluster develops as an interconnected cyst and is encapsulated by a pair of somatic support cells, called cyst cells. Recent work in the lab has uncovered that communication between these somatic cyst cells and the spermatids is necessary to direct the separation of spermatids within a cyst during the late stages of spermatogenesis. To better understand the roles of the somatic support cells, this project will identify factors required in cyst cells to complete sperm development. For this project, we will specifically impair the functions of particular candidate genes only in the support cells of the testis to examine the impact on sperm formation. Because the process of spermatogenesis is similar across species, this work will not only aid in understanding cellular collaboration in Drosophila spermatogenesis, but it will contribute to our understanding of spermatogenesis in general.

Tumor suppressor genes are essential for cellular function in multicellular organisms, but many Tumor Suppressor Genes (TSGs) and tumor suppressing mechanisms remain unknown or their mechanisms poorly understood, particularly as they function in vivo. In the Duncan lab, we study planarian flatworms, which are best known for their remarkable regenerative abilities. In addition, they share many striking similarities to cancer cells, including genomic patterns that are known to mark TSGs in human cells, e.g. wide peaks of histone H3 K4 trimethylation (H3K4me3). We have analyzed the patterns of H3K4me3 signal across the planarian genome and identified many loci with this pattern. Moreover, we have identified the enzyme, Set1, that is largely responsible for creating this signature in planarian stem cells. Noteably, depletion of set1 in planarians induces stem cell phenotypes that suggest loss of TSG function, including hyperproliferation and an abnormal response to DNA damage. In his KBRIN project this summer, Zach is cloning genes we have identified as Set1 targets, i.e. potential TSGs. He is then creating dsRNA from these cloned constructs, delivering it to planarian animals, and assessing the effects. He is using both live animal imaging and immunofluorescence to screen for genes that cause phenotypes relevant to tumor suppression. This work will help discover new roles for known genes and uncover the functions of unknown planarian genes.

Comparative neuroanatomy of various tactile foraging ducks. With tactile foraging ducks utilizing a bill to locate resources, research into mechanoreception is prevalent. The duck bill skin contains Herbst and Grandry corpuscles, which are multicellular non-neuronal structures, equivalent to Pacinian and Meisner corpuscles in human glabrous skin. As with mammalian corpuscles, in the duck bill sensory neurons interact with these corpuscles detect vibration and pressure. Various species of ducks have different foraging abilities, raising the question of whether this is reflected in the anatomy and/or function of structures that sense touch in the bill. The current study focuses on how densely populated the two types of corpuscles are in duck bills of seven species, to give insight as to whether the corpuscle density and size varies based upon foraging abilities. With results still underway, the goal is to be able to measure the density of corpuscles per/mm^2 in the bill skin and average corpuscle diameter, to correlate this with bone morphology and functional responses from trigeminal neurons from these species (Schneider et al., 2019). In addition, previous studies have recently shown that the lamellar cells of Herbst and Grandry corpuscles in late-stage embryos can be excited by mechanical stimulation (Nikolaev et al., 2020). Therefore, in parallel we are investigating the developmental time-course of expression of the force-gated ion channel piezo2 in these cells using in situ hybridization and qPCR. Understanding how these structures relate to tactile sensitivity can provide insight to how human mechanoreception forms and discriminates touch.

Development of Research Tools to Study the Role of PRL-3 in Cancer. PRL-3 or PTP4A3 is an oncogenic phosphatase upregulated in a multitude of cancers with roles in tumorigenesis and metastasis. Despite the remarkable number of studies deciphering great insights into the physiological roles of PRL-3, structural challenges and high degree of homology between its family members remain challenging to therapeutically target PRL-3. Therefore, development of new tools to bypass these challenges are needed. Towards this end Nanobodies have emerged as an important research tool and has potential to be implemented in therapeutics as well. In the current project a bacterial immunotoxin PE38 will be fused to the nanobody that is highly specific to PRL-3 by the traditional insertional cloning into a bacterial expression construct pSKB3. The construct is then purified as a Immunotoxin fused to nanobody that has the ability to specifically bind to PRL-3 and potentially be toxic to the cancer cells. This strategy if successful can be used as a translational approach to therapeutically target PRL-3.

PAS domain containing protein kinase (Pask) is essential in the detection of and respective response regarding environmental signals from within the stem cell niche. The ability to phosphorylate Wdr5, a strictly nuclear protein and eventual epigenetic activator of transcription, is key to the function of Pask. However, in normal cell conditions, Pask remains in the cytosol of the cell, waiting to perform its function. Here, we examine some of the possible mechanisms by which Pask is able to migrate to the nucleus. Without the migration back into the cytosol, stem cells keep differentiating and this has detrimental effects on homeostasis of the cell, therefore making this mechanism incredibly critical to the function of PasK and by extension, the differentiation of stem cells.

Neural crest cells are responsible for forming multiple tissues in the body. The periocular mesenchyme is a subgroup of neural crest cells that forms the anterior segment of the eye, which includes the lens, cornea, and the iris. In the Famulski lab, zebrafish were used as the model organism to study these periocular mesenchyme cells on the molecular level. The periocular mesenchyme cells were isolated and a single cell transcriptome analysis identified the genes highly expressed by the cells. Si:ch211-251b21.1 and hgd were two of these upregulated genes. Si:ch211-251b21.1 is suspected to be a glutamate receptor of the kainate family, while hgd is involved in pigment synthesis. Knocking out both genes with the CRISPR Cas9 system resulted in a particular phenotype that included maldevelopment of the anterior segment. My project will focus on the role of the potential glutamate receptor si:ch211-251b21.1 for eye development. The subgroup of glutamate receptors in zebrafish embryos will be inhibited with drugs known from previous studies. These drugs include CNQX, UBP 302, Kainate acid, and sym2081. CNQX and UBP 302 are Kainate receptor antagonists, that will block the glutamate receptor function. Kainate acid and sym2081 are Kainate receptor agonist which could produce an enhanced effect of the kainate receptor on the cell. After drug exposure, in situ hybridization and antibody stainings will be performed to examine if the drugs had an impact on the expression and function of si:ch211-251b21.1. The expected result includes a similar phenotype from the CRISPR Cas9 knockout and changes in expression of the si:ch211-251b21.1 gene.

Genetic analysis of soma-germline communication during spermatid differentiation
Sperm are perhaps the most highly specialized and modified cells in the animal world. They are specifically designed to be efficient couriers of the male’s genetic information. Animal sperm vary in shapes and sizes to carry out this task. Nonetheless, the general process of sperm production is largely similar from insects to mammals. Construction of a sperm is accomplished with the assistance of other cells in the testis. While many studies have uncovered genes that are active in sperm during their development, little is known about the genes and functions of the support cells in the testis. In the fruit fly, Drosophila melanogaster, each 64-spermatid cluster develops as an interconnected cyst and is encapsulated by a pair of somatic support cells, called cyst cells. Recent work in the lab has uncovered that communication between these somatic cyst cells and the spermatids is necessary to direct the separation of spermatids within a cyst during the late stages of spermatogenesis. To better understand the roles of the somatic support cells, this project will identify factors required in cyst cells to complete sperm development. For this project, we will specifically impair the functions of particular candidate genes only in the support cells of the testis to examine the impact on sperm formation. Because the process of spermatogenesis is similar across species, this work will not only aid in understanding cellular collaboration in Drosophila spermatogenesis, but it will contribute to our understanding of spermatogenesis in general.

The channels for transmitting electrical activity along neurons is similar from squid to humans with sodium currents accounting for the upstroke of an action potential and potassium channels for the rapid and delayed ionic flux for the repolarization. Pharmacological agents tetraethylammonium chloride (TEA) and 4-aminopyridine (4-AP) block different subsets of voltage gated potassium (K+) channels. The chordotonal organs in crab limbs are a model of proprioceptive sensation and have rapidly- and slowly-adapting sensory neurons. Since 4-AP is used clinically for amyotrophic lateral sclerosis (ALS) and Multiple sclerosis (MS) treatments, a better understanding of its action on proprioceptive models can aid in understanding the potential effects in mammalian systems. To assess the action of these blockers on the function of proprioceptive sensory neurons, the neurons were evoked by movements associated with the joint while applying these compounds individually as well as in combination. Both 4-AP and TEA decreased activity individually, as well as when combined together. Full function was not returned in most preparations after the 4-AP and TEA were washed out individually or from the cocktail exposure. Thus, the potassium channels are sensitive to both blockers in this marine crustacean model. It appears the action is on electrical induction and conduction within the axons. This crab crustacean proprioceptive model can be used for future intensive investigations, building on the results presented, in the pharmacology of the mechanosensitive channels and neuronal activity.