Biomedical subjects

S L Amacher

Publications and source records attributed to S L Amacher.

11 recordsLinked to original sources

PubMed2001

Zebrafish deadly seven functions in neurogenesis.

In a genetic screen, we isolated a mutation that perturbed motor axon outgrowth, neurogenesis, and somitogenesis. Complementation tests revealed that this mutation is an allele of deadly seven (des). By creating genetic mosaics, we demonstrate that the motor axon defect is non-cell autonomous. In addition, we show that the pattern of migration for some neural crest cell populations is aberrant and crest-derived dorsal root ganglion neurons are misplaced. Furthermore, our analysis reveals that des mutant embryos exhibit a neurogenic phenotype. We find an increase in the number of primary motoneurons and in the number of three hindbrain reticulospinal neurons: Mauthner cells, RoL2 cells, and MiD3cm cells. We also find that the number of Rohon-Beard sensory neurons is decreased whereas neural crest-derived dorsal root ganglion neurons are increased in number supporting a previous hypothesis that Rohon-Beard neurons and neural crest form an equivalence group during development. Mutations in genes involved in Notch-Delta signaling result in defects in somitogenesis and neurogenesis. We found that overexpressing an activated form of Notch decreased the number of Mauthner cells in des mutants indicating that des functions via the Notch-Delta signaling pathway to control the production of specific cell types within the central and peripheral nervous systems.

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PubMed1999

Transcriptional regulation during zebrafish embryogenesis.

Many zebrafish transcription factors have been isolated, and the challenge at present is to uncover the genes and pathways they regulate. The wealth of developmental mutants available for study and recent advances in zebrafish transgenic technology have allowed identification of putative transcriptional regulatory pathways, as well as characterization of promoter interactions at a molecular level.

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PubMed1998

Molecular identification of spadetail: regulation of zebrafish trunk and tail mesoderm formation by T-box genes.

Inhibition of fibroblast growth factor (FGF) signaling prevents trunk and tail formation in Xenopus and zebrafish embryos. While the T-box transcription factor Brachyury (called No Tail in zebrafish) is a key mediator of FGF signaling in the notochord and tail, the pathways activated by FGF in non-notochordal trunk mesoderm have been uncertain. Previous studies have shown that the spadetail gene is required for non-notochordal trunk mesoderm formation; spadetail mutant embryos have major trunk mesoderm deficiencies, but relatively normal tail and notochord development. We demonstrate here that spadetail encodes a T-box transcription factor with homologues in Xenopus and chick. Spadetail is likely to be a key mediator of FGF signaling in trunk non-notochordal mesoderm, since spadetail expression is regulated by FGF signaling. Trunk and tail development are therefore dependent upon the complementary actions of two T-box genes, spadetail and no tail. We show that the regulatory hierarchy among spadetail, no tail and a third T-box gene, tbx6, are substantially different during trunk and tail mesoderm formation, and propose a genetic model that accounts for the regional phenotypes of spadetail and no tail mutants.

Amino Acid Sequence↗

PubMed1998

Zebrafish paraxial protocadherin is a downstream target of spadetail involved in morphogenesis of gastrula mesoderm.

Zebrafish paraxial protocadherin (papc) encodes a transmembrane cell adhesion molecule (PAPC) expressed in trunk mesoderm undergoing morphogenesis. Microinjection studies with a dominant-negative secreted construct suggest that papc is required for proper dorsal convergence movements during gastrulation. Genetic studies show that papc is a close downstream target of spadetail, gene encoding a transcription factor required for mesodermal morphogenetic movements. Further, we show that the floating head homeobox gene is required in axial mesoderm to repress the expression of both spadetail and papc, promoting notochord and blocking differentiation of paraxial mesoderm. The PAPC structural cell-surface protein may provide a link between regulatory transcription factors and the actual cell biological behaviors that execute morphogenesis during gastrulation.

Amino Acid Sequence↗

PubMed1998

Promoting notochord fate and repressing muscle development in zebrafish axial mesoderm.

Cell fate decisions in early embryonic cells are controlled by interactions among developmental regulatory genes. Zebrafish floating head mutants lack a notochord; instead, muscle forms under the neural tube. As shown previously, axial mesoderm in floating head mutant gastrulae fails to maintain expression of notochord genes and instead expresses muscle genes. Zebrafish spadetail mutant gastrulae have a nearly opposite phenotype; notochord markers are expressed in a wider domain than in wild-type embryos and muscle marker expression is absent. We examined whether these two phenotypes revealed an antagonistic genetic interaction by constructing the double mutant. Muscle does not form in the spadetail;floating head double mutant midline, indicating that spadetail function is required for floating head mutant axial mesoderm to transfate to muscle. Instead, the midline of spadetail;floating head double mutants is greatly restored compared to that of floating head mutants; the floor plate is almost complete and an anterior notochord develops. In addition, we find that floating head mutant cells can make both anterior and posterior notochord when transplanted into a wild-type host, showing that enviromental signals can override the predisposition of floating head mutant midline cells to make muscle. Taken together, these results suggest that repression of spadetail function by floating head is critical to promote notochord fate and prevent midline muscle development, and that cells can be recruited to the notochord by environmental signals.

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PubMed1997

Genetic interactions in zebrafish midline development.

Mutational analyses have shown that the genes no tail (ntl, Brachyury homolog), floating head (flh, a Not homeobox gene), and cyclops (cyc) play direct and essential roles in the development of midline structures in the zebrafish. In both ntl and flh mutants a notochord does not develop, and in cyc mutants the floor plate is nearly entirely missing. We made double mutants to learn how these genes might interact. Midline development is disrupted to a greater extent in cyc;flh double mutants than in either cyc or flh single mutants; their effects appear additive. Both the notochord and floor plate are completely lacking, and other phenotypic disturbances suggest that midline signaling functions are severely reduced. On the other hand, trunk midline defects in flh;ntl double mutants are not additive, but are most often similar to those in ntl single mutants. This finding reveals that loss of ntl function can suppress phenotypic defects due to mutation at flh, and we interpret it to mean that the wild-type allele of ntl (ntl+) functions upstream to flh in a regulatory hierarchy. Loss of function of ntl also strongly suppresses the floor plate deficiency in cyc mutants, for we found trunk floor plate to be present in cyc;ntl double mutants. From these findings we propose that ntl+ plays an early role in cell fate choice at the dorsal midline, mediated by the Ntl protein acting to antagonize floor plate development as well as to promote notochord development.

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PubMed1997

Loss of cerebum function ventralizes the zebrafish embryo.

Recent studies implicate ventrally derived signals, in addition to dorsal ones emanating from the organizer, in patterning the vertebrate gastrula. We have identified five overlapping deficiencies that uncover the zebrafish cerebum locus and dramatically alter dorsal-ventral polarity at gastrulation. Consistent with the properties of experimentally ventralized amphibian embryos, cerebum mutants exhibit reduced neurectodermal gene expression domains and an increase in derivatives of ventral mesoderm. Structures derived from paraxial and lateral mesoderm also are reduced; however, dorsal axial mesodermal derivatives, such as the hatching gland and notochord, are largely spared. The pleiotropic action of cerebum deficiencies, and the differential response of affected tissues, suggest that the cerebum gene may normally function as an inhibitor of ventralizing signals, a function previously ascribed to Noggin and Chordin in Xenopus. Analysis of the cerebum phenotype provides genetic evidence for the existence of ventralizing signals in the zebrafish gastrula and for antagonists of those signals.

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PubMed1993

A dosage-dependent suppressor of a temperature-sensitive calmodulin mutant encodes a protein related to the fork head family of DNA-binding proteins.

The cmd1-1 mutation of calmodulin causes temperature-sensitive growth in Saccharomyces cerevisiae. We have isolated a dosage-dependent suppressor of cmd1-1, designated HCM1. Twentyfold overexpression of HCM1 permits strains carrying cmd1-1 to grow at temperatures up to and including 34 degrees C but does not suppress the lethality of either cmd1-1 at higher temperatures or the deletion of CMD1. Thus, overexpression of HCM1 does not bypass the requirement for calmodulin but enhances the ability of the mutant calmodulin to function. HCM1 is not essential for growth, but deletion of HCM1 exacerbates the phenotype of a strain carrying cmd1-1. HCM1 is located on chromosome III, which was recently sequenced. Our results correct errors in the published DNA sequence. The putative polypeptide encoded by HCM1 is 564 amino acids long and has a predicted molecular weight of 63,622. Antisera prepared against Hcm1p detect a protein that is overproduced in yeast strains overexpressing HCM1 and has an apparent molecular mass of 65 kDa. Eighty-six amino acid residues in the N terminus of Hcm1p show 50% identity with a DNA-binding region of the fork head family of DNA-binding proteins. When fused to the DNA-binding domain of Gal4p, residues 139 to 511 of Hcm1p can act as a strong activator of transcription. However, overexpression of HCM1 does not affect the expression of calmodulin. Furthermore, Hcm1p does not bind to calmodulin in a gel overlay assay. Thus, overexpression of HCM1 enhances calmodulin function by an apparently indirect mechanism.

Amino Acid Sequence↗

PubMed1993

Multiple regulatory elements contribute differentially to muscle creatine kinase enhancer activity in skeletal and cardiac muscle.

We have used transient transfections in MM14 skeletal muscle cells, newborn rat primary ventricular myocardiocytes, and nonmuscle cells to characterize regulatory elements of the mouse muscle creatine kinase (MCK) gene. Deletion analysis of MCK 5'-flanking sequence reveals a striated muscle-specific, positive regulatory region between -1256 and -1020. A 206-bp fragment from this region acts as a skeletal muscle enhancer and confers orientation-dependent activity in myocardiocytes. A 110-bp enhancer subfragment confers high-level expression in skeletal myocytes but is inactive in myocardiocytes, indicating that skeletal and cardiac muscle MCK regulatory sites are distinguishable. To further delineate muscle regulatory sequences, we tested six sites within the MCK enhancer for their functional importance. Mutations at five sites decrease expression in skeletal muscle, cardiac muscle, and nonmuscle cells. Mutations at two of these sites, Left E box and MEF2, cause similar decreases in all three cell types. Mutations at three sites have larger effects in muscle than nonmuscle cells; an A/T-rich site mutation has a pronounced effect in both striated muscle types, mutations at the MEF1 (Right E-box) site are relatively specific to expression in skeletal muscle, and mutations at the CArG site are relatively specific to expression in cardiac muscle. Changes at the AP2 site tend to increase expression in muscle cells but decrease it in nonmuscle cells. In contrast to reports involving cotransfection of 10T1/2 cells with plasmids expressing the myogenic determination factor MyoD, we show that the skeletal myocyte activity of multimerized MEF1 sites is 30-fold lower than that of the 206-bp enhancer. Thus, MyoD binding sites alone are not sufficient for high-level expression in skeletal myocytes containing endogenous levels of MyoD and other myogenic determination factors.

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PubMed1990

Thrombospondin gene expression is associated with mitogenesis in 3T3 cells: induction by basic fibroblast growth factor.

Growth factor-depleted Swiss 3T3 cells responded to basic fibroblast growth factor (bFGF) with a burst of mitogenesis and with a rapid and marked increase in thrombospondin (TS) mRNA levels. mRNA levels for the alpha 1 chain of type I collagen and for fibronectin were unaffected. At early times following stimulation (0-2 h), "superinduction" of TS mRNA by inhibition of protein synthesis with cycloheximide was not observed, and the increase in TS mRNA could be attributed primarily to an increase in transcription rate of the TS gene. However, at later times (4-8 h) the combination of cycloheximide and bFGF superinduced TS mRNA levels, suggesting the existence of a labile inhibitor of transcription or a short-lived RNase that might be produced in response to prolonged treatment with bFGF. In contrast to its stimulatory effect on 3T3 cells, bFGF did not stimulate the proliferation of mouse muscle BC3H1 cells nor did it cause an increase in TS mRNA levels, but BC3H1 cells do respond to bFGF by inhibition of myogenic differentiation. We propose, on the basis of these and other findings, that TS facilitates the progression of some anchorage-dependent cells through the cell cycle.

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PubMed1989

Glucocorticoid-regulated and constitutive trafficking of proteolytically processed cell surface-associated glycoproteins in wild type and variant rat hepatoma cells.

Glucocorticoids regulate the trafficking of mouse mammary tumor virus (MMTV) glycoproteins to the cell surface in the rat hepatoma cell line M1.54, but not in the immunoselected sorting variant CR4. To compare the localization of MMTV glycoproteins to another proteolytically processed glycoprotein, both wild type M1.54 cells and variant CR4 cells were transfected with a human insulin receptor (hIR) expression vector, pRSVhIR. The production of cell surface hIR was monitored in dexamethasone-treated and -untreated wild type M1.54 and variant CR4 cells by indirect immunofluorescence, direct plasma membrane immunoprecipitation, and by [125I] insulin binding. In both wild type and variant rat hepatoma cells, hIR were localized at the cell surface in the presence or in the absence of 1 microM dexamethasone. In contrast, the glucocorticoid-regulated trafficking of cell surface MMTV glycoproteins occurred only in wild type M1.54 cells. We conclude that the hIR, which undergoes posttranslational processing reactions similar to MMTV glycoproteins, does not require glucocorticoids to be transported to the plasma membrane and is representative of a subset of cell surface glycoproteins whose trafficking is constitutive in rat hepatoma cells. Thus, MMTV glycoproteins and hIR provide specific cell surface markers to characterize the glucocorticoid-regulated and constitutive sorting pathways.

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