Stephanie A. Connors

Bio: Stephanie A. Connors is an academic researcher from University of Pennsylvania. The author has contributed to research in topics: Gastrulation & Zebrafish. The author has an hindex of 8, co-authored 8 publications receiving 2209 citations.

Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway.

Abstract: In the accompanying report (Visconti, P.E., Bailey, J.L., Moore, G.D., Pan, D., Olds-Clarke, P. and Kopf, G.S. (1995) Development, 121, 1129–1137) we demonstrated that the tyrosine phosphorylation of a subset of mouse sperm proteins of M(r) 40,000-120,000 was correlated with the capacitation state of the sperm. The mechanism by which protein tyrosine phosphorylation is regulated in sperm during this process is the subject of this report. Cauda epididymal sperm, when incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin do not display the capacitation-associated increases in protein tyrosine phosphorylation of this subset of proteins. This NaHCO3, CaCl2 or bovine serum albumin requirement for protein tyrosine phosphorylation can be completely overcome by the addition of biologically active, but not inactive, cAMP analogues. Addition of the active cAMP analogues to sperm incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin overcomes the inability of these media to support capacitation, as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. The effects of the cAMP analogues to enhance protein tyrosine phosphorylation and to promote capacitation appears to be at the level of the cAMP-dependent protein kinase (PKA), since two specific inhibitors of this enzyme (H-89 and Rp-cAMPS) block the capacitation-dependent increases in protein tyrosine phosphorylation in sperm incubated in media supporting capacitation. Capacitation, as assessed by the aforementioned endpoints, also appears to be inhibited by H-89 in a concentration-dependent manner. These results provide further evidence for the interrelationship between protein tyrosine phosphorylation and the appearance of the capacitated state in mouse sperm. They also demonstrate that both protein tyrosine phosphorylation and capacitation appear to be regulated by cAMP/PKA. Up-regulation of protein tyrosine phosphorylation by cAMP/PKA in sperm is, to our knowledge, the first demonstration of such an interrelationship between tyrosine kinase/phosphatase and PKA signaling pathways.

Equivalent genetic roles for bmp7/snailhouse and bmp2b/swirl in dorsoventral pattern formation

Abstract: A bone morphogenetic protein (BMP) signaling pathway acts in the establishment of the dorsoventral axis of the vertebrate embryo. Here we demonstrate the genetic requirement for two different Bmp ligand subclass genes for dorsoventral pattern formation of the zebrafish embryo. From the relative efficiencies observed in Bmp ligand rescue experiments, conserved chromosomal synteny, and isolation of the zebrafish bmp7 gene, we determined that the strongly dorsalized snailhouse mutant phenotype is caused by a mutation in the bmp7 gene. We show that the original snailhouse allele is a hypomorphic mutation and we identify a snailhouse/bmp7 null mutant. We demonstrate that the snailhouse/bmp7 null mutant phenotype is identical to the presumptive null mutant phenotype of the strongest dorsalized zebrafish mutant swirl/bmp2b, revealing equivalent genetic roles for these two Bmp ligands. Double mutant snailhouse/bmp7; swirl/bmp2b embryos do not exhibit additional or stronger dorsalized phenotypes, indicating that these Bmp ligands do not function redundantly in early embryonic development. Furthermore, overexpression experiments reveal that Bmp2b and Bmp7 synergize in the ventralization of wild-type embryos through a cell-autonomous mechanism, suggesting that Bmp2b/Bmp7 heterodimers may act in vivo to specify ventral cell fates in the zebrafish embryo.

Dorsal and intermediate neuronal cell types of the spinal cord are established by a BMP signaling pathway

Abstract: We have studied the role of Bmp signaling in patterning neural tissue through the use of mutants in the zebrafish that disrupt three different components of a Bmp signaling pathway: swirl/bmp2b, snailhouse/bmp7 and somitabun/smad5. We demonstrate that Bmp signaling is essential for the establishment of the prospective neural crest and dorsal sensory Rohon-Beard neurons of the spinal cord. Moreover, Bmp signaling is necessary to limit the number of intermediate-positioned lim1+ interneurons of the spinal cord, as observed by the dramatic expansion of these prospective interneurons in many mutant embryos. Our analysis also suggests a positive role for Bmp signaling in the specification of these interneurons, which is independent of Bmp2b/Swirl activity. We found that a presumptive ventral signal, Hh signaling, acts to restrict the amount of dorsal sensory neurons and trunk neural crest. This restriction appears to occur very early in neural tissue development, likely prior to notochord or floor plate formation. A similar early role for Bmp signaling is suggested in the specification of dorsal neural cell types, since the bmp2b/swirl and bmp7/snailhouse genes are only coexpressed during gastrulation and within the tail bud, and are not found in the dorsal neural tube or overlying epidermal ectoderm. Thus, a gastrula Bmp2b/Swirl and Bmp7/Snailhouse-dependent activity gradient may not only act in the specification of the embryonic dorsoventral axis, but may also function in establishing dorsal and intermediate neuronal cell types of the spinal cord.

Neuronal specification in the spinal cord: inductive signals and transcriptional codes

Abstract: Neural circuits are assembled with remarkable precision during embryonic development, and the selectivity inherent in their formation helps to define the behavioural repertoire of the mature organism. In the vertebrate central nervous system, this developmental program begins with the differentiation of distinct classes of neurons from progenitor cells located at defined positions within the neural tube. The mechanisms that specify the identity of neural cells have been examined in many regions of the nervous system and reveal a high degree of conservation in the specification of cell fate by key signalling molecules.

The snail superfamily of zinc-finger transcription factors.

Abstract: The Snail superfamily of zinc-finger transcription factors is involved in processes that imply pronounced cell movements, both during embryonic development and in the acquisition of invasive and migratory properties during tumour progression. Different family members have also been implicated in the signalling cascade that confers left–right identity, as well as in the formation of appendages, neural differentiation, cell division and cell survival.

Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism

Abstract: Bone morphogenetic protein (BMP) signals coordinate developmental patterning and have essential physiological roles in mature organisms. Here we describe the first known small-molecule inhibitor of BMP signaling—dorsomorphin, which we identified in a screen for compounds that perturb dorsoventral axis formation in zebrafish. We found that dorsomorphin selectively inhibits the BMP type I receptors ALK2, ALK3 and ALK6 and thus blocks BMP-mediated SMAD1/5/8 phosphorylation, target gene transcription and osteogenic differentiation. Using dorsomorphin, we examined the role of BMP signaling in iron homeostasis. In vitro, dorsomorphin inhibited BMP-, hemojuvelin- and interleukin 6–stimulated expression of the systemic iron regulator hepcidin, which suggests that BMP receptors regulate hepcidin induction by all of these stimuli. In vivo, systemic challenge with iron rapidly induced SMAD1/5/8 phosphorylation and hepcidin expression in the liver, whereas treatment with dorsomorphin blocked SMAD1/5/8 phosphorylation, normalized hepcidin expression and increased serum iron levels. These findings suggest an essential physiological role for hepatic BMP signaling in iron-hepcidin homeostasis.

Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation.

Abstract: The molecular basis of mammalian sperm capacitation, defined functionally as those processes that confer on the sperm the acquisition of fertilization-competence either in vivo in the female reproductive tract or in vitro, is poorly understood. We demonstrate here that capacitation of caudal epididymal mouse sperm in vitro is accompanied by a time-dependent increase in the protein tyrosine phosphorylation of a subset of proteins of M(r) 40,000-120,000. Incubation of sperm in media devoid of bovine serum albumin, CaCl2 or NaHCO3, components which individually are required for capacitation, prevent the sperm from undergoing capacitation as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. In each of these cases the protein tyrosine phosphorylation of the subset of capacitation-associated proteins does not occur. Protein tyrosine phosphorylation of these particular proteins, as well as sperm capacitation, can be recovered in media devoid of each of these three constituents (bovine serum albumin, CaCl2 or NaHCO3) by adding back the appropriate component in a concentration-dependent manner. The requirement of NaHCO3 for these phosphorylations is not due to an alkalinization of intracellular sperm pH or to an increase in media pH. Caput epididymal sperm, which lack the ability to undergo capacitation in vitro, do not display this capacitation-dependent subset of tyrosine phosphorylated proteins in complete media even after extended incubation periods, and do not fertilize metaphase II-arrested eggs in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential Effects of Culture on Imprinted H19 Expression in the Preimplantation Mouse Embryo

Abstract: The H19 gene is imprinted with preferential expression from the maternal allele. The putative imprinting control region for this locus is hypermethylated on the repressed paternal allele. Although maternal-specific expression of H19 is observed in mouse blastocysts that develop in vivo, biallelic expression has been documented in embryos and embryonic stem cells experimentally manipulated by in vitro culture conditions. In this study the effect of culture on imprinted H19 expression and methylation was determined. After culture of 2-cell embryos to the blastocyst stage in Whitten’s medium, the normally silent paternal H19 allele was aberrantly expressed, whereas little paternal expression was observed following culture in KSOM containing amino acids (KSOM1AA). Analysis of the methylation status of a CpG dinucleotide located in the upstream imprinting control region revealed a loss in methylation in embryos cultured in Whitten’s medium but not in embryos cultured in KSOM1AA. Thus, H19 expression and methylation were adversely affected by culture in Whitten’s medium, while the response of H19 to culture in KSOM1AA approximated more closely the in vivo situation. It is unlikely that biallelic expression of H19 following culture in Whitten’s medium is a generalized effect of lower methylation levels, since the amount of DNA methyltransferase activity and the spatial distribution of Dnmt1 protein were similar in in vivo-derived and cultured embryos. Moreover, imprinted expression of Snrpn was maintained following culture in either medium, indicating that not all imprinted genes are under the same stringent imprinting controls. The finding that culture conditions can dramatically, but selectively, affect the expression of imprinted genes provides a model system for further study of the linkage between DNA methylation and gene expression.