Although the relationship between the structure, function, and physiology of organs is well documented, the mechanisms by which cells collectively coordinate into three-dimensional tissues and organ components remains unknown.The countless factors that inform the morphogenesis of mammalian organs poses a challenge to understand organogenesis from first principles. However, the Malpighian tubules of the fruit fly Drosophila offer an excellent model system for investigating this question due to their rapid development, relative simplicity, and the degree to which scientists can manipulate variables that affect their development. These tubules are the renal equivalent of the fruit fly excretory system; further, many of the genes involved in sculpting these tubules are conserved from flies to humans. One conserved gene is the fly homolog of β-catenin, which is known to play an essential role in cell-cell adhesion. The goal of my research is to define how β-catenin impacts organ morphogenesis. To do this, I use fluorescence microscopy and live imaging to compare wildtype Drosophila to those with decreased β-catenin expression. Using tissue-specific fluorescent protein tagging, I can differentiate Malpighian tubule cells from other embryonic cells under the microscope so that their shapes can be analyzed, and I control the level of β-catenin expression specifically in Malpighian tubule cells using RNAi. Due to β-catenin’s integral role in cell-cell adhesion, I expect to find localization of β-catenin to the cell membranes of the tubules, with high concentration along membranes undergoing the greatest adhesion or motion, and interrupted tubule morphogenesis in reduced-expression lines. I also suspect that cells may completely fail to adhere and will be unable to transmit tension effectively along the tissue. The results of this experiment will contribute to our understanding not only of Malpighian tubule morphogenesis, but of one of the components of morphogenesis in general.

How do organs have such consistent and reproducible shape, form, and volume? One factor of this complex phenomena is cell-cell adhesion. Cell-cell adhesion plays a vital role in organ formation, as it is an essential driver of cell shape, cell arrangements, and tissue structure. To determine the role of adhesion in organ formation, I define the role of E-Cadherin, a cell-cell junction projection that adheres neighboring cells. The developing renal system of Drosophila, Malpighian tubules, are an excellent system because I can selectively manipulate expression of E-Cadherin in the organ and can utilize fluorescence microscopy to observe how these changes affect tubule morphogenesis. I observe where the adhesion protein is located during organ growth, and what happens to organ growth when expression of the adhesion protein is reduced. To track the dynamic localization of E-Cadherin, I take measurements of specific location of E-Cadherin between cells and concentration of E-Cadherin throughout organ development. I expect the concentration of E-Cadherin to increase during elongation, and that it will be enriched in more looped parts of the organ. To define the requirement of E-cadherin during organ formation, I use RNA interference to reduce E-Cadherin expression. Because of how vital E-Cadherin is in other developmental morphogenetic processes, I expect a decrease of expression to have profound impacts, leading to severe organ developmental defects. I measure these defects by comparing cell shape change and organ shape in control and E-Cadherin reduced organs. The results of this study will not only help us understand Malpighian tubule morphogenesis, but it will also help us understand organogenesis more generally. Elucidating the precise mechanisms behind cell behavior, shape, and cell-cell interaction has important human health implications and will enable work in many other fields such as cancer, regenerative treatments, tissue growth, and organ synthesis.

Infants perceive speech and acquire language amidst noisy and complex auditory environments. Thus, elucidation of the cognitive mechanisms governing speech perception under noisy conditions is crucial. Cortical encoding of the speech envelope has been one approach used to study speech-in-noise perception in adults. For infants, research shows that Infant Directed Speech (IDS) facilitates cortical encoding of the speech envelope in quiet conditions more than adult direct speech. However, it is unclear whether infants are able to track the IDS speech envelope amidst competing speech. To investigate this, we recorded the neural responses from 40 typically-hearing infants (20 seven-month-olds, 20 eleven-month-olds) to continuous IDS using electroencephalography (EEG) in three conditions: Quiet, Co-located Noise, and Separated Noise. The target stimuli consisted of naturally recorded IDS produced by two female English speakers. The noise stimuli consisted of a four-person babble constructed from audiobooks read by 2 male and 2 female English speakers. We presented stimuli at an overall level of 70 dB SPL via speakers placed at 0°, +90°, and -90° azimuth to infants sitting on a caregiver’s lap in a sound-attenuated booth. Our team analyzed EEG signals using the Multivariate Temporal Response Function (mTRF) toolbox in MATLAB. This backward modeling approach assesses whether the stimulus envelope can be reconstructed based on the recorded neural responses. Reconstruction accuracies greater than chance were observed in all three conditions for the majority of infants, suggesting that we were able to decode the speech envelope in both quiet and noise. Participants demonstrated the capacity to process speech, even amidst competing auditory stimuli, emphasizing speech perception competencies from an early developmental stage. These results support using the envelope model and mTRF method as a feasible method for investigating the development of speech-in-noise perception in infants and young children.

Science is rapidly evolving, yet its advances do not enter classrooms at the same rate. Systems Education Experiences (SEE) is a program in the Baliga Lab at the Institute for Systems Biology (ISB) that accelerates the transfer of scientific knowledge and practices to classrooms. One active area of Baliga Lab research is elucidating the level of resilience organisms have, when faced with complex environmental changes. My role is to design laboratory experiments that investigate this with the model organism Halobacterium salinarum (Halo) and to connect this to broader rules governing natural systems for use in high school classrooms. My first experiment probes the resiliency of Halo with the introduction of a combination of stressors (salt and hydrogen peroxide) and its recovery after population collapse. The second measures the long term phenotypic changes in the population. I wanted to see if after being exposed to a new environment if there was an advantage to having gas vesicles and if it is an irreversible trait that allows Halo to be resilient across a variety of environmental conditions. This relates to broadly applicable rules governing resilience across many systems. This project serves as a model for how all organisms respond to stress. Combinations of stressors in human lives can make us less resilient. However, strategies to quickly prepare, respond, and react can improve outcomes for individuals and the overall population. This project connects to a goal of K-12 science education which is to not just teach academic concepts but to equip students with knowledge that can be applied to all parts of life. Our knowledge on the mechanisms that control how organisms respond to stress is extremely limited. By understanding the biological stress response we can promote resilience in the earth's most vulnerable systems in the wake of climate change.

The human genome is associated with numerous variations, some of which can be pathogenic. Any variations larger than 50 base pairs (bp) are considered structural variants (SVs), and SVs impacting copy number of the genome are termed copy number variants (CNVs). By studying CNVs, we can understand the underlying mechanisms of damage and repair within DNA, which can help work towards prevention and cure in other fields, such as cancer genomics. My project investigates a CNV on chromosome 2 of a pediatric patient, who presents with tetralogy of Fallot, global development delay, and multiple other congenital anomalies. Using array comparative genomics hybridization (aCGH), we identified a 3.2 megabase (Mb) complex genomic rearrangement (CGR) spanning the 2q31 region of the proband’s chromosome 2. Detailed analysis of the short-read whole genome sequencing (WGS) allowed us to locate the exact coordinates of each junction, or the beginning and end points of each extra copy. PacBio long-read genome sequencing and optical genome mapping detected the same junctions and facilitated confirmation of the overall structure. The CGR can be characterized as a duplication-triplication-duplication-triplication-duplication (DUP-TRP-DUP-TRP-DUP), meaning this segment of the genome contains a segment of alternating 1 and 2 extra copies of this region. The CGR is de novo, or not inherited from the proband’s parents. Due to the nature of this variant, it is likely to be impacting the phenotype of our patient. To establish a genotype-phenotype correlation, I did a literature review of patients with overlapping CGRs, comparing their phenotypes to our proband, as well as reviewing any known disease-associated genes in the region using the online catalog of human genes and genetic disorders (OMIM). Patient and disease comparison revealed the extreme rarity of our patient’s CGR, leading us to believe the phenotype of the proband results from impact of multiple genes in the affected region.

The heart is one of the most mechanically active organs in the body. In a mechanism known as the “Bainbridge Reflex”, the heart rate accelerates in response to the mechanical stretch induced by the increase in venous return. The cardiac pacemaker controls heart rate, and while stretch-activated channels have been identified in cardiac tissue, their molecular identity remains unknown. We hypothesize that PIEZO channels are the molecular determinant of the stretch-dependent heart rate acceleration responsible for the Bainbridge reflex. Using quantitative polymerase chain reaction (qPCR), we assessed the presence of Piezo1 and Piezo2 transcripts in the pacemaker, atrium, and ventricle of the mouse heart. Our findings revealed that both Piezo1 and Piezo2 are present in the three regions with significantly higher expression in the pacemaker and atria. Combining immunohystochemistry, tissue clearing, and super-resolution microscopy, we analyzed the distribution of Piezo1 and Piezo2 in mouse pacemaker explants. Our results show that Piezo2 is uniformly expressed in the pacemaker and surrounding atrial tissue, whereas Piezo1 exhibits higher expression levels outside the pacemaker. These results were further confirmed at the single-cell level, with immunostaining of Piezo1 and Piezo2 in isolated pacemaker cells (HCN4+) and transitional cells (HCN4-). We observed similar expression levels of Piezo2 in both cell types and increased Piezo1 expression in transitional cells. In addition, we observed distinct localization patterns for Piezo1 and Piezo2 at the subcellular level. Piezo1 predominantly localizes to the sarcolemma, while Piezo2 exhibits a striated distribution that colocalizes alternately with both the Z- and the M- line of the sarcomere. Given this pattern, half of the Piezo2 bands colocalize with the RyR. These results set the starting point to evaluate the functional role of PIEZO channels in the cardiac pacemaker.