Zebrafish kit mutation reveals primary and secondary regulation of melanocyte development during fin stripe regeneration

Fin regeneration in adult zebrafish is accompanied by re-establishment of the pigment stripes. To understand the mechanisms underlying fin stripe regeneration and regulation of normal melanocyte stripe morphology, we investigated the origins of melanocytes in the regenerating fin and their requirement for the kit receptor tyrosine kinase. Using pre-existing melanin as a lineage tracer, we show that most fin regeneration melanocytes develop from undifferentiated precursors, rather than from differentiated melanocytes. Mutational analysis reveals two distinct classes of regeneration melanocytes. First, an early regeneration class develops dependent on kit function. In the absence of kit function and kit-dependent melanocytes, a second class of melanocytes develops at later stages of regeneration. This late kit-independent class of regeneration melanocytes has little or no role in wild-type fin stripe development, thus revealing a secondary mode for regulation of fin stripes. Expression of melanocyte markers in regenerating kit mutant fins suggests that kit normally acts after mitf and before dct to promote development of the primary kit-dependent melanocytes. kit-dependent and kit-independent melanocytes are also present during fin stripe ontogeny in patterns similar to those observed during regeneration.

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Requirements for the kit receptor tyrosine kinase during regeneration of zebrafish fin melanocytes

Development ◽

10.1242/dev.128.11.1943 ◽

2001 ◽

Vol 128 (11) ◽

pp. 1943-1949 ◽

Cited By ~ 6

Author(s):

John F. Rawls ◽

Stephen L. Johnson

Keyword(s):

Stem Cell ◽

Embryonic Development ◽

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Cell Populations ◽

Temperature Sensitive ◽

Kit Mutation ◽

Fin Regeneration ◽

Kit Receptor ◽

Stem Cell Populations

Embryonic neural crest-derived melanocytes and their precursors express the kit receptor tyrosine kinase and require its function for their migration and survival. However, mutations in kit also cause deficits in melanocytes that make up adult pigment patterns, including melanocytes that re-establish the zebrafish fin stripes during regeneration. As adult melanocytes in mice and zebrafish are generated and maintained by stem cell populations that are presumably established during embryonic development, it has been proposed that adult phenotypes in kit mutants result from embryonic requirements for kit. We have used a temperature-sensitive zebrafish kit mutation to show that kit is required during adult fin regeneration to promote melanocyte differentiation, rather than during embryonic stages to establish their stem cell precursors. We also demonstrate a transient role for kit in promoting the survival of newly differentiated regeneration melanocytes.

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PKCδ plays opposite roles in growth mediated by wild-type Kit and an oncogenic Kit mutant

Blood ◽

10.1182/blood-2004-04-1450 ◽

2005 ◽

Vol 105 (5) ◽

pp. 1923-1929 ◽

Cited By ~ 13

Author(s):

Tanya Jelacic ◽

Diana Linnekin

Keyword(s):

Receptor Tyrosine Kinase ◽

Catalytic Domain ◽

Dominant Negative ◽

Wild Type ◽

Aspartic Acid Residue ◽

Kit Receptor ◽

Murine Mast Cell ◽

Oncogenic Form ◽

In Cells

AbstractThe Kit receptor tyrosine kinase is critical for normal hematopoiesis. Mutation of the aspartic acid residue encoded by codon 816 of human c-kit or codon 814 of the murine gene results in an oncogenic form of Kit. Here we investigate the role of protein kinase Cδ (PKCδ) in responses mediated by wild-type murine Kit and the D814Y mutant in a murine mast cell-like line. PKCδ is activated after wild-type (WT) Kit binds stem cell factor (SCF), is constitutively active in cells expressing the Kit catalytic domain mutant, and coprecipitates with both forms of Kit. Inhibition of PKCδ had opposite effects on growth mediated by wild-type and mutant Kit. Both rottlerin and a dominant-negative PKCδ construct inhibited the growth of cells expressing mutant Kit, while SCF-induced growth of cells expressing wild-type Kit was not inhibited. Further, overexpression of PKCδ inhibited growth of cells expressing wild-type Kit and enhanced growth of cells expressing the Kit mutant. These data demonstrate that PKCδ contributes to factor-independent growth of cells expressing the D814Y mutant, but negatively regulates SCF-induced growth of cells expressing wild-type Kit. This is the first demonstration that PKCδ has different functions in cells expressing normal versus oncogenic forms of a receptor.

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Autoinhibition of the Kit Receptor Tyrosine Kinase by the Cytosolic Juxtamembrane Region

Molecular and Cellular Biology ◽

10.1128/mcb.23.9.3067-3078.2003 ◽

2003 ◽

Vol 23 (9) ◽

pp. 3067-3078 ◽

Cited By ~ 112

Author(s):

Perry M. Chan ◽

Subburaj Ilangumaran ◽

Jose La Rose ◽

Avijit Chakrabartty ◽

Robert Rottapel

Keyword(s):

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Regulatory Function ◽

Wild Type ◽

Juxtamembrane Domain ◽

Juxtamembrane Region ◽

Phosphorylated Form ◽

Oncogenic Mutations ◽

Kit Receptor ◽

Oncogenic Form

ABSTRACT Genetic studies have implicated the cytosolic juxtamembrane region of the Kit receptor tyrosine kinase as an autoinhibitory regulatory domain. Mutations in the juxtamembrane domain are associated with cancers, such as gastrointestinal stromal tumors and mastocytosis, and result in constitutive activation of Kit. Here we elucidate the biochemical mechanism of this regulation. A synthetic peptide encompassing the juxtamembrane region demonstrates cooperative thermal denaturation, suggesting that it folds as an autonomous domain. The juxtamembrane peptide directly interacted with the N-terminal ATP-binding lobe of the kinase domain. A mutation in the juxtamembrane region corresponding to an oncogenic form of Kit or a tyrosine-phosphorylated form of the juxtamembrane peptide disrupted the stability of this domain and its interaction with the N-terminal kinase lobe. Kinetic analysis of the Kit kinase harboring oncogenic mutations in the juxtamembrane region displayed faster activation times than the wild-type kinase. Addition of exogenous wild-type juxtamembrane peptide to active forms of Kit inhibited its kinase activity in trans, whereas the mutant peptide and a phosphorylated form of the wild-type peptide were less effective inhibitors. Lastly, expression of the Kit juxtamembrane peptide in cells which harbor an oncogenic form of Kit inhibited cell growth in a Kit-specific manner. Together, these results show the Kit kinase is autoinhibited through an intramolecular interaction with the juxtamembrane domain, and tyrosine phosphorylation and oncogenic mutations relieved the regulatory function of the juxtamembrane domain.

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Substrate phosphorylation specificity of the human c-kit receptor tyrosine kinase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)54869-9 ◽

1991 ◽

Vol 266 (30) ◽

pp. 19908-19916 ◽

Cited By ~ 2

Author(s):

R. Herbst ◽

R. Lammers ◽

J. Schlessinger ◽

A. Ullrich

Keyword(s):

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Kit Receptor ◽

Substrate Phosphorylation

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A proliferation gradient between proximal and msxb-expressing distal blastema directs zebrafish fin regeneration

Development ◽

10.1242/dev.129.11.2607 ◽

2002 ◽

Vol 129 (11) ◽

pp. 2607-2617 ◽

Cited By ~ 9

Author(s):

Alex Nechiporuk ◽

Mark T. Keating

Keyword(s):

High Rate ◽

Proliferating Cells ◽

Homogeneous Population ◽

Fin Regeneration ◽

New Model ◽

Blastema Formation ◽

Phosphohistone H3 ◽

Slow Cycling ◽

Early Regeneration ◽

Regeneration Blastema

Previous studies of zebrafish fin regeneration led to the notion that the regeneration blastema is a homogeneous population of proliferating cells. Here, we show that the blastema consists of two components with markedly distinct proliferation properties. During early blastema formation, proliferating cells are evenly distributed. At the onset of regenerative outgrowth, however, blastemal cells are partitioned into two domains. Proximal blastemal cells proliferate at a high rate, shifting from a median G2 of more than 6 hours to approximately 1 hour. By contrast, the most distal blastemal cells do not proliferate. There is a gradient of proliferation between these extremes. Using bromodeoxyuridine incorporation and anti-phosphohistone H3 labeling, we find a 50-fold difference in proliferation across the gradient that extends approximately 50 μm, or ten cell diameters. We show that during early regeneration, proliferating blastemal cells express msxb, a homeodomain transcriptional repressor. While msxb is widely expressed among proliferating cells during blastema formation, its expression becomes restricted to a small number of non-proliferating, distal blastemal cells during regenerative outgrowth. Bromodeoxyuridine pulse-chase experiments show that distal and proximal blastemal cells are formed from proliferating, msxb-positive blastemal cells, not from preexisting slow-cycling cells. These data support the idea that blastema formation results from dedifferentiation of intraray mesenchymal cells. Based on these findings, we propose a new model of zebrafish fin regeneration in which the function of non-proliferating, msxb-expressing, distal blastemal cells is to specify the boundary of proliferation and provide direction for regenerative outgrowth.

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An Allelic Series of Mutations in the Kit ligand Gene of Mice. I. Identification of Point Mutations in Seven Ethylnitrosourea-Induced KitlSteel Alleles

Genetics ◽

10.1093/genetics/162.1.331 ◽

2002 ◽

Vol 162 (1) ◽

pp. 331-340 ◽

Cited By ~ 1

Author(s):

S Rajaraman ◽

W S Davis ◽

A Mahakali-Zama ◽

H K Evans ◽

L B Russell ◽

...

Keyword(s):

Receptor Tyrosine Kinase ◽

Stem Cell Factor ◽

Genetic Resource ◽

Point Mutations ◽

Exon Skipping ◽

Missense Mutations ◽

Allelic Series ◽

Kit Ligand ◽

Kit Receptor ◽

Induced Mutants

Abstract An allelic series of mutations is an extremely valuable genetic resource for understanding gene function. Here we describe eight mutant alleles at the Steel (Sl) locus of mice that were induced with N-ethyl-N-nitrosourea (ENU). The product of the Sl locus is Kit ligand (or Kitl; also known as mast cell growth factor, stem cell factor, and Steel factor), which is a member of the helical cytokine superfamily and is the ligand for the Kit receptor tyrosine kinase. Seven of the eight ENU-induced KitlSl alleles, of which five cause missense mutations, one causes a nonsense mutation and exon skipping, and one affects a splice site, were found to contain point mutations in Kitl. Interestingly, each of the five missense mutations affects residues that are within, or very near, conserved α-helical domains of Kitl. These ENU-induced mutants should provide important information on structural requirements for function of Kitl and other helical cytokines.

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Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2523-2535.1988 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2523-2535

Author(s):

J H Hegemann ◽

J H Shero ◽

G Cottarel ◽

P Philippsen ◽

P Hieter

Keyword(s):

Saccharomyces Cerevisiae ◽

Mitotic Chromosome ◽

Mutational Analysis ◽

Point Mutations ◽

Vector System ◽

Chromosome Loss ◽

Wild Type ◽

Sequence Elements ◽

Chromosome Fragments

Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.

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Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris

Biological Procedures Online ◽

10.1186/s12575-020-00138-0 ◽

2020 ◽

Vol 22 (1) ◽

Author(s):

Roghayyeh Baghban ◽

Safar Farajnia ◽

Younes Ghasemi ◽

Reyhaneh Hoseinpoor ◽

Azam Safary ◽

...

Keyword(s):

Pichia Pastoris ◽

Proteolytic Activity ◽

Mutational Analysis ◽

Expression System ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Vitreomacular Adhesion ◽

Autolytic Activity

Abstract Background Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases. Results The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations. Conclusions The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

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Isolation and Characterization of Mutations in theEscherichia coli Regulatory Protein XapR

Journal of Bacteriology ◽

10.1128/jb.181.14.4397-4403.1999 ◽

1999 ◽

Vol 181 (14) ◽

pp. 4397-4403 ◽

Cited By ~ 22

Author(s):

Casper Jørgensen ◽

Gert Dandanell

Keyword(s):

Amino Acids ◽

Purine Nucleoside Phosphorylase ◽

Mutational Analysis ◽

Regulatory Protein ◽

Three Dimensional ◽

Dimensional Structure ◽

Wild Type ◽

Isolation And Characterization ◽

In The Wild

ABSTRACT In this work, the LysR-type protein XapR has been subjected to a mutational analysis. XapR regulates the expression of xanthosine phosphorylase (XapA), a purine nucleoside phosphorylase inEscherichia coli. In the wild type, full expression of XapA requires both a functional XapR protein and the inducer xanthosine. Here we show that deoxyinosine can also function as an inducer in the wild type, although not to the same extent as xanthosine. We have isolated and characterized in detail the mutants that can be induced by other nucleosides as well as xanthosine. Sequencing of the mutants has revealed that two regions in XapR are important for correct interactions between the inducer and XapR. One region is defined by amino acids 104 and 132, and the other region, containing most of the isolated mutations, is found between amino acids 203 and 210. These regions, when modelled into the three-dimensional structure of CysB from Klebsiella aerogenes, are placed close together and are most probably directly involved in binding the inducer xanthosine.

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