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Generating RFX6 Mutants via CRISPR/Cas9 to Model Mitchell-Riley Syndrome in Xenopus tropicalis

Swathi Balaji¹ and Marko Horb²

¹The University of Chicago, Chicago, IL, USA, ²National Xenopus Resource and Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA, USA

RFX6 is a transcription factor crucial for anterior endoderm and pancreas development with an identical DNA binding domain between humans and Xenopus. The RFX6 gene is associated with Mitchell-Riley Syndrome, characterized by neonatal diabetes, intestinal atresia, pancreatic hypoplasia, and gallbladder agenesis. In a previous study, morpholinos have been used to block RFX6 translation in Xenopus laevis, causing down-regulation of foregut and pancreas markers, including foxa2 and insulin. Here, we investigate whether a gene knockout via CRISPR/Cas9 will result in similar phenotypic effects in Xenopus tropicalis and hypothesize that genomic changes will produce similar phenotypes.

Four single guide RNAs (sgRNAs) were designed to target the RFX6 DNA-binding domain, assessed in silico, synthesized, and microinjected with Cas9 enzyme and Texas Red into Xenopus tropicalis embryos. Sequencing the genomic DNA of injected embryos has shown that these four sgRNAs were not effective at editing the RFX6 gene. More sgRNAs will be designed and tested in vivo. In situ hybridizations have been performed to assess insulin expression patterns in Stage 41 Control embryos, and anti-sense DIG-labeled RNA probes are being synthesized for the foxa2 and insulin markers for future in situ hybridizations to compare phenotypes in control and RFX6 knockout embryos.

Once effective sgRNAs are identified, we will inject them into embryos, assess phenotypic effects in founders, raise F0 animals to adulthood, and then cross them to obtain homozygous RFX6 null mutants, which will serve as models to further understand disease progression and pancreas development.

Jeff Metcalf Summer Undergraduate Research Fellowship – The University of Chicago

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Studying the Role of RNA Editing in Regenerating Octopus Bimaculoides

N.B. Coffey^1,2, J.F. Diaz Quiroz², J.J.C. Rosenthal²

¹The University of Chicago, ²Bell Center, Marine Biological Laboratory

RNA editing is a process in which genetic information can be altered at the RNA level. Editing occurs when the enzyme Adenosine Deaminase Acting on RNA changes Adenosine to Inosine. This process is interesting because in coding regions of mRNA, Inosine is read as Guanosine during translation; thus, editing changes the amino acid sequence and possibly protein function. While RNA editing rarely occurs in coding regions in vertebrates, recoding events occur several orders of magnitude higher in cephalopods. In addition to their incredible RNA editing capabilities, cephalopods also have the capacity to regenerate fully functional arms after amputation. Given that cephalopods can both recode proteins through RNA editing and regenerate, our study’s goal was to illuminate whether RNA editing may be involved in arm regeneration in Octopus Bimaculoides. To do so, we looked for differences in editing patterns (editing sites and percentage of editing) in genes involved in regulating stemness during arm regeneration. 12 juveniles were amputated on their right first arm on day 0 and tissue was collected for RNA extraction to have the basal levels of editing in the genes of interest. To detect changes in editing during regeneration, the regenerating tip of three animals was collected for RNA extraction 1, 3, 11 and 25 day(s) after amputation, as well as the left first arm as a control. In preliminary results, we observed a trend for the transcription factor Sox2 where control arms displayed higher levels of editing than regenerating arms on day one. Interestingly, this trend was reversed at day three, where regenerating arms had higher levels of editing than controls. Though the study of other genes, more animals, and other time points is required to draw conclusion, the results strongly suggest that RNA editing could play an active role in arm regeneration in Octopus Bimaculoides.

Jeff Metcalf Summer Undergraduate Research Fellowship – The University of Chicago

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Assessing calcification of Astrangia under different temperature and physiological conditions using Scanning Electron Microscopy

Zoe Dellaert^1,2, Dr. Loretta Roberson²

¹University of Chicago, Chicago, IL, ²Marine Biological Laboratory, Woods Hole, MA

Symbiotic and aposymbiotic colonies of Astrangia poculata were reared for four weeks in seawater at 10ºC, 20ºC, or 27ºC. Buoyant weight measurements were used to investigate the effects of temperature and symbiont state on growth rate. Scanning electron microscopy (SEM) was used to describe how these physiological and environmental conditions are reflected in the crystal structure of the coral skeletons. As reflected in both SEM images and buoyant weight data, aposymbiotic colonies in all temperatures and all colonies kept in 10ºC showed higher occurrences of bioerosion than other colonies. The buoyant weight data also revealed that symbiont state and temperature both significantly affect growth rates. SEM of corals kept at 27ºC revealed that symbiont absence appears to result in a lower density of calcium carbonate at growing septa tips. Furthermore, the crystal structure of growing septa tips appears to be different in colonies kept at different temperatures. This is the first study examining the skeleton of Astrangia using SEM, so these results unlock new insights into the skeletons of temperate corals. In today’s changing climate, tropical corals face higher frequencies of warmer ocean temperatures, ocean acidification, and coral bleaching events. This study has the possibility to inform further studies regarding the effects of symbiont state and temperature on the calcification of these integral at-risk corals. This study can also provide insight into historical coral health and symbiosis based on the crystal structures of coral skeletons from the past.

Jeff Metcalf Summer Undergraduate Research Fellowship – The University of Chicago

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Origins of deep sea fatty acids assessed using compound specific stable isotopes

Marianna Karagiannis^1,2, JC Weber², and Maureen Conte²

¹University of Chicago, ²The Ecosystems Center, Marine Biological Laboratory

The oceans’ biological pump is the transfer of material and energy from the surface to the deep ocean via biotic interactions within ecosystems. Critical in both atmospheric and oceanic processes, it is responsible for controlling atmospheric carbon dioxide uptake, the cycling of elements and nutrients, and providing a food source for deep sea organisms. Lipid biomarkers are powerful tools to identify the sources and transformations of carbon in such ecosystems, but questions remain regarding transport and synthesis. We analyzed carbon isotopic signatures in lipids collected from the Sargasso Sea to determine whether polyunsaturated fatty acids (PUFAs) found in the deep ocean are produced by phytoplankton and transported down, or whether there is a significant deep ocean source. We found a rapid increase in δ13C from the production zone to 500 m, and a general trend of increasing δ13C with depth. This can potentially be explained by isotopic fractionation during degradation, but does not appear to indicate a bacterial source of PUFAs in the deep ocean. While much work remains to be done to determine if these trends continue, and to more precisely identify sources, these results provide a significant basis of understanding the role of deep sea communities in the global carbon cycle.

Jeff Metcalf Summer Undergraduate Research Fellowship – The University of Chicago

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Quantifying camouflage: how well do cephalopods resemble the color of the background in the eyes of different predators?

Jaida Kenana^1,2, Derya Akkaynak², Roger Hanlon³

¹University of Chicago, ²Princeton University, ³Marine Biological Laboratory

An animal’s effective ability to use camouflage and remain undetected by predators is due largely to coloration. Cephalopods such as Octopus vulgaris seem to have effective camouflage against a large range of predators with various combinations of cone receptors. However, this has rarely been studied quantitatively. We aim to quantify animal coloration and compare it to substrates within its immediate environment under the perspectives of various predators with differing cone receptors. In doing so, we answer the questions: “What does a camouflage prey look like solely in the color space of predator vision” and “What quantifiable differences in color are present in cephalopods and their immediate surrounds?” These questions are explored by analyzing RGB images of Octopus vulgaris camouflage on different coral reef habitats such as Puerto Rico, Little Cayman, and Bonaire in the Caribbean Sea. A caveat to this, however, is that captured images introduce non-uniform variables to the image quality and its perception. To control for the discrepancies in captured images, ImageJ and the Image Calibration and Analysis Toolbox is used to extract and quantify the coloration data from these images. We determined that camouflaged octopuses resemble their environment in such a way that allows them to remain undetected, in terms of color, in the eyes of monochromatic and dichromatic predators. However, due to insufficient data, it is still relatively inconclusive as to how camouflaged prey look like solely in the color space of predator vision.

Jeff Metcalf Summer Undergraduate Research Fellowship – The University of Chicago

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Hydrogel enables injection of squid embryos for CRISPR-Cas9 genome editing and other genetic tools in cephalopods

Atreyo Pal¹, Eric Edsinger²

¹Biological Sciences Division, University of Chicago, ²Marine Biological Laboratory

Cephalopods, are unique invertebrates due to their large brains – with more than 500 million neurons. Studying their neural circuits has been a prime target for many neurobiologists. Despite extensive documentation of cephalopod neural processes, there has yet to be a targeted attempt to build genome editing tools to study circuits in their brains. This study focuses on developing the tools required for a thorough interrogation of neural circuits in the squid species Doriteuthis pealeii via CRISPR-Cas9 knockout of Pax-6 and Rhodopsin genes which govern the visual pathway. For this we have developed two different methods of in vitro injection into squid embryos – one which is a variant on the Giraldez method and the other which is a novel technique of embedding embryos in hydrogel, and is an exciting possibility to be applied to pygmy squid Idiosepius notoides, where traditional injection methods have proven unsuccessful. For the first injection protocol, we built hydrogel holders (20% w/v) with an aperture such that embryos just fit inside and injected (a) Di-I and dextrin dyes to fluorescently label the embryos to establish a proof of concept for the injection and (b) CRISPR-Cas9 complex for Pax6/Rhodopsin knockout. All injected cells were cultured in agarose and seawater as they developed to the hatchling stage. The cells labelled with Di-I showed the expected fluorescence and developed normally, while the CRISPR injected embryos were phenotyped and genotyped eventually after full development. For the alternate novel hydrogel embedding injection method, we overcame a major limitation of traditional injections because we no longer have to snip and damage the chorion, which makes for an easier and scalable injection technique. Breaking the barrier of genome editing in cephalopods will open up exciting possibilities about investigating the response of neural circuits to varied novel responses, like artificially inducing colored skin pigmentation via optogenetics on the visual pathway.

Jeff Metcalf Summer Undergraduate Research Fellowship – The University of Chicago

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Effects of Actin Polymerization Inhibition on Vesicular Exocytosis and Recovery

William A. Ramos^{1, 2}, Leonard K. Kaczmarek^{2, 3}, Kambiz N. Alavian^{2, 3, 4}, Elizabeth A. Jonas^{2, 3}

¹The College of the University of Chicago; ²Marine Biological Laboratory ³Yale University School of Medicine; ⁴Imperial College London, Department of Medicine

Evidence suggests that actin polymerization may be necessary for re-supply of synaptic vesicles after strong stimulation of the presynaptic terminal that depletes these vesicles. Re-supply of vesicles may occur through re-endocytosis of neurotransmitters or the movement of vesicles from farther up the neuron to the synapse. Latrunculin B (LatB), a potent actin polymerization inhibitor that prevents polymerization of f-actin and sequesters g-actin, had previously been studied to play a role in hippocampal synapses, but had yet to be studied in Squid stellate ganglion synapse. Electrophysiological experiments were used to determine the effects of latrunculin B on release of neurotransmitter and resupply of synaptic vesicles as assayed by the slope of excitatory post synaptic potentials (EPSPs). The depolarization slope of the EPSPs was determined to be decreased in LatB-exposed synapses and recovery of the EPSP was delayed after strong stimulation (tetanus) Rundown of the EPSPs during a tetanus was also determined to occur earlier in synapses treated with LatB. Our data suggest that there could be a relationship between actin dynamics and synaptic vesicle re-supply during and after a prolonged tetanus in Loligo pealeii (longfin inshore squid) presynapse. The mechanism by which this occurs has yet to be elucidated.��

The University of Chicago, McCarter Family Scholarship

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Vitamin C transporter gene regulation in early developmental stages of Porites astreoides

Phoebe Seltzer¹, Eldad Gutner-Hoch², Loretta Roberson²

¹The University of Chicago, Chicago, IL 60637, ²Marine Biological Laboratory, Woods Hole, MA 02543

The mechanisms by which corals form their extracellular skeletal matrix remains poorly understood, despite their importance for both the coral’s structure and the transport of necessary molecules. Potentially due to its role as a cofactor in collagen formation, a Vitamin C Transporter gene (SLC23A) had previously been found to be highly upregulated in recently settled larvae—the stage at which the coral’s inorganic matrix begins to form. We sought to analyze the regulation of SLC23A in the prevalent and hardy Caribbean coral Porites astreoides during the swimming larval stage and the settled larval stage, with no skeletal formation occurring during the former and the skeletal formation commencing in the latter. Considering increasing global oceanic temperatures, we utilized Porites astreoides to further uncover how the gene regulation changes throughout the larval life stages of the coral and determine possible effects of elevated thermal stress on the regulation of SLC23A. Using reverse-transcriptase quantitative PCR, a significant upregulation was found between 1, 3, and 10 days post-settlement as well as between 0 and 10 post-spawning. A significant decrease in expression was also found for day 10 settled larvae under heat-stressed conditions. This suggests that Vitamin C is an essential molecule utilized in the formation of the coral’s skeletal matrix in the early stages after settlement, and increasing temperatures may hinder its transport and diminish skeletal growth.

Jeff Metcalf Summer Undergraduate Research Fellowship – The University of Chicago

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Nitrogen load regime change: changes in atmospheric deposition drive trajectory of nitrogen loads in Buzzards Bay estuaries

Claire Valva¹, Ivan Valiela ², Javier Lloret²

¹The University of Chicago, Chicago IL, ²91�鶹����, Woods Hole, MA

Land-derived nitrogen (N) supply determines primary production, water quality, and eutrophication to estuarine systems. To assess the contributions of atmospheric deposition, fertilizer, and waste water nitrogen loads to estuaries, and to determine the fate and effects of nitrogen as it moves through estuarine systems, we examined the changing nitrogen loads and water quality of 23 of the estuaries in Buzzards Bay, MA. Nitrate, chlorophyll, and salinity concentrations were measured from 1992 to 2016 in each estuary and N loads were determined from 1985 to 2015. In Buzzards Bay estuaries, 83% of nitrogen loads increased until 2000 and 78% of nitrogen loads decreased after 2000. This regime shift was primarily caused by decreased atmospheric deposition of nitrogen to Cape Cod from 1998 forward. The responses to this shift included decreased nitrate concentrations from 2002 - 2016 and a slight increase in chlorophyll concentrations from 1992 to 2016. Both nitrate and chlorophyll concentrations were lowest in the saltiest reaches of these estuaries. The effect of changes in N loads on nitrate concentrations were most evidenced in the low salinity reaches of estuaries, and were not significant in higher salinity waters within estuaries. This indicated that nitrogen inputs were from the watershed, and that water quality in Buzzards Bay have not changed across decades.��

This work was supported by the Jeff Metcalf Summer Undergraduate Research Fellowship of the University of Chicago and EPA SNEP. We would also like to thank the Buzzards Bay Coalition for data on water quality for these estuaries.

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Potassium Channel Organizes Actin Structure of the Presynaptic Terminal

Jessica Xia¹, Shobana Subramanian², Elizabeth A. Jonas^2,4, Leonard K. Kaczmarek^3,4

¹University of Chicago, Chicago, IL, ²Departments of Internal Medicine and Neuroscience, Yale University School of Medicine, New Haven, CT, ³Departments of Pharmacology and Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT, ⁴Marine Biological Laboratory, Woods Hole, MA

Spinocerebellar ataxia type 13 (SCA13) is a neurodegenerative disease that primarily affects the cerebellum and auditory brainstem nuclei, is associated with motor symptoms, and impairs sound localization. SCA13 is autosomal dominant and caused by mutations in KCNC3, the gene that encodes a voltage-gated potassium channel subunit called Kv3.3. Because of their ability to repolarize action potentials rapidly, Kv3.3 channels are highly expressed in regions of the auditory system that require cells to fire at high rates. G592R, a mutation associated with late-onset SCA13, is a substitution mutation that occurs in a region of the C-terminal of the Kv3.3 channel containing a chain of prolines. This one amino acid change results in a functioning potassium channel but disrupts its interaction with actin. Previous studies of wild-type Kv3.3, Kv3.3 knock-out (KO), and G592R knock-in (KI) mice have shown that Kv3.3 influences certain components of endocytosis and found that these channels interact with Arp2/3 to prompt actin polymerization. We have tested the hypothesis that Kv3.3 channels regulate actin structure in the medial nucleus of the trapezoid body (MNTB), a region with a particularly large presynaptic terminal called the Calyx of Held. Using fluorescent-tagged phalloidin, which specifically binds to filamentous actin, we stained slices of mouse brain containing the MNTB to observe differences in actin organization among Calyces of Held from wild-type, Kv3.3 KO, and G592R KI mice. Through super-resolution microscopy, it was found that the actin structure of the synapse is disrupted in Kv3.3 KO and G592R KI mice, indicating that the Kv3.3 channel organizes the structure of actin at the presynaptic terminal.

Jeff Metcalf Summer Undergraduate Research Fellowship – The University of Chicago

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Photoreceptor characterization as evidence for retinal specialization and visual adaptation in an elasmobranch

Kevin Zhao¹, Lena M. Herbst³, and Lydia M. Mathgër²

¹University of Chicago, Chicago, IL, USA, ²Marine Biological Laboratory, Woods Hole, MA, USA, University of Massachusetts Amherst, Amherst, MA, USA

The pure rod distribution in the retina, and the frilled shape of the pupil of the little skate, Leucoraja erinacea, are among the most unique characteristics of the visual system of this benthic, elasmobranch fish. Much of this animal’s specific optical anatomy remains unexplored, however, and studying these characteristics provides insight into the visual demands and adaptive specialization of this animal. In this project, we aim to provide the first detailed examination of a particular part of this anatomy, the photoreceptor. Two skate retinas were examined: (1) Using light microscopy techniques, one retina was cut into 38 specimens, for which orientation and location were carefully documented. Semi-thin sections (~65 µm thickness) were cut, stained with Toluidine blue and imaged so we could record photoreceptor length, width and angle with respect to the retinal surface. (2) The other retina was whole-mounted, imaged and the number of photoreceptors was counted for 205 sites in order to calculate the density distribution. We found that photoreceptor morphology and distribution varied across the retina, with a distinct horizontal visual streak pattern of increased photoreceptor density which indicates differences in sampling ability.

Jeff Metcalf Summer Undergraduate Research Fellowship – The University of Chicago