# The Role of Auxilin, a Clathrin Uncoating Chaperone, in Congenital Heart Disease and Left-Right Patterning

> **NIH NIH F31** · YALE UNIVERSITY · 2021 · $30,882

## Abstract

Project Summary/Abstract
Congenital heart disease (CHD) afflicts approximately 1% of live births and is among the most life-threatening
of the birth defects. Patients with heterotaxy (HTX), improper left-right (LR) patterning and orientation of the
visceral organs, are at high risk of CHD. Whole exome sequencing of patient-parent trios revealed many
candidate CHD/HTX genes. Among these is DNAJC6, which codes for the neuronal-specific clathrin-coat
HSP70 chaperone, Auxilin. Although auxilin has been studied in the context of clathrin vesicle uncoating and
Juvenile Parkinson's, its role in embryonic development and CHD/HTX has not. A preliminary loss of function
(LOF) screen revealed that DNAJC6 depletion causes abnormal cardiac looping in 15% of embryos, and
abnormal pitx2c expression in 40% of embryos. These indicate abnormal LR patterning. No previous studies
have linked DNAJC6 with LR patterning or heart morphogenesis in early development. Thus, the goal of this
proposal is to elucidate the molecular mechanism by which auxilin (DNAJC6) leads to abnormal LR
patterning that can lead to CHD/HTX. The first aim will use LOF experiments to determine when in the LR
patterning cascade DNAJC6 has its earliest effects by visualizing dand5 expression in the left-right organizer.
Determination of spatiotemporal DNAJC6 expression in early development in will focus subsequent
mechanistic analysis. The second aim will analyze whether DNAJC6 LOF affects LR patterning by disrupting
endocytosis. Fluorescence and TEM microscopy will reveal whether endocytosis, clathrin-coated vesicle
uncoating, and clathrin pool maintenance are affected in auxilin-depleted embryos. Phenocopy and DNAJC6
LOF LR patterning defect rescue experiments with clathrin-mediated endocytosis components will indicate
whether auxilin's role in LR patterning is clathrin-dependent. The final aim will investigate whether auxilin
impacts LR patterning by disturbing Wnt or Notch signaling, both of which are affected by clathrin-mediated
endocytosis. Luciferase reporters and western blots for downstream outputs (β-catenin for Wnt and the notch
intracellular domain for Notch) will demonstrate which signaling cascade is activated. Phenocopy and LOF
rescue experiments with downstream signaling inhibitors and components will indicate whether auxilin's role in
LR patterning is Wnt or Notch dependent. Together, these experiments will elucidate the role of auxilin
(DNAJC6) in left-right patterning, cardiac development, and CHD pathogenesis. This will aid future patient
diagnosis and outcomes as their treatment can be targeted to genotype in addition to CHD phenotype. This
application also outlines the applicant's training plan in advanced coursework, research mentorship, new
techniques, and professional skills including written and oral data presentation. This training plan will equip the
applicant with the skills to pursue a career as an independent research scientist and professor.

## Key facts

- **NIH application ID:** 10214682
- **Project number:** 5F31HL149246-03
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Delfina Pearledith Gonzalez
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $30,882
- **Award type:** 5
- **Project period:** 2019-09-01 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10214682

## Citation

> US National Institutes of Health, RePORTER application 10214682, The Role of Auxilin, a Clathrin Uncoating Chaperone, in Congenital Heart Disease and Left-Right Patterning (5F31HL149246-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10214682. Licensed CC0.

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