2001 Warkany lecture: to die or not to die, the role of apoptosis in normal and abnormal mammalian development

Cell death is a common and reproducible feature of the development of many mammalian tissues/organs. Two well-known examples of programmed cell death (PCD) are the cell deaths associated with fusion of the neural folds and removal of interdigital mesenchymal cells during digit formation. Like normal development, abnormal development is also associated with increased cell death in tissues/organs that develop abnormally after exposure to a wide variety of teratogens. At least in some instances, teratogens induce cell death in areas of normal PCD, suggesting that there is a link between programmed and teratogen-induced cell death. Although researchers recognized early on that cell death is an integral part of both normal and abnormal development, little was known about the mechanisms of cell death. In 1972, Kerr et al. ('72) showed conclusively that cell deaths, induced in a variety of contexts, followed a reproducible pattern, which they termed apoptosis. The next breakthrough came in the 1980s when Horvitz and his colleagues identified specific cell death genes (ced) that controlled PCD in the roundworm, Caenorhabditis elegans (C. elegans). Identification of ced genes in the roundworm quickly led to the isolation of their mammalian homologues. Subsequent research in the 1990s led to the identification of a cadre of proteins controlling cell death in mammals, i.e., receptors/ligands, caspases, cytochrome c, Apaf-1, Bcl-2 family proteins, and IAPs. Two major pathways of apoptosis have now been elucidated, the receptor-mediated and the mitochondrial apoptotic pathways. The latter pathway, induced by a wide variety of toxic agents, is activated by the release of cytochrome c from mitochondria. Cytochrome c then facilitates the activation of a caspase cascade involving caspase-9 and -3. Activation of these caspases results in the cleavage of a variety of cellular proteins leading to the orderly demise of the cell. Work from my laboratory in the last 5 years has shown that teratogens, such as hyperthermia, 4-hydroperoxycyclophosphamide, and staurosporine, induce cell death in day 9 mouse embryos by activating the mitochondrial apoptotic pathway, i.e., mitochondrial release of cytochrome c, activation of caspase-9 and -3, inactivation of poly (ADP-ribose) polymerase (PARP), and systematic degradation of DNA. Our work, as well as the work of others, has also shown that different tissues within the early post implantation mammalian embryo are differentially sensitive to the cell death inducing potential of teratogens, from exquisite sensitivity of cells in the developing central nervous system to complete resistance of cells in the developing heart. More importantly, we have shown that the resistance of heart cells is directly related to the failure to activate the mitochondrial apoptotic pathway in these cells. Thus, whether a cell dies in response to a teratogen and therefore contributes to the pathogenesis culminating in birth defects, depends, at least in part, by the cell's ability to regulate the mitochondrial apoptotic pathway. Future research aimed at understanding this regulation should provide insight not only into the mechanism of teratogen-induced cell death but also the role of cell death in the genesis of birth defects.     

Computer simulation of acidosis-induced abnormal repolarization and repetitive activity in dog Purkinje fibers

Equations described by Mc Allister et al. (1975) were used to simulate the abnormal repolarization phase of the action potential (early after depolarization or hump), which often occurs in isolated dog Purkinje fibers submitted to acid media and can result in re-excitation. The development of humps was simulated by modifying the īK2-Em relationship in such a way that the resulting repolarizing current was increased at low membrane potential and decreased at higher (more negative) membrane potential. Beyond a certain size, humps triggered single, or multiple re-excitations. Hump size was very sensitive to small changes in īK2 and iK1, moderately sensitive to changes in īx1 and almost insensitive to changes in ix2. Negative shifts in the s infinity-Em relationship induced slowing of the basic frequency and only a small decrease in hump size. Decreasing gsi and gNa exerted inhibitory effects on hump development, the latter effect being larger than the former. The steady-state inward sodium current resulting from the overlap of m infinity-Em and h infinity-Em relationships appeared of particular importance in the development of humps.     

Fixed cervical count and the origin of the mammalian diaphragm

Why is mammalian cervical count fixed across the historically long and ecologically broad mammalian radiation? Multiple lines of evidence, considered together, suggest a link between fixed cervical count and the muscularization of the diaphragm, a key innovation in mammalian history. We test this hypothesis by documenting the anteroposterior (AP) movement of the diaphragm, a lateral plate derivative, relative to that of the somitic thoracolumbar transition in unusually patterned mammals, by comparing the temporal occurrence of an osteological proxy for the diaphragm and fixed cervical counts in the fossil record, and by quantifying morphological differentiation within the mammalian cervical series. We then integrate these anatomical observations with details of diaphragm function and development to propose a sequence of innovations in mammalian evolution that could have led to fixed cervical count. We argue that the novel commitment of migratory muscle precursor cells (MMPs) of mid‐cervical somites to a fate in the abaxial diaphragm defined these somites as a new and uniquely mammalian modular subunit. We further argue that the coordination of primaxial abaxial patterning constrained subsequent AP migration of the forelimb, thereby secondarily fixing cervical count.     

Clonal analysis of early mammalian development

Various extrinsic markers have been used to label single cells in the early mouse embryo. However, they are appropriate only for short-term experiments because of their susceptibility to dilution. Studies on cell lineage and commitments have therefore depended mainly on exploiting genes as markers by combining cells from embryos that differ in genotype at particular loci. Tissue recombination and transplantation experiments using such indelible intrinsic markers have enabled the fate of different cell populations in the blastocyst to be determined with reasonable precision. The trophectoderm and inner cell mass (i.c.m.) give rise to distinct complementary groups of tissues in the later conceptus, as do the primitive endodermal and primitive ectodermal components of the more mature i.c.m. When cloned by blastocyst injection, single i.c.m. cells colonize only those parts of host conceptuses that are derived from their tissue of origin. Thus, while clonal descendants of early i.c.m. cells can contribute to all tissues other than those of trophectodermal origin, primitive endodermal and primitive ectodermal clones are restricted, respectively, to the extraembryonic endoderm versus all i.c.m. derivatives except the extraembryonic endoderm. Interestingly, individual primitive ectoderm cells can include both germ cells and somatic cells among their mitotic descendants. By using the genetically determined presence versus absence of cytoplasmic malic enzyme activity as a cell marker, the deployment of clones has been made visible in situ in whole-mount preparations of extraembryonic membranes. Very little mixing of donor and host cells was seen in either the endoderm of the visceral yolk sac or the mesodermal and ectodermal layers of the amnion. In contrast, mosaicism in the parietal endoderm was so fine grained that, in all except 1 of 15 fields from several specimens that were analysed, the arrangement of donor and host cells did not differ significantly from that expected on the basis of their random association.     

Canonical analysis of cells in normal and abnormal cervical smears

Over 4,000 cells from 105 normal and 96 abnormal uterine cervical scrapes were prepared according to the UCLA monolayer procedure, stained by a routine Papanicolaou method and visually classified by two cytopathologists and a technologist into seven classes: parabasal, metaplastic, mild dysplasia, moderate dysplasia, severe dysplasia, carcinoma in situ and invasive carcinoma. Canonical analysis was used to correlate effects-coded class membership variables with 23 cell features derived from digital image analysis. In general, nuclear texture measures derived from linear combinations of run-length correlations along with features derived from a Markov transitional probability matrix provided the best predictors of cell class. After cells were divided into benign (moderate dysplasia or less) and malignant (severe dysplasia or worse) groups, discriminant analysis correctly classified 84% of the benign cells and 91% of the malignant cells.     

Using mouse models to understand normal and abnormal urogenital tract development

The central objective of diabetes research and management is to restore the deficient secretion of insulin, thereby restoring a state of euglycemia and minimizing short- and long-term risks associated with poor glucose control. The development of the artificial pancreas seeks to imitate the action of the pancreatic beta cell by employing closed-loop control to respond to glycemic excursions by appropriately infusing appropriate amounts of insulin. This article examines progress towards implementing an artificial pancreas in the context of the pancreatic islet as the ideal model for controlling blood glucose. Physiologic insulin secretion will form our foundation for considering the technical design elements relevant to electromechanically imitating the beta cell. The most recent clinical trials using closed-loop control are reviewed and this modality is compared to other curative approaches including islet cell transplantation and preservation. Finally, the potential of the artificial pancreas as a method to adequately reestablish euglycemia is considered.     

Embryonic stem cell differentiation and the analysis of mammalian development

Molecular and cellular analysis of early mammalian development is compromised by the experimental inaccessibility of the embryo. Pluripotent embryonic stem (ES) cells are derived from and retain many properties of the pluripotent founder population of the embryo, the inner cell mass. Experimental manipulation of these cells and their environment in vitro provides an opportunity for the development of differentiation systems which can be used for analysis of the molecular and cellular basis of embryogenesis. In this review we discuss strengths and weaknesses of the available ES cell differentiation methodologies and their relationship to events in vivo. Exploitation of these systems is providing novel insight into embryonic processes as diverse as cell lineage establishment, cell progression during differentiation, patterning, morphogenesis and the molecular basis for cell properties in the early mammalian embryo.     

Early development of the primordial mammalian diaphragm and cellular mechanisms of nitrofen‐induced congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) is a frequently occurring cause of neonatal respiratory distress and is associated with high mortality and long‐term morbidity. Evidence from animal models suggests that CDH has its origins in the malformation of the pleuroperitoneal fold (PPF), a key structure in embryonic diaphragm formation. The aims of this study were to characterize the embryogenesis of the PPF in rats and humans, and to determine the potential mechanism that leads to abnormal PPF development in the nitrofen model of CDH. Analysis of rat embryos, and archived human embryo sections, allowed the timeframe of PPF formation to be determined for both species, thus delineating a critical period of diaphragm development in relation to CDH. Experiments on nitrofen‐exposed NIH 3T3 cells in vitro led us to hypothesize that nitrofen might cause diaphragmatic hernia in vivo by two possible mechanisms: through decreased cell proliferation or by inducing apoptosis. Data from nitrofen‐exposed rat embryos indicates that the primary mechanism of nitrofen teratogenesis in the PPF is through decreased cell proliferation. This study provides novel insight into the embryogenesis of the PPF in rats and humans, and it indicates that impaired cell proliferation might contribute to abnormal diaphragm development in the nitrofen model of CDH. Birth Defects Research (Part A) 2010. © 2009 Wiley‐Liss, Inc.     

Computer simulation and analysis of the reaction pathway of triosephosphate isomerase

A theoretical approach designed for chemical reactions in the condensed phase is used to determine the energy along the reaction path of the enzyme triosephosphate isomerase. The calculations address the role of the enzyme in lowering the barrier to reaction and provide a decomposition into specific residue contributions. The results suggest that, although Lys-12 is most important, many other residues within 16 A of the substrate contribute and that histidine-95 as the imidazole/imidazolate pair could act as an acid/base catalyst.