scholarly journals Copb2 is essential for embryogenesis and hypomorphic mutations cause human microcephaly

2017 ◽  
Author(s):  
Andrew DiStasio ◽  
Ashley Driver ◽  
Kristen Sund ◽  
Milene Donlin ◽  
Ranjith M. Muraleedharan ◽  
...  
Keyword(s):  
Neural Development ◽  
Null Alleles ◽  
Recessive Mutation ◽  
Allelic Series ◽  
Primary Microcephaly ◽  
Congenital Brain Malformation ◽  
Wd40 Domain ◽  
Clinical Interest ◽  
Layer V ◽  
Potential Pathogenicity

AbstractPrimary microcephaly is a congenital brain malformation characterized by a head circumference less than three standard deviations below the mean for age and sex and results in moderate to severe mental deficiencies and decreased lifespan. We recently studied two children with primary microcephaly in an otherwise unaffected family. Exome sequencing identified an autosomal recessive mutation leading to an amino acid substitution in a WD40 domain of the highly conserved Coatomer Protein Complex, Subunit Beta 2 (COPB2). To study the role of Copb2 in neural development, we utilized genome editing technology to generate an allelic series in the mouse. Two independent null alleles revealed that Copb2 is essential for early stages of embryogenesis. Mice homozygous for the patient variant (Copb2R254C/R254C) appear to have a grossly normal phenotype, likely due to differences in corticogenesis between the two species. Strikingly, mice heterozygous for the patient mutation and a null allele (Copb2R254C/Znf) show a severe perinatal phenotype including low neonatal weight, significantly increased apoptosis in the brain, and death within the first week of life. Immunostaining of the Copb2R254C/Znf brain revealed a reduction in layer V (CTIP2+) neurons, while the overall cell density of the cortex is unchanged. Moreover, disruption of Copb2 in mouse neurospheres resulted in reduced proliferation. These results identify a general requirement for COPB2 in embryogenesis and a specific role in corticogenesis. We further demonstrate the utility of CRISPR-Cas9 generated mouse models in the study of potential pathogenicity of variants of potential clinical interest.

scholarly journals Molecular Characterization of Pax62Neu Through Pax610Neu: An Extension of the Pax6 Allelic Series and the Identification of Two Possible Hypomorph Alleles in the Mouse Mus musculus

Genetics ◽  
2001 ◽  
Vol 159 (4) ◽  
pp. 1689-1700
Author(s):  
Jack Favor ◽  
Heiko Peters ◽  
Thomas Hermann ◽  
Wolfgang Schmahl ◽  
Bimal Chatterjee ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Characterization ◽  
Amino Acid Substitution ◽  
Gene Dosage ◽  
Three Dimensional ◽  
Null Alleles ◽  
Target Sequence ◽  
Allelic Series ◽  
Phenotypic Analysis ◽  
Major Interest

Abstract Phenotype-based mutagenesis experiments will increase the mouse mutant resource, generating mutations at previously unmarked loci as well as extending the allelic series at known loci. Mapping, molecular characterization, and phenotypic analysis of nine independent Pax6 mutations of the mouse recovered in mutagenesis experiments is presented. Seven mutations result in premature termination of translation and all express phenotypes characteristic of null alleles, suggesting that Pax6 function requires all domains to be intact. Of major interest is the identification of two possible hypomorph mutations: Heterozygotes express less severe phenotypes and homozygotes develop rudimentary eyes and nasal processes and survive up to 36 hr after birth. Pax64Neu results in an amino acid substitution within the third helix of the homeodomain. Three-dimensional modeling indicates that the amino acid substitution interrupts the homeodomain recognition α-helix, which is critical for DNA binding. Whereas cooperative dimer binding of the mutant homeodomain to a paired-class DNA target sequence was eliminated, weak monomer binding was observed. Thus, a residual function of the mutated homeodomain may explain the hypomorphic nature of the Pax64Neu allele. Pax67Neu is a base pair substitution in the Kozak sequence and results in a reduced level of Pax6 translation product. The Pax64Neu and Pax67Neu alleles may be very useful for gene-dosage studies.

ISOLATION AND GENETIC CHARACTERIZATION OF CELL-LINEAGE MUTANTS OF THE NEMATODE CAENORHABDITIS ELEGANS

Genetics ◽  
1980 ◽  
Vol 96 (2) ◽  
pp. 435-454 ◽  
Author(s):  
H Robert Horvitz ◽  
John E Sulston
Keyword(s):  
Caenorhabditis Elegans ◽  
Cell Lineage ◽  
Embryonic Cell ◽  
Null Alleles ◽  
Recessive Mutation ◽  
Incomplete Penetrance ◽  
Nematode Caenorhabditis Elegans ◽  
Cell Lineages ◽  
Cell Divisions ◽  
Recessive Mutations

ABSTRACT Twenty-four mutants that alter the normally invariant post-embryonic cell lineages of the nematode Caenorhabditis elegans have been isolated and genetically characterized. In some of these mutants, cell divisions fail that occur in wild-type animals; in other mutants, cells divide that do not normally do so. The mutants differ in the specificities of their defects, so that it is possible to identify mutations that affect some cell lineages but not others. These mutants define 14 complementation groups, which have been mapped. The abnormal phenotype of most of the cell-lineage mutants results from a single recessive mutation; however, the excessive cell divisions characteristic of one strain, CB1322, require the presence of two unlinked recessive mutations. All 24 cell-lineage mutants display incomplete penetrance and/or variable expressivity. Three of the mutants are suppressed by pleiotropic suppressors believed to be specific for null alleles, suggesting that their phenotypes result from the complete absence of gene activity.

scholarly journals GENETIC ANALYSIS OF THREE DOMINANT FEMALE-STERILE MUTATIONS LOCATED ON THE X CHROMOSOME OF DROSOPHILA MELANOGASTER

Genetics ◽  
1983 ◽  
Vol 105 (2) ◽  
pp. 309-325
Author(s):  
D Busson ◽  
M Gans ◽  
K Komitopoulou ◽  
M Masson
Keyword(s):  
X Chromosome ◽  
Germ Line ◽  
Female Sterility ◽  
Recessive Mutation ◽  
Wild Type Allele ◽  
Wild Type ◽  
Allelic Series ◽  
Type Allele ◽  
Dominant Female ◽  
Female Germ Line

ABSTRACT Three dominant female-sterile mutations were isolated following ethyl methanesulfonate (EMS) mutagenesis. Females heterozygous for two of these mutations show atrophy of the ovaries and produce no eggs (ovoD  1) or few eggs (ovoD  2); females heterozygous for the third mutation, ovoD  3, lay flaccid eggs. All three mutations are germ line-dependent and map to the cytological region 4D-E on the X chromosome; they represent a single allelic series. Two doses of the wild-type allele restore fertility to females carrying ovoD  3 and ovoD  2, but females carrying ovoD  1 and three doses of the wild-type allele remain sterile. The three mutations are stable in males but are capable of reversion in females; reversion of the dominant mutations is accompanied by the appearance, in the same region, of a recessive mutation causing female sterility. We discuss the utility of these mutations as markers of clones induced in the female germ line by mitotic recombination as well as the nature of the mutations.

scholarly journals FlpStop, a tool for conditional gene control in Drosophila

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yvette E Fisher ◽  
Helen H Yang ◽  
Jesse Isaacman-Beck ◽  
Marjorie Xie ◽  
Daryl M Gohl ◽  
...  
Keyword(s):  
Neural Development ◽  
Gene Disruption ◽  
Neuronal Excitability ◽  
Null Alleles ◽  
Acid Decarboxylase ◽  
Specific Gene ◽  
Cell Type ◽  
Gabaergic Neurotransmission ◽  
Drosophila Research ◽  
Voltage Gated Ion Channels

Manipulating gene function cell type-specifically is a common experimental goal in Drosophila research and has been central to studies of neural development, circuit computation, and behavior. However, current cell type-specific gene disruption techniques in flies often reduce gene activity incompletely or rely on cell division. Here we describe FlpStop, a generalizable tool for conditional gene disruption and rescue in post-mitotic cells. In proof-of-principle experiments, we manipulated apterous, a regulator of wing development. Next, we produced conditional null alleles of Glutamic acid decarboxylase 1 (Gad1) and Resistant to dieldrin (Rdl), genes vital for GABAergic neurotransmission, as well as cacophony (cac) and paralytic (para), voltage-gated ion channels central to neuronal excitability. To demonstrate the utility of this approach, we manipulated cac in a specific visual interneuron type and discovered differential regulation of calcium signals across subcellular compartments. Thus, FlpStop will facilitate investigations into the interactions between genes, circuits, and computation.

scholarly journals Mutations in the bli-4 (I) locus of Caenorhabditis elegans disrupt both adult cuticle and early larval development.

Genetics ◽  
1991 ◽  
Vol 129 (1) ◽  
pp. 95-102 ◽  
Author(s):  
K Peters ◽  
J McDowall ◽  
A M Rose
Keyword(s):  
Caenorhabditis Elegans ◽  
Adult Stage ◽  
Null Alleles ◽  
Recessive Mutation ◽  
Complex Locus ◽  
In Trans ◽  
Essential Function ◽  
Early Larval Development ◽  
I Gene ◽  
New Alleles

Abstract The bli-4 (I) gene of Caenorhabditis elegans had been previously defined by a single recessive mutation, e937, which disrupts the structure of adult-stage cuticle causing the formation of fluid-filled separations of the cuticle layers, or blisters. We report the identification of 11 new alleles of bli-4, all early larval lethals, including an allele induced by transposon mutagenesis. Nine of the lethal alleles failed to complement the blistered phenotype of e937; two alleles, s90 and h754, complement e937. The complementing alleles arrested development somewhat later than the noncomplementing alleles, which blocked just prior to hatching. We conclude that bli-4 is a complex locus with an essential function late in embryogenesis. We investigated the blistered phenotype of e937 through interactions with other mutations that alter worm morphology or cuticle structure. Recessive and dominant epistasis of several dumpy mutations over the blistered phenotype was observed. Using two heterochronic mutations that alter the developmental stage at which adult cuticle is expressed, we observed that adult worms that lack an adult-stage cuticle could not express blisters. However, late larval worms that expressed the adult cuticle did not express blisters either. It seems likely that the presence of the adult cuticle is necessary, but not sufficient, for blister expression. Blistering resulting from e937 is more severe in trans to null alleles, indicating that e937 is hypomorphic. We postulate that the adult-specific blistering is due to an altered or reduced function of bli-4 gene product in the adult cuticle.(ABSTRACT TRUNCATED AT 250 WORDS)

Dosage requirement of Pitx2 for development of multiple organs

Development ◽  
1999 ◽  
Vol 126 (20) ◽  
pp. 4643-4651 ◽  
Author(s):  
P.J. Gage ◽  
H. Suh ◽  
S.A. Camper
Keyword(s):  
Heart Development ◽  
Threshold Level ◽  
The Body ◽  
Null Alleles ◽  
Allelic Series ◽  
Heart Tube ◽  
Rieger Syndrome ◽  
Multiple Organs ◽  
Abdominal Organs ◽  
Left Right Asymmetry

Pitx2 is a homeodomain transcription factor that is mutated in Rieger syndrome, a haploinsufficiency disorder affecting eyes and teeth. Pitx2 also has a postulated role in left-right axis determination. We assessed the requirements for Pitx2 directly by generating hypomorphic and null alleles. Heterozygotes for either allele have eye abnormalities consistent with Rieger syndrome. The ventral body wall fails to close in embryos homozygous for the null allele, leaving the heart and abdominal organs externalized and the body axis contorted. In homozygotes for either allele, the heart tube undergoes normal, rightward looping and the stomach is positioned normally. In contrast, homozygotes for both alleles exhibit right isomerization of the lungs. Thus, Pitx2 is required for left-right asymmetry of the lungs but not other organs. Homozygotes for either allele exhibit septal and valve defects, and null homozygotes have a single atrium proving that a threshold level of Pitx2 is required for normal heart development. Null homozygotes exhibit arrest of pituitary gland development at the committed Rathke pouch stage and eye defects including optic nerve coloboma and absence of ocular muscles. This allelic series establishes that Pitx2 is required for the development of mulitple organs in a dosage-sensitive manner.

Fine Structure of Platelet Interaction with a New Microcrystalline Collagen Preparation

1974 ◽  
Vol 32 ◽  
pp. 58-59
Author(s):  
W. H. Zucker ◽  
R. G. Mason
Keyword(s):  
Fine Structure ◽  
Platelet Aggregation ◽  
Hydrochloric Acid ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Hemostatic Agent ◽  
Native Collagen ◽  
Fibrillar Morphology ◽  
Clinical Interest ◽  
New Form

Platelet adhesion initiates platelet aggregation and is an important component of the hemostatic process. Since the development of a new form of collagen as a topical hemostatic agent is of both basic and clinical interest, an ultrastructural and hematologic study of the interaction of platelets with the microcrystalline collagen preparation was undertaken.In this study, whole blood anticoagulated with EDTA was used in order to inhibit aggregation and permit study of platelet adhesion to collagen as an isolated event. The microcrystalline collagen was prepared from bovine dermal corium; milling was with sharp blades. The preparation consists of partial hydrochloric acid amine collagen salts and retains much of the fibrillar morphology of native collagen.

The Ultrastructure of Myocardial Cells in Normal and Cardiac Lethal Mutant Mexican Axolotls, (Ambystoma Mexicanum)

1970 ◽  
Vol 28 ◽  
pp. 62-63
Author(s):  
Larry F. Lemanski ◽  
Eldridge M. Bertke ◽  
J. T. Justus
Keyword(s):  
Fine Structure ◽  
Heart Development ◽  
Osmium Tetroxide ◽  
Picric Acid ◽  
Ambystoma Mexicanum ◽  
Recessive Mutation ◽  
Acid Mixture ◽  
Myocardial Cells ◽  
Lethal Mutant ◽  
Mexican Axolotl

A recessive mutation has been recently described in the Mexican Axolotl, Ambystoma mexicanum; in which the heart forms structurally, but does not contract (Humphrey, 1968. Anat. Rec. 160:475). In this study, the fine structure of myocardial cells from normal (+/+; +/c) and cardiac lethal mutant (c/c) embryos at Harrison's stage 40 was compared. The hearts were fixed in a 0.1 M phosphate buffered formaldehyde-glutaraldehyde-picric acid-styphnic acid mixture and were post fixed in 0.1 M s-collidine buffered 1% osmium tetroxide. A detailed study of heart development in normal and mutant embryos from stages 25-46 will be described elsewhere.

Oligodendrocytes in Developing Hameter Cerebral Cortex

1976 ◽  
Vol 34 ◽  
pp. 126-127
Author(s):  
MB. Tank Buschmann
Keyword(s):  
Cerebral Cortex ◽  
Optic Nerve ◽  
Frontal Cortex ◽  
Osmium Tetroxide ◽  
Sodium Cacodylate Buffer ◽  
Sodium Cacodylate ◽  
Tissue Samples ◽  
Cacodylate Buffer ◽  
Layer V ◽  
Adult Hamster

Development of oligodendrocytes in rat corpus callosum was described as a sequential change in cytoplasmic density which progressed from light to medium to dark (1). In rat optic nerve, changes in cytoplasmic density were not observed, but significant changes in morphology occurred just prior to and during myelination (2). In our study, the ultrastructural development of oligodendrocytes was studied in newborn, 5-, 10-, 15-, 20-day and adult frontal cortex of the golden hamster (Mesocricetus auratus).Young and adult hamster brains were perfused with paraformaldehyde-glutaraldehyde in sodium cacodylate buffer at pH 7.3 according to the method of Peters (3). Tissue samples of layer V of the frontal cortex were post-fixed in 2% osmium tetroxide, dehydrated in acetone and embedded in Epon-Araldite resin.

The Pancreas: Current Contributions of Clinical Interest

1960 ◽  
Vol 39 (4) ◽  
pp. 469-496 ◽  
Author(s):  
Thomas A. Johnson ◽  
Martin H. Kalser
Keyword(s):  
Clinical Interest
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