Leigh Ann Samsa

Many have raised concerns about the reproducibility of biomedical research. In this Perspective, the authors address this “reproducibility crisis” by distilling discussions around reproducibility into a simple guide to facilitate understanding of the topic. Reproducibility applies both within and across studies. The following questions address reproducibility within studies: “Within a study, if the investigator repeats the data management and analysis, will she get an identical answer?” and “Within a study, if someone else starts with the same raw data, will she draw a similar conclusion?” Contrastingly, the following questions address reproducibility across studies: “If someone else tries to repeat an experiment as exactly as possible, will she draw a similar conclusion?” and “If someone else tries to perform a similar study, will she draw a similar conclusion?” Many elements of reproducibility from clinical trials can be applied to preclinical research (e.g., changing the culture of preclinical research to focus more on transparency and rigor). For investigators, steps toward improving reproducibility include specifying data analysis plans ahead of time to decrease selective reporting; more explicit data management and analysis protocols; and increasingly detailed experimental protocols, which allow others to repeat experiments. Additionally, senior investigators should take greater ownership of the details of their research (e.g., implementing active laboratory management practices, such as random audits of raw data [or at least reduced reliance on data summaries], more hands-on time overseeing experiments, and encouraging a healthy skepticism from all contributors). These actions will support a culture where rigor + transparency = reproducibility.

Regeneration of intestinal epithelium is fueled by a heterogeneous population of rapidly proliferating stem cells (ISCs) found in the base of the small intestine and colonic crypts. ISCs populations can be enriched by fluorescence-activated cell sorting (FACS) based on expression of combinatorial cell surface markers, and fluorescent transgenes. Conventional ISC culture is performed by embedding single ISCs or whole crypt units in a matrix and culturing in conditions that stimulate or repress key pathways to recapitulate ISC niche signaling. Cultured ISCs form organoid, which are spherical, epithelial monolayers that are self-renewing, self-patterning, and demonstrate the full complement of intestinal epithelial cell lineages. However, this conventional “bulk” approach to studying ISC biology is often semiquantitative, low throughput, and masks clonal effects and ISC phenotypic heterogeneity. Our group has recently reported the construction, long-term biocompatibility, and use of microfabricated cell raft arrays (CRA) for high-throughput analysis of single ISCs and organoids. CRAs are composed of thousands of indexed and independently retrievable microwells, which in combination with time-lapse microscopy and/or gene-expression analyses are a powerful tool for studying clonal ISC dynamics and micro-niches. In this protocol, we describe how CRAs are used as an adaptable experimental platform to study the effect of exogenous factors on clonal stem cell behavior.

Cardiac trabeculae are mesh-like muscular structures within ventricular walls. Subtle perturbations in trabeculation are associated with many congenital heart diseases (CHDs), and complete failure to form trabeculae leads to embryonic lethality. Despite the severe consequence of an absence of trabecular formation, the exact function of trabeculae remains unclear. Since ErbB2 signaling plays a direct and essential role in trabecular initiation, in this study, we utilized the erbb2 zebrafish mutant as a model to address the function of trabeculae in the heart. Intriguingly, we found that the trabeculae-deficient erbb2 mutant develops a hypertrophic-like (HL) phenotype that can be suppressed by inhibition of Target of Rapamycin (TOR) signaling in a similar fashion to adult mammalian hearts subjected to mechanical overload. Further, cell transplantation experiments demonstrated that erbb2 mutant cells in an otherwise wildtype heart did not undergo hypertrophy, indicating that erbb2 mutant HL phenotypes are due to a loss of trabeculae. Together, we propose that trabeculae serve to enhance contractility and that defects in this process lead to wall-stress induced hypertrophic remodeling.

The Neuregulin‐1 (Nrg1)/ErbB pathway plays multiple, critical roles in early cardiac and nervous system development and has been implicated in both heart and nerve repair processes. However, the early embryonic lethality of mouse Nrg1 mutants precludes an analysis of Nrg1's function in later cardiac development and homeostasis. In this study, we generated a novel nrg1 null allele targeting all known isoforms of nrg1 in zebrafish and examined cardiac structural and functional parameters throughout development. We found that zebrafish nrg1 mutants instead survived until young adult stages when they exhibited reduced survivorship. This coincided with structural and functional defects in the developing juvenile and young adult hearts, as demonstrated by reduced intracardiac myocardial density, cardiomyocyte cell number, swimming performance and dysregulated heartbeat. Interestingly, nrg1 mutant hearts were missing long axons on the ventricle surface by standard length (SL) 5 mm, which preceded juvenile and adult cardiac defects. Given that the autonomic nervous system normally exerts fine control of cardiac output through this nerve plexus, these data suggest that Nrg1 may play a critical role in establishing the cardiac nerve plexus such that inadequate innervation leads to deficits in cardiac maturation, function and survival.

A genetic component to familial mitral valve prolapse (MVP) has been proposed for decades. Despite this, very few genes have been linked to MVP. Herein is described a four-generation pedigree with numerous individuals affected with severe MVP, some at strikingly young ages. A detailed clinical evaluation performed on all affected family members demonstrated a spectrum of MVP morphologies and associated phenotypes. Linkage analysis failed to identify strong candidate loci, but revealed significant regions, which were investigated further using whole-exome sequencing of one of the severely affected family members. Whole-exome sequencing identified variants in this individual that fell within linkage analysis peak regions, but none was an obvious pathogenic candidate. Follow up segregation analysis of all exome-identified variants was performed to genotype other affected and unaffected individuals in the family, but no variants emerged as clear pathogenic candidates. Two notable variants of uncertain significance in candidate genes were identified: p.I1013S in PTPRJ at 11p11.2 and FLYWCH1 p.R540Q at 16p13.3. Neither gene has been previously linked to MVP in humans, although PTPRJ mutant mice display defects in endocardial cushions, which give rise to the cardiac valves. PTPRJ and FLYWCH1 expression was detected in adult human mitral valve cells, and in-silico analysis of these variants suggests they may be deleterious. However, neither variant segregated completely with all of the affected individuals in the family, particularly when 'affected' was broadly defined. While a contributory role for PTPRJ and FLYWCH1 in this family cannot be excluded, the study results underscored the difficulties involved in uncovering the genomic contribution to MVP, even in apparently Mendelian families.

Background—: The genetic variation underlying many heritable forms of cardiovascular disease is incompletely understood, even in patients with strong family history or early age at onset. Methods and Results—: We used whole exome sequencing to detect pathogenic variants in 55 patients with suspected monogenic forms of cardiovascular disease. Diagnostic analysis of established disease genes identified pathogenic variants in 21.8% of cases and variants of uncertain significance in 34.5% of cases. Three patients harbored heterozygous nonsense or splice-site variants in the nucleoporin genes NUP37 , NUP43 , and NUP188 , which have not been implicated previously in cardiac disease. We also identified a heterozygous splice site variant in the nuclear envelope gene SYNE1 in a child with severe dilated cardiomyopathy that underwent transplant, as well as in his affected father. To confirm a cardiovascular role for these candidate genes in vivo, we used morpholinos to reduce SYNE1, NUP37, and NUP43 gene expression in zebrafish. Morphant embryos displayed cardiac abnormalities, including pericardial edema and heart failure. Furthermore, lymphoblasts from the patient carrying a SYNE1 splice-site variant displayed changes in nuclear morphology and protein localization that are consistent with disruption of the nuclear envelope. Conclusions—: These data expand the repertoire of pathogenic variants associated with cardiovascular disease and validate the diagnostic and research use of whole exome sequencing. We identify NUP37 , NUP43 , and NUP188 as novel candidate genes for cardiovascular disease, and suggest that dysfunction of the nuclear envelope may be an under-recognized component of inherited cardiac disease in some cases.

The Neuregulin-1 (Nrg1) signaling pathway has been widely implicated in many aspects of heart development including cardiac trabeculation. Cardiac trabeculation is an important morphogenetic process where clusters of ventricular cardiomyocytes extrude and expand into the lumen of the ventricular chambers. In mouse, Nrg1 isoforms containing an immunoglobulin-like (IgG) domain are essential for cardiac trabeculation through interaction with heterodimers of the epidermal growth factor-like (EGF-like) receptors ErbB2/ErbB4. Recent reports have underscored the importance of Nrg1 signaling in cardiac homeostasis and disease, however, placental development has precluded refined evaluation of the role of this pathway in mammals. ErbB2 has been shown to have a developmentally conserved role in cardiac trabeculation in zebrafish, a vertebrate model organism with completely external development, but the requirement for Nrg1 has not been examined. We found that among the multiple Nrg1 isoforms, the IgG domain-containing, type I Nrg1 (nrg1-I) is the only isoform detectable in the heart. Then, using CRISPR/Cas9 gene editing, we targeted the IgG domain of Nrg1 to produce novel alleles, nrg1^nc28 and nrg1^nc29, encoding nrg1-I and nrg1-II truncations. Our results indicated that zebrafish deficient for nrg1-I developed trabeculae in an ErbB2-dependent manner. Further, these mutants survive to reproductive adulthood with no overt cardiovascular defects. We also found that additional EGF-like ligands were expressed in the zebrafish heart during development of trabeculae. Together, these results suggest that Nrg1 is not the primary effector of trabeculation and/or that other EGF-like ligand(s) activates the ErbB2/ErbB4 pathway, either through functioning as the primary ligand or acting in a redundant manner. Overall, our work provides an example of cross-species differences in EGF family member requirements for an evolutionary conserved process.