Molecular Mechanisms of Neural Plasticity Following Anterior-Posterior (AP) Axis Rotation in the Central Nervous System of African Clawed Frog, Xenopus laevis

Abstract: Our preliminary research has discovered that at early (gastrula) stages of development, Xenopus laevis embryos are able to recover from a near complete rotation of their anterior-posterior axis; by late gastrula stages this ability is largely lost. RNA-Seq analysis of RNA extracted from embryos with rotated tissues generated a list of differentially expressed candidate genes that might account for such differences in healing capacity. This project will characterize the roles of these genes with the goal of defining the molecular mechanisms governing neural plasticity following physical perturbations.

Experiences and Preliminary Data: I began working in Dr. Saha’s lab the start of my sophomore year. I first worked on my Monroe project, “Localization of Neural Progenitor Cells and Differentiated Neurons in Xenopus laevis CNS during Secondary Neurogenesis”. In September 2016, I successfully completed and presented it. The experimental techniques I employed in my Monroe project, which I have now mastered, are mostly the same as the ones I’ll be using in my Honors project. These two years of research experience, including a full-time summer, will greatly expedite my Honors project research.

After finishing my previous project in Summer 2016, I started working on the AP Axis Rotation project, which has generated a significant amount of preliminary data for my proposed research. The data show that while embryos with presumptive neural tissue rotated at mid gastrula stages (St. 11.5) recover almost completely, embryos with neural tissue rotated at late gastrula stages (St. 12.5) failed to recover. Analysis of our data shows that this recovery take place gradually throughout embryogenesis. It also suggests that the signaling environment could also play a key role, since late gastrula donor tissue placed into early neurula hosts show a higher level of recovery than early neural tissue into late gastrula hosts. We are in the process of writing a paper about these exciting findings for publication in a peer-reviewed journal, and I am a co-author. We are currently planning to submit the paper within the next month.

The experiments described above established that embryos do have this profound ability to recover during a narrow period of development. In order to uncover the molecular mechanisms regulating this ability to recover, that is, plasticity, we performed RNA-Seq, a technique that examines all expressed genes in selected tissues. The RNA-Seq resulted in a list of differentially expressed genes that may be important in regulating the ability of the embryo to recover from perturbations. After examining the list of genes, we selected the following list of candidate genes that might contribute to the neural plasticity of Xenopus laevis for further research. These genes all play some role in regulating cell growth and differentiation, a process that has already been shown to be involved in embryonic response to other perturbations.

1) EGFL6: EGF-like-domain, multiple 6; 2) OSGIN1: oxidative stress induced growth inhibitor 1; 3) KIF5A: kinesin family member 5 A; 4) PRDM13: PR domain containing 13; 5) PROM1: prominin 1; 6) PTBP3: polypyrimidine tract binding protein 3; 7) PTFLA: pancreas specific transcription factor, 1a; 8) STAT3.2: signal transducer and activator of transcription 3 (acute-phase response factor), gene 2

Methodology: The role of these genes in regulating neural plasticity will be examined using the following methods.

Embryos: Fertilized Xenopus laevis embryos are obtained and separated into donor and host groups. Donor embryos are bilaterally injected with Fldx fluorescent tracer during 2-cell stage. All embryos are raised to mid or late gastrula stages (11.5 or 12.5).

AP Axis Rotation: One donor and one host embryo of desired stages are devitillinated. Using glass needle and forceps, the presumptive neural ectoderm of host embryo is removed and discarded. Fldx labeled presumptive neural ectoderm of donor embryo is removed using the same method. For rotated embryos, the donor tissue is transferred to the host embryo after a 180°rotation along the anterior-posterior axis; for control embryos, the donor tissue is transferred to the host embryo as it was originally. They are then covered under a piece of glass so the donor tissue can incorporate into the host embryo. When incorporation is complete, the glass is removed and embryos would grow until desired stages. After they are screened under fluorescent scope to ensure donor tissue has been incorporated, they will be imaged, fixed in 1X MEMFA, and stored in ethanol in -20°C.

Double In Situ Hybridization (ISH): Antisense ISH probes of candidate genes are synthesized. The experimental embryos then undergo a double ISH. The first hybridization will show us the expression patterns of candidate genes; the second hybridization will show us the localization of donor tissues. Following the double ISH, these embryos are imaged and fixed again.

Histology: Embryos that underwent double ISH are embedded in paraffin and transversely sectioned at 18μm. All sections will then be imaged.

Analysis: All whole mount and sectioned images of embryos will be analyzed for gene expression and co-localization with donor tissue. The level of colocalization between candidate genes and donor tissues will be rated on a numerical scale using a set of predetermined standards, the same one as we used to generate preliminary data. Through statistical tests, genes that likely contribute to neural plasticity of Xenopus laevis after AP rotation are discovered.

Functional Analysis: To confirm and further investigate the roles of the candidate genes during Xenopus laevis development, overexpression and knockdown/knockout experiments will be performed to manipulate their expression. For overexpression experiments, embryos will be injected with capped mRNA of the candidate genes; the candidate genes will be knocked out by CRISPR-Cas, or knocked down by Morpholino injections. Then the embryonic response to these manipulations will be studied.

Significance: Certain vertebrates, like Xenopus laevis, exhibit an incredible degree of neural plasticity and regenerative ability following physical perturbation; however, although we share a vast majority of genes with Xenopus laevis, this phenomenon doesn’t occur in humans. By discovering the molecular mechanisms of neural plasticity following AP axis rotation in Xenopus laevis, we can compare that to the cells in human Central Nervous System (CNS) and determine why humans don’t have this regenerative ability. The research would also lay groundwork for the future advances of neurological and regenerative medicine, so that cells in human CNS would also gain this neural plasticity. Patients suffering trauma in nervous systems can potentially have their neurons regenerated to regain their bodily function. Alzheimer’s may also be treated, since the malfunctioning neurons can now be replaced by the newly induced, healthy cells. In general, this project would be a significant step in the progress of developmental and medical neurobiology.

Future Goals: After working in Dr. Saha’s research lab for almost two years, I am determined more than ever to pursue a career in molecular biology research, especially in areas closely related human health and medicine. This will involve years of rigorous graduate work for my PhD, as well as post-doctoral research. By doing an Honors thesis, which is quite similar to graduate-level research in format, time investment and difficulty, it would not only advance my knowledge and research skills as a science student, but also prepare me for the intensive works that lie ahead. Most importantly, by finishing this project, publishing the results, and presenting the findings at the Society of Neuroscience and Developmental Biology meetings, I could help make important progress in our basic understanding of regeneration and regenerative medicine for neural degenerative diseases and spinal cord injuries, two of the major causes of morbidity and mortality in our population.