The Influence of Neural Tube-derived Factors on Differentiation of Neural Crest Cells In Vitro.I. Histochemical Study on the Appearance of Adrenergic Cells

The neural crest gives rise to numerous derivatives including adrenergic and cholinergic neurons, supportive cells of the nervous system, and melanocytes. In tissue culture, neural crest cells explanted from both cranial and trunk regions were found to differentiate into adrenergic neuroblasts or into pigmented cells when grown in medium containing 10% chick embryo extract. When the embryo extract concentration was lowered to 2%, no catecholamine-containing cells (as assayed by formaldehyde-induced fluorescence) were detected, although pigment cells were observed. These results suggest the presence of a factor(s) in embryo extract that promotes or supports adrenergic differentiation. To examine the possible tissue sources of this factor(s), neural tube, notochord, or somite cells were used to condition medium containing 2% embryo extract. When neural crest cells were grown in medium conditioned by neural tube cells, adrenergic neuroblasts were observed in all cultures. However, somite- and notochord conditioned medium did not support adrenergic differentiation. In addition, medium supplemented with extracts derived from central nervous system components did support adrenergic expression, whereas medium supplemented with embryo extract from which the neural tissue was removed did not. Direct contact with neural tube cell ghost membranes was unable to substitute for high embryo extract concentrations or for neural tube-conditioned medium. These results suggest that the neural tube makes a diffusible factor(s) that will support adrenergic differentiation of neural crest cells

Environmental Estrogens Alter Early Development in Xenopus laevis

A growing number of environmental toxicants found in pesticides, herbicides, and industrial solvents are believed to have deleterious effects on development by disrupting hormone-sensitive processes. We exposed Xenopus laevis embryos at early gastrula to the commonly encountered environmental estrogens nonylphenol, octylphenol, and methoxychlor, the antiandrogen, p,p-DDE, or the synthetic androgen, 17 alpha-methyltestosterone at concentrations ranging from 10 nM to 10 microM and examined them at tailbud stages (approximately 48 hr of treatment). Exposure to the three environmental estrogens, as well as to the natural estrogen 17 beta-estradiol, increased mortality, induced morphologic deformations, increased apoptosis, and altered the deposition and differentiation of neural crest-derived melanocytes in tailbud stage embryos. Although neural crest-derived melanocytes were markedly altered in embryos treated with estrogenic toxicants, expression of the early neural crest maker Xslug, a factor that regulates both the induction and subsequent migration of neural crest cells, was not affected, suggesting that the disruption induced by these compounds with respect to melanocyte development may occur at later stages of their differentiation. Co-incubation of embryos with the pure antiestrogen ICI 182,780 blocked the ability of nonylphenol to induce abnormalities in body shape and in melanocyte differentiation but did not block the effects of methoxychlor. Our data indicate not only that acute exposure to these environmental estrogens induces deleterious effects on early vertebrate development but also that different environmental estrogens may alter the fate of a specific cell type via different mechanisms. Finally, our data suggest that the differentiation of neural crest-derived melanocytes may be particularly sensitive to the disruptive actions of these ubiquitous chemical contaminants

Loss of Hand2 in a population of Periostin lineage cells results in pronounced bradycardia and neonatal death

The Periostin Cre (Postn-Cre) lineage includes endocardial and neural crest derived mesenchymal cells of the cardiac cushions, neural crest-derived components of the sympathetic and enteric nervous systems, and cardiac fibroblasts. In this study, we use the Postn-Cre transgenic allele to conditionally ablate Hand2 (H2CKO). We find that Postn-Cre H2CKOs die shortly after birth despite a lack of obvious cardiac structural defects. To ascertain the cause of death, we performed a detailed comparison of the Postn-Cre lineage and Hand2 expression at mid and late stages of embryonic development. Gene expression analyses demonstrate that Postn-Cre ablates Hand2 from the adrenal medulla as well as the sphenopalatine ganglia of the head. In both cases, Hand2 loss-of-function dramatically reduces expression of Dopamine Beta Hydroxylase (Dbh), a gene encoding a crucial catecholaminergic biosynthetic enzyme. Expression of the genes Tyrosine Hydroxylase (Th) and Phenylethanolamine N-methyltransferase (Pnmt), which also encode essential catecholaminergic enzymes, were severely reduced in postnatal adrenal glands. Electrocardiograms demonstrate that 3-day postnatal Postn-Cre H2CKO pups exhibit sinus bradycardia. In conjunction with the aforementioned gene expression analyses, these results strongly suggest that the observed postnatal lethality occurs due to a catecholamine deficiency and subsequent heart failure

Generative modeling of the enteric nervous system employing point pattern analysis and graph construction

We describe a generative network model of the architecture of the enteric nervous system (ENS) in the colon employing data from images of human and mouse tissue samples obtained through confocal microscopy. Our models combine spatial point pattern analysis with graph generation to characterize the spatial and topological properties of the ganglia (clusters of neurons and glial cells), the inter-ganglionic connections, and the neuronal organization within the ganglia. We employ a hybrid hardcore-Strauss process for spatial patterns and a planar random graph generation for constructing the spatially embedded network. We show that our generative model may be helpful in both basic and translational studies, and it is sufficiently expressive to model the ENS architecture of individuals who vary in age and health status. Increased understanding of the ENS connectome will enable the use of neuromodulation strategies in treatment and clarify anatomic diagnostic criteria for people with bowel motility disorders.Comment: 17 pages, 5 figure

Targeted deletion of Hand2 in cardiac neural crest-derived cells influences cardiac gene expression and outflow tract development

AbstractThe basic helix–loop–helix DNA binding protein Hand2 has critical functions in cardiac development both in neural crest-derived and mesoderm-derived structures. Targeted deletion of Hand2 in the neural crest has allowed us to genetically dissect Hand2-dependent defects specifically in outflow tract and cardiac cushion independent of Hand2 functions in mesoderm-derived structures. Targeted deletion of Hand2 in the neural crest results in misalignment of the aortic arch arteries and outflow tract, contributing to development of double outlet right ventricle (DORV) and ventricular septal defects (VSD). These neural crest-derived developmental anomalies are associated with altered expression of Hand2-target genes we have identified by gene profiling. A number of Hand2 direct target genes have been identified using ChIP and ChIP-on-chip analyses. We have identified and validated a number of genes related to cell migration, proliferation/cell cycle and intracellular signaling whose expression is affected by Hand2 deletion in the neural crest and which are associated with development of VSD and DORV. Our data suggest that Hand2 is a multifunctional DNA binding protein affecting expression of target genes associated with a number of functional interactions in neural crest-derived cells required for proper patterning of the outflow tract, generation of the appropriate number of neural crest-derived cells for elongation of the conotruncus and cardiac cushion organization. Our genetic model has made it possible to investigate the molecular genetics of neural crest contributions to outflow tract morphogenesis and cell differentiation

P. aeruginosa SGNH Hydrolase-Like Proteins AlgJ and AlgX Have Similar Topology but Separate and Distinct Roles in Alginate Acetylation

The O-acetylation of polysaccharides is a common modification used by pathogenic organisms to protect against external forces. Pseudomonas aeruginosa secretes the anionic, O-acetylated exopolysaccharide alginate during chronic infection in the lungs of cystic fibrosis patients to form the major constituent of a protective biofilm matrix. Four proteins have been implicated in the O-acetylation of alginate, AlgIJF and AlgX. To probe the biological function of AlgJ, we determined its structure to 1.83 Å resolution. AlgJ is a SGNH hydrolase-like protein, which while structurally similar to the N-terminal domain of AlgX exhibits a distinctly different electrostatic surface potential. Consistent with other SGNH hydrolases, we identified a conserved catalytic triad composed of D190, H192 and S288 and demonstrated that AlgJ exhibits acetylesterase activity in vitro. Residues in the AlgJ signature motifs were found to form an extensive network of interactions that are critical for O-acetylation of alginate in vivo. Using two different electrospray ionization mass spectrometry (ESI-MS) assays we compared the abilities of AlgJ and AlgX to bind and acetylate alginate. Binding studies using defined length polymannuronic acid revealed that AlgJ exhibits either weak or no detectable polymer binding while AlgX binds polymannuronic acid specifically in a length-dependent manner. Additionally, AlgX was capable of utilizing the surrogate acetyl-donor 4-nitrophenyl acetate to catalyze the O-acetylation of polymannuronic acid. Our results, combined with previously published in vivo data, suggest that the annotated O-acetyltransferases AlgJ and AlgX have separate and distinct roles in O-acetylation. Our refined model for alginate acetylation places AlgX as the terminal acetlytransferase and provides a rationale for the variability in the number of proteins required for polysaccharide O-acetylation

Prisoners’ Families’ Research: Developments, Debates and Directions

After many years of relative obscurity, research on prisoners’ families has gained significant momentum. It has expanded from case-oriented descriptive analyses of family experiences to longitudinal studies of child and family development and even macro analyses of the effects on communities in societies of mass incarceration. Now the field engages multi-disciplinary and international interest although it arguably still remains on the periphery of mainstream criminological, psychological and sociological research agendas. This chapter discusses developments in prisoners’ families’ research and its positioning in academia and practice. It does not aim to provide an all-encompassing review of the literature rather it will offer some reflections on how and why the field has developed as it has and on its future directions. The chapter is divided into three parts. The first discusses reasons for the historically small body of research on prisoners’ families and for the growth in research interest over the past two decades. The second analyses patterns and shifts in the focus of research studies and considers how the field has been shaped by intersecting disciplinary interests of psychology, sociology, criminology and socio-legal studies. The final part reflects on substantive and ethical issues that are likely to shape the direction of prisoners’ families’ research in the future

Mechanisms and perspectives on differentiation of autonomic neurons

AbstractNeurons share many features in common but are distinguished by expression of phenotypic characteristics that define their specific function, location, or connectivity. One aspect of neuronal fate determination that has been extensively studied is that of neurotransmitter choice. The generation of diversity of neuronal subtypes within the developing nervous system involves integration of extrinsic and intrinsic instructive cues resulting in the expression of a core set of regulatory molecules. This review focuses on mechanisms of growth and transcription factor regulation in the generation of peripheral neural crest-derived neurons. Although the specification and differentiation of noradrenergic neurons are the focus, I have tried to integrate these into a larger picture providing a general roadmap for development of autonomic neurons. There is a core of DNA binding proteins required for the development of sympathetic, parasympathetic, and enteric neurons, including Phox2 and MASH1, whose specificity is regulated by the recruitment of additional transcriptional regulators in a subtype-specific manner. For noradrenergic neurons, the basic helix-loop-helix DNA binding protein HAND2 (dHAND) appears to serve this function. The studies reviewed here support the notion that neurotransmitter identity is closely linked to other aspects of neurogenesis and reveal a molecular mechanism to coordinate expression of pan-neuronal genes with cell type-specific genes

Neural Tube-Derived Factors Influence Differentiation of Neural Crest Cells in Vitro: Effects on Activity of Neurotransmitter Biosynthetic Enzymes

Previously, we have demonstrated that a factor present in chick embryo extract or medium conditioned by neural tube cells supports adrenergic differentiation of some neural crest cells in vitro. These studies have been extended here to examine the effects of this factor(s) on the development of enzymes involved in neurotransmitter biosynthesis. The time course of expression of choline acetyltransferase (ChAT), a marker for cholinergic cells, and dopamine-β-hydroxylase (DBH), a marker for adrenergic cells, was examined in neural crest cell cultures grown under three conditions: (1) in medium containing 10% embryo extract, (2) in medium containing 2% embryo extract, and (3) in medium containing 2% embryo extract that was conditioned by neural tube cells (NTCM). Significant levels of DBH activity were measured in neural crest cell cultures grown in 10% embryo extract containing medium or in NTCM, while only low levels were present in cultures grown in medium containing 2% embryo extract. In contrast, ChAT activity was inhibited by NTCM in comparison to levels in both 10 and 2% embryo extract containing medium. As a preliminary characterization of the factor(s) present in chick embryo extract, we have fractionated embryo extract and find that a pool of 10 kDa or less can support adrenergic differentiation of some neural crest cells. These results suggest that low molecular weight factors present in embryo extract and NTCM support adrenergic expression of neural crest cells, whereas NTCM suppresses cholinergic expression