Brent McCright

Notch signaling regulates bile duct morphogenesis in mice.

BACKGROUND Alagille syndrome is a developmental disorder caused predominantly by mutations in the Jagged1 (JAG1) gene, which encodes a ligand for Notch family receptors. A characteristic feature of Alagille syndrome is intrahepatic bile duct paucity. We described previously that mice doubly heterozygous for Jag1 and Notch2 mutations are an excellent model for Alagille syndrome. However, our previous study did not establish whether bile duct paucity in Jag1/Notch2 double heterozygous mice resulted from impaired differentiation of bile duct precursor cells, or from defects in bile duct morphogenesis. METHODOLOGY/PRINCIPAL FINDINGS Here we characterize embryonic biliary tract formation in our previously described Jag1/Notch2 double heterozygous Alagille syndrome model, and describe another mouse model of bile duct paucity resulting from liver-specific deletion of the Notch2 gene. CONCLUSIONS/SIGNIFICANCE Our data support a model in which bile duct paucity in Notch pathway loss of function mutant mice results from defects in bile duct morphogenesis rather than cell fate specification.

The contribution of Notch1 to nephron segmentation in the developing kidney is revealed in a sensitized Notch2 background and can be augmented by reducing Mint dosage

We previously determined that Notch2, and not Notch1, was required for forming proximal nephron segments. The dominance of Notch2 may be conserved in humans, since Notch2 mutations occur in Alagille syndrome (ALGS) 2 patients, which includes renal complications. To test whether mutations in Notch1 could increase the severity of renal complications in ALGS, we inactivated conditional Notch1 and Notch2 alleles in mice using a Six2-GFP::Cre. This BAC transgene is expressed mosaically in renal epithelial progenitors but uniformly in cells exiting the progenitor pool to undergo mesenchymal-to-epithelial transition. Although delaying Notch2 inactivation had a marginal effect on nephron numbers, it created a sensitized background in which the inactivation of Notch1 severely compromised nephron formation, function, and survival. These and additional observations indicate that Notch1 in concert with Notch2 contributes to the morphogenesis of renal vesicles into S-shaped bodies in a RBP-J-dependent manner. A significant implication is that elevating Notch1 activity could improve renal functions in ALGS2 patients. As proof of principle, we determined that conditional inactivation of Mint, an inhibitor of Notch-RBP-J interaction, resulted in a moderate rescue of Notch2 null kidneys, implying that temporal blockage of Notch signaling inhibitors downstream of receptor activation may have therapeutic benefits for ALGS patients.

Functional conservation of Notch1 and Notch2 intracellular domains

The Notch receptor is a key component of a highly conserved signaling pathway that regulates cell fate determination during development. In Drosophila, where Notch signaling was first identified and studied, there is only one Notch receptor. In contrast, mammals have four Notch receptor genes, Notch1‐4. Notch1 and Notch2 are both required for embryo viability, are widely expressed in mammals, and are structurally conserved. It is presently unknown if these two receptors are functionally redundant or if they have unique capabilities related to differences in their amino acid sequences. In contrast to the rest of the molecule, the amino acid sequences of a large region of the Notch intracellular domain are not highly conserved and thus may be able to interact with distinct transcription factors and mediate the expression of different sets of genes. To determine if the function of this region is conserved, the last 426 amino acids of the Notch2 receptor have been replaced with the corresponding region of Notch1 in mice by using gene targeting. We have determined that even though the amino acid sequences of this region are only 37% identical (137/426), the C‐terminal region of the Notch1 intracellular domain can functionally replace that of Notch2 in vivo.

The protein phosphatase 2A B56γ regulatory subunit is required for heart development.

Background: Protein Phosphatase 2A (PP2A) function is controlled by regulatory subunits that modulate the activity of the catalytic subunit and direct the PP2A complex to specific intracellular locations. To study PP2As role in signal transduction pathways that control growth and differentiation in vivo, a transgenic mouse lacking the B56γ regulatory subunit of PP2A was made. Results: Lack of PP2A activity specific to the PP2A‐B56γ holoenzyme, resulted in the formation of an incomplete ventricular septum and a decrease in the number of ventricular cardiomyocytes. During cardiac development, B56γ is expressed in the nucleus of α‐actinin‐positive cardiomyocytes that contain Z‐bands. The pattern of B56γ expression correlated with the cardiomyocyte apoptosis we observed in B56γ‐deficient mice during mid to late gestation. In addition to the cardiac phenotypes, mice lacking B56γ have a decrease in locomotive coordination and gripping strength, indicating that B56γ has a role in controlling PP2A activity required for efficient neuromuscular function. Conclusions: PP2A‐B56γ activity is required for efficient cardiomyocyte maturation and survival. The PP2A B56γ regulatory subunit controls PP2A substrate specificity in vivo in a manner that cannot be fully compensated for by other B56 subunits. Developmental Dynamics 243:778–790, 2014.

Synopsis of the Food and Drug Administration-National Institute of Standards and Technology co-sponsored "In Vitro Analyses of Cell/Scaffold Products" Workshop.

Complex, dynamic mixtures of cells and structural components, known as cell=scaffold products, are in development as therapeutics for the repair, replacement, and regeneration of a wide variety of tissues damaged by acute, chronic, degenerative, or congenital diseases. These types of tissue-engineered products hold the potential to treat many diseases and injuries that currently do not have effective treatments. Cell=scaffold products are manufactured using complex regimens of cell expansion, materials processing, and cell–biomaterial integration. A critical step toward commercial availability of cell=scaffold-based therapeutics is the establishment of methods to produce a product that can be manufactured in a consistent and reliable manner. To effectively treat patients’ needs, manufacturing processes must be designed to achieve desired, pre-defined product criteria and characteristics. Product inconsistency may also contribute to clinical studies yielding ambiguous data and hinder market approval by the Food and Drug Administration (FDA). Manufacturing processes will vary between different types of cell=scaffold products, but there may be commonalities in the types of product testing used to evaluate product consistency, safety, and potency. Product developers and the FDA face technological challenges in evaluation of cell=scaffold constructs because these products consist of multiple components that must function together to form the final product. To generate discussion on cell=scaffold product evaluation and to promote the initiation of clinical studies and progression to marketing approval of these products, the FDA and the National Institutes of Standards and Technology (NIST) convened a workshop titled ‘‘In Vitro Analyses of Cell=Scaffold Products’’ (Washington, DC, December 2007). This workshop was designed and organized to meet the following two goals. 1) Identify and discuss what questions should be asked and addressed when evaluating cell=scaffold products in preparation for clinical study. 2) Identify and discuss what test methods are available and what analytical methods and processes should be further researched, developed, and standardized to determine the safety, purity, potency, and consistency of cell=scaffold products.

Translation of Regenerative Medicine Products Into the Clinic in the United States: FDA Perspective

Abstract The field of regenerative medicine encompasses a breathtaking array of interdisciplinary scientific approaches with the promise of delivering future therapies to meet current unmet medical needs for patients. Increasingly more of these innovative products are being translated into human clinical trials in the United States, and general familiarity of the FDA is important to efficiently navigate the process. The basics of FDA history, organization, and processes are described herein for those new to clinical translation, with more detailed content added regarding approval pathways, regulations, guidances, and select special topics of relevance to regenerative medicine. In addition to the cumulative experience of previous products, the FDA regulatory approach to medical products evaluation includes an ongoing assessment of how the science of those products informs regulatory policy. FDA engages in ongoing dialogue with the scientific community and product sponsors to continue to develop science-based regulatory review policies that are robust and predictable in order to meet the needs of the challenging array of products that are on the horizon.

PP2A-B56γ is required for an efficient spindle assembly checkpoint

ABSTRACT The Spindle Assembly Checkpoint (SAC) is part of a complex feedback system designed to ensure that cells do not proceed through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. The formation of the kinetochore complex and the implementation of the SAC are regulated by multiple kinases and phosphatases. BubR1 is a phosphoprotein that is part of the Cdc20 containing mitotic checkpoint complex that inhibits the APC/C so that Cyclin B1 and Securin are not degraded, thus preventing cells going into anaphase. In this study, we found that PP2A in association with its B56γ regulatory subunit, are needed for the stability of BubR1 during nocodazole induced cell cycle arrest. In primary cells that lack B56γ, BubR1 is prematurely degraded and the cells proceed through mitosis. The reduced SAC efficiency results in cells with abnormal chromosomal segregation, a hallmark of transformed cells. Previous studies on PP2As role in the SAC and kinetochore formation were done using siRNAs to all 5 of the B56 family members. In our study we show that inactivation of only the PP2A-B56γ subunit can affect the efficiency of the SAC. We also provide data that show the intracellular locations of the B56 subunits varies between family members, which is consistent with the hypothesis that they are not completely functionally redundant.

Regulatory considerations of stem and progenitor cell-based products: US Food and Drug Administration

Abstract: This chapter focuses on US Food and Drug Administration (FDA) regulatory considerations for stem/progenitor cell-based products (S/PCPs) intended to treat, mitigate or cure disease. Information critical to the regulatory decision-making process for determining the safety and efficacy of investigational S/PCPs is described. Included are sections that cover cellular product manufacturing and characterization, preclinical testing and clinical trial design. Increased regulatory complexity associated with stem/progenitor cell-based combination products is also discussed.