Biology subjects

Clark, E. M.

Publications and source records attributed to Clark, E. M..

3 recordsLinked to original sources

bioRxiv2025

Stress-Responsive Transcriptomic Signatures in Human iPSC-Derived Microglia Reveal Links to Alzheimer's Disease Risk Genes

Commercially available human iPSC lines with inducible CRISPR interference (CRISPRi) systems offer scalable platforms for gene function studies. One widely available line, the AICS-0090 dCas9-KRAB iPSC line developed by the Allen Institute, has been extensively validated for genomic integrity and stem cell potency. However, its utility in modeling specialized immune cell types such as microglia -- and in assessing their functional responses to disease-relevant stimuli -- has not been fully established. Here, we evaluated the AICS-0090 line for its ability to differentiate into microglia-like cells, support efficient gene knockdown, and respond to environmental stressors. We assessed its differentiation capacity by qPCR, flow cytometry, and immunocytochemistry, confirming reproducible expression of microglial surface markers at early and late timepoints. Gene knockdown efficiency was validated both at the single-gene level and in pooled CRISPRi screens. Focusing on functional responsiveness, we exposed the microglia-like cells to two distinct stimuli: amyloid-{beta} (A{beta}), a disease-associated trigger in Alzheimers disease, and lipopolysaccharide (LPS), a classical inflammatory signal. Transcriptomic and functional analyses revealed stimulus-specific responses: A{beta} induced limited activation of stress and inflammation pathways, whereas LPS elicited broader transcriptional reprogramming and cytokine release. Signatures from both conditions partially overlapped with ex vivo human and mouse microglial states. Together, these findings support the use of the AICS-0090 dCas9-KRAB iPSC-derived microglia-like cells as a flexible and tractable model for gene function interrogation under defined inflammatory contexts, with potential for future applications in neuroimmune modeling and perturbation-based screening.

neuroscience↗

bioRxiv2020

Complementary and divergent roles for Ctage5 and Tango1 in zebrafish

Coat protein complex II (COPII) factors mediate cargo export from the endoplasmic reticulum (ER), but bulky collagens and lipoproteins are too large for traditional COPII vesicles. Mammalian CTAGE5 and TANGO1 have been well characterized individually as specialized cargo receptors at the ER that function with COPII coats to facilitate trafficking of bulky cargoes. Here, we present a genetic interaction study in zebrafish of deletions in ctage5, tango1, or both to investigate their potential distinct and complimentary functions. We found that Ctage5 and Tango1 have different roles related to organogenesis, collagen versus lipoprotein trafficking, stress-pathway activation, and survival. While deletion of both ctage5 and tango1 compounded phenotype severity, deletion of either factor alone revealed novel tissue specific defects in the building of heart, muscle, lens, and intestine, in addition to the previously described roles in the development of neural and cartilage tissues. Together, our results suggest that Ctage5 and Tango1 have overlapping, but also divergent roles in tissue development and homeostasis. SummaryIn this genetic study Ctage5 and Tango1 endoplasmic reticulum cargo receptors were investigated together in vivo for the first time. Cell differentiation, survival, trafficking, and stress pathway activation were investigated.

cell biology↗

bioRxiv2019

Establishment and validation of an endoplasmic reticulum stress reporter to monitor zebrafish ATF6 activity in development and disease

Induction of endoplasmic reticulum (ER) stress is associated with diverse developmental and degenerative diseases. Modified ER homeostasis causes activation of conserved stress pathways at the ER called the unfolded protein response (UPR). ATF6 is a transcription factor activated during ER stress as part of a coordinated UPR. ATF6 resides at the ER, and upon activation is transported to the Golgi apparatus where it is cleaved by proteases to create an amino-terminal cytoplasmic fragment (ATF6f). ATF6f translocates to the nucleus to activate transcriptional targets. Here, we describe establishment and validation of zebrafish reporter lines for ATF6 activity. These transgenic lines are based on a defined and multimerized ATF6 consensus site which drives either eGFP or destabilized eGFP (d2GFP), enabling dynamic study of ATF6 activity during development and disease. The results show that the reporter is specific for the ATF6 pathway, active during development, and induced in disease models known to engage UPR. Specifically, during development, ATF6 activity is highest in the lens, skeletal muscle, fins, and gills. The reporter is also activated by common chemical inducers of ER stress including tunicamycin, thapsigargin, and brefeldin A, as well as by heat shock. In both an ALS and a cone dystrophy model, ATF6 reporter expression is induced in spinal cord interneurons or photoreceptors, respectively, suggesting a role for ATF6 response in multiple neurodegenerative diseases. Collectively our results show these ATF6 reporters can be used to monitor ATF6 activity changes throughout development and in zebrafish models of disease.\n\nSummary StatementWe have established and validated transgenic zebrafish reporter lines to quantitatively measure the ATF6 branch of the endoplasmic reticulum stress pathway in development and disease.

cell biology↗