Brueckner Laboratory Research
Our Laboratory Research includes studies regarding:
- Cilia in Left-Right Development
- Cilia in Cardiac Development
- Genetic Control in Human Heterotoxy
Cilia in Left-Right Development
The development of non-random asymmetry along the left-right axis is a unique feature of vertebrate development. Defects in this process in mouse and man commonly affect the development of the heart and result in severe congenital cardiac anomalies.Our goal is to understand the mechanism by which embryonic cilia create and signal left-right positional information, and to investigate whether cilia have essential roles in other developmental processes.
We have previously shown that the vertebrate LR axis is initiated at the mammalian node late in gastrulation. Dynein-driven motility of monocilia found on node cells generates directional flow of extraembryonic fluid, termed "nodal flow". The direction of nodal flow is determined by the inherent chirality of the cilium itself, and artificial reversal of nodal flow is able to reverse the LR axis. Nodal flow sets up an asymmetric calcium signal found in the cells at the left border of the node.
The development of LR asymmetry is also abnormal in mice with defects in the polycystin gene Pkd2, which functions in kidney monocilia as a mechanotransducer by mediating an intracellular calcium signal in response to fluid flow in the renal tubule. We have shown that Polycystin-2 protein localizes to a subset of node monocilia that are non-motile. This data suggests that embryonic cilia may be required to both create and sense nodal flow.
The major focus of our interest is: How is the asymmetric perinodal calcium signal transmitted to the developing organs to result in asymmetric morphogenesis?
LR Development
The embryo is apparently symmetric at e7.75. The node, located at the tip of the embryo (shown in green) is ciliated and generates leftward nodal flow. At e8.25 (0–3 somites), the previously symmetric perinodal expression of the growth factor nodal becomes asymmetric (shown in blue). Shortly thereafter at e8.5, a large region of asymmetric nodal expression appears in the left lateral plate mesoderm (shown in blue). Several other genes also become asymmetrically expressed at this time, including the transcription factor Pitx2, which is expressed in a more posterior region of lateral plate mesoderm abutting the heart primordia, and eventually in the heart itself. At e8.5, the embryo turns, the heart forms its characteristic rightward (D) loop, and molecular asymmetry is converted to asymmetric organogenesis of the lungs, heart, spleen and gut. Normal LR development results in situs solitus, and is highlighted by the gray box at the left of the figure. When LR development does not proceed normally, a range of outcomes is possible. Mirror-image reversal of all the organs is called situs inversus, and is usually not associated with significant pathology. Complete failure to break bilateral symmetry is thought to result in left isomerism (and the development of bilateral multi-lobed lungs and bilateral spleens) or right isomerism (bilateral bi- or uni-lobed lungs and absent spleen). The isomerisms have associated severe complex cardiac malformations.
Beating cilia produces directional flow across the node and causes asymmetric propagation of calcium signals.
Cilia in Cardiac Development
Cross sections, H&E stained, of the inflow (posterior region of the heart) of e9.5 wild-type, lrdΔneo/Δneo, Pkd2-/- and Kif3a-/- embryos embryos. A. Overview images of wild type, lrdΔneo/Δneo, Pkd2-/-, and Kif3a-/- hearts including the atrium (A), endocardial cushions (ECC), and ventricle (V). B. Endocardial cushions, arrow indicates cushion mesenchyme. C. Higher-magnification view of the trabecular and compact myocardium (indicated by arrow), and the surrounding pericardium showing trabeculations in the WT and lrdΔneo/Δneo sections, decreased trabeculations in the Pkd2-/- section, and none in the Kif3a-/- section.
Since cilia function as mechanosensors in other fluid-filled organs they could also be fluid flow sensors in heart development. We have shown that cilia are found in the mouse embryo heart at e8.5 – e12.5, and that heart development in mice with absent cilia is much more severely affected than in mice with paralyzed cilia and isolated abnormalities of LR development.
We are using mice with conditional mutations affecting cilia structure and function to generate mice with cardiac-specific ciliary abnormalities in order to investigate the mechanism by which cardiac cilia function directly in heart morphogenesis.
Genetic Control in Human Heterotaxy
We are collaborating with the laboratory of Richard Lifton in the Dept. of Genetics at Yale to perform a large-scale genomic analysis of human patients with heterotaxy. Analysis of copy-number variation in a cohort of patients with Htx identified >40 novel candidate genes for human Htx. These genes cluster in several developmental pathways, some of which have not previously been linked to LR development.
We are validating the role of these genes in animal systems including mouse, Xenopus and zebrafish, and beginning more detailed analysis of their function within the molecular framework established for LR development.