Research & Publications
The goal of our work is to determine the genetic cause and developmental mechanisms underlying congenital heart disease and, in particular, the function of cilia in heart development. The research aims to bridge research in the basic developmental biology mechanisms underlying development of the embryonic left-right axis with clinical pediatric cardiology and cardiac genetics. The laboratory has been integral in understanding the cellular and molecular mechanism underlying vertebrate LR asymmetry, beginning with the discovery that the axonemal dynein left-right dynein is essential for the development of vertebrate left-right asymmetry. We then demonstrated that a combination of motile and immotile cilia establishes an early asymmetric calcium signal that is essential to normal LR development of the heart. As part of the Pediatric Cardiac Genomics Consortium (PCGC), we are now combining our understanding of the basic biology underlying left-right development with state-of-the-art genomics to a more comprehensive understanding of human congenital disease, in particular, human heterotaxy. We are focusing on the ability to directly test putative genetic causes of human CHD identified from genomic analysis of patient DNA in animal model systems including mouse and zebrafish.
Although our medical and surgical management of patients with congenital heart disease has made tremendous progress in the past 25 years, the understanding of why CHD develops remains relatively limited. One challenge in the care of patients with CHD is that different patients with anatomically very similar disease can have greatly disparate long-term outcomes. The mechanism underlying any individual patient’s CHD may have as of yet unknown impact on how well they do, and it is hoped that eventually it will become possible to tailor medical management and surgery more specifically based on an individual’s combination of anatomical abnormality and underlying developmental defect. If ciliary defects are responsible for some human CHD, it may become possible to treat the later manifestations of the disease, such as progressive cardiac valve dysfunction observed in many adult patients who have had successful surgery for their CHD, with drugs aimed at restoring more normal ciliary signaling.
Specialized Terms: Development of left-right asymmetry; Heterotaxy syndrome; Kartagener syndrome; Situs inversus
Extensive Research Description
The current research foci of the Brueckner Lab are:
1. Understanding the cellular and molecular mechanism underlying vertebrate LR asymmetry
My laboratory first discovered that the axonemal dynein left-right dynein (lrd) is essential for the development of vertebrate left-right asymmetry (Supp et al, Nature 1997). Lrd powers directional beating of cilia at the left-right organizer and breaks bilateral symmetry by creating directional flow of extraembryonic fluid. Importantly, in a collaboration with the labs of Clifford Tabin and H. Joseph Yost, we identified that the ciliated left-right organizer is conserved throughout vertebrates (Essner et al, Nature, 2002); this observation has allowed us to move seamlessly between model organism systems including mouse and zebrafish for our continuing work on left-right development. My laboratory then addressed the question of how directional flow is sensed to connect cilia motility to asymmetric organogenesis. We demonstrated that polycystin-2 containing immotile cilia sense directional flow to initiate asymmetric signaling linking the events at the left-right organizer with subsequent asymmetric gene expression and heart development (McGrath et al, Cell 2003). In order to address the question of the molecular mechanism by which polycystin initiates asymmetric signaling in response to directional flow of extraembryonic fluid, we then developed a method to target genetically-encoded calcium indicators specifically into cilia in living embryos. This approach allowed us to see asymmetric, polycystin2-dependent intraciliary calcium waves at the left-right organizer of living zebrafish that are the earliest molecular asymmetry in the vertebrate embryo (Yuan et al, Current Biology, 2015). We are continuing to develop technology to permit live imaging of intraciliary calcium in zebrafish and mouse embryos (Yuan and Brueckner, Methods Mol Bio, 2016). Current work is focused on the link between calcium signaling and asymmetric organ development utilizing genetically encoded calcium reporters targeted to cilia in embryos and cultured cells. We are testing the hypothesis that mechanical stimuli trigger intraciliary calcium via the polycystin complex, and that asymmetric calcium is determinative for the development of left-right asymmetry. In addition, we are exploring the potential role of the Ankyrin-repeat protein Inversin as a link between intraciliary calcium and asymmetric molecular signaling. Finally, we are exploring the role of intracardiac cilia in heart development where we propose that they also function as mechanosensors, but now integrate mechanical signals such as contractile and hemodynamic forces with transcriptional control of cardiac morphogenesis.
2. Understanding the genetic architecture of congenital heart disease
The question remains, however, how understanding the novel and remarkable mechanism by which cilia drive left-right and cardiac development connects to patients with congenital heart disease. To this end, we have established collaboration with Richard Lifton and the Yale Center for Genome Analysis, and have become part of the Pediatric Cardiac Genomics Consortium (PCGC). We began by showing that copy-number variations underlie ~10-15% of human heterotaxy (Fakhro et al, PNAS, 2011). The PCGC has recruited ~13,000 patients with CHD so far and aims to apply current genomic approaches in order to develop a more global understanding of the genetics of congenital heart disease. The initial analysis of 362 patients with severe CHD demonstrated that de-novo mutations underlie ~10% of CHD, and implicated chromatin remodeling as a heretofore unrecognized molecular mechanism in CHD (Zaidi et al, Nature, 2013). Expansion of the sequenced patient cohort to over 1,100 patients showed a link between the genetic cause of CHD and neurodevelopmental outcome (Homsey et al, Science, 2015). Analysis of mutations identified from CHD patients has already lead us to new insights into the mechanism of early heart development, including how the glycosylation enzyme GALNT11 modulates NOTCH signaling in determining cilia identity in the development of LR asymmetry (Boskovski et al, Nature, 2013), and how the NIMA-like kinase Nek2 balances ciliogenesis and resorption (Endicott et al, Development, 2015). Finally, increasing the size of the studied CHD cohort coupled with a novel computational approach for the first time allowed the unbiased identification of inherited variants contributing to human disease, and identified that mutations affecting cilia genes contribute directly to human CHD (Jin et al, Nature Genetics, 2017). Work is ongoing to begin to connect genotype with clinical outcome and reconnect the developmental biology work with clinical pediatric cardiology.
Current work is focused on expanding the understanding of the genetic underpinnings of congenital heart disease through large-scale genomic analyses of patients with CHD, including exploration of inherited contributions to CHD, and potential multigenic inheritance.
3. The role of Chromatin regulation in cilia and cardiac development.
Exome sequencing of 2,426 parent-offspring trios with severe CHD in the offspring identified a highly significant excess of de-novo dominant mutations in chromatin remodeling genes including 11 patients with mutations affecting monoubiquitylation of Histone H2BK120. The mechanisms linking mutation to disease are unknown. In our preliminary work, knockdown of five CHD-associated chromatin remodeling genes in Xenopus produced an entirely unexpected phenotype: in addition to abnormal cardiac development, cilia structure and/or motility were abnormal. This mirrored the phenotype of a patient with mutation in RNF20, who had Htx and primary ciliary dyskinesia (PCD) with absent inner dynein arms (Robson et al, PNAS, 2019). RNF20 is an E3 ubiquitin ligase that ubiquitylates H2BK120. Rnf20 has previously been implicated in differentiation of embryonic stem cells; however, there is no known role for RNF20 and H2BK120 ubiquitylation in ciliary biology, LR patterning, or cardiac development. These observations raise the question why haploinsufficiency for ubiquitously required epigenetic regulation affects heart development, and cilia function, in particular. We propose that RNF20 functions in cardiac development first through control of the transcriptional program directing cilia biogenesis and LR patterning, and then through direct function in the heart. Current experiments are testing the mechanism of Rnf20-mediated H2BK120 ubiquitylation in mouse embryos and cultured cardiomyocytes using ChIPseq and RNAseq analyses.
Cardiology; Genetics; Heart Diseases; Kartagener Syndrome; Situs Inversus; Heterotaxy Syndrome