Analysis of Combinatorial Loss-of-Function Mutants in the Arabidopsis Ethylene Receptors Reveals That the ers1 etr1 Double Mutant Has Severe Developmental Defects That Are EIN2 Dependent

Anne E. Hall; Anthony B. Bleecker

doi:10.1105/tpc.013060

mRNA Surveillance Complex PELOTA-HBS1 Regulates Phosphoinositide-Dependent Protein Kinase1 and Plant Growth

Plant Physiology ◽

10.1093/plphys/kiab199 ◽

2021 ◽

Author(s):

Wei Kong ◽

Shutang Tan ◽

Qing Zhao ◽

De-Li Lin ◽

Zhi-Hong Xu ◽

...

Keyword(s):

Quality Control ◽

Messenger Rna ◽

Loss Of Function ◽

Yeast Saccharomyces Cerevisiae ◽

Developmental Defects ◽

Mrna Surveillance ◽

Dna Insertion ◽

Dependent Protein ◽

Heterodimeric Complex ◽

Severe Growth

Abstract The quality control system for messenger RNA (mRNA) is fundamental for cellular activities in eukaryotes. To elucidate the molecular mechanism of 3’-Phosphoinositide-Dependent Protein Kinase1 (PDK1), a master regulator that is essential throughout eukaryotic growth and development, we employed a forward genetic approach to screen for suppressors of the loss-of-function T-DNA insertion double mutant pdk1.1 pdk1.2 in Arabidopsis thaliana. Notably, the severe growth attenuation of pdk1.1 pdk1.2 was rescued by sop21 (suppressor of pdk1.1 pdk1.2), which harbours a loss-of-function mutation in PELOTA1 (PEL1). PEL1 is a homologue of mammalian PELOTA and yeast (Saccharomyces cerevisiae) DOM34p, which each form a heterodimeric complex with the GTPase HBS1 (HSP70 SUBFAMILY B SUPPRESSOR1, also called SUPERKILLER PROTEIN7, SKI7), a protein that is responsible for ribosomal rescue and thereby assures the quality and fidelity of mRNA molecules during translation. Genetic analysis further revealed that a dysfunctional PEL1-HBS1 complex failed to degrade the T-DNA-disrupted PDK1 transcripts, which were truncated but functional, and thus rescued the growth and developmental defects of pdk1.1 pdk1.2. Our studies demonstrated the functionality of a homologous PELOTA-HBS1 complex and identified its essential regulatory role in plants, providing insights into the mechanism of mRNA quality control.

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Inner Ear and Muscle Developmental Defects in Smpx-Deficient Zebrafish Embryos

International Journal of Molecular Sciences ◽

10.3390/ijms22126497 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6497

Author(s):

Anna Ghilardi ◽

Alberto Diana ◽

Renato Bacchetta ◽

Nadia Santo ◽

Miriam Ascagni ◽

...

Keyword(s):

Hearing Loss ◽

Inner Ear ◽

Hair Cells ◽

Muscle Fibers ◽

Underlying Mechanism ◽

Loss Of Function ◽

Zebrafish Model ◽

Developmental Defects ◽

Inner Ear Development ◽

Syndromic Hearing Loss

The last decade has witnessed the identification of several families affected by hereditary non-syndromic hearing loss (NSHL) caused by mutations in the SMPX gene and the loss of function has been suggested as the underlying mechanism. In the attempt to confirm this hypothesis we generated an Smpx-deficient zebrafish model, pointing out its crucial role in proper inner ear development. Indeed, a marked decrease in the number of kinocilia together with structural alterations of the stereocilia and the kinocilium itself in the hair cells of the inner ear were observed. We also report the impairment of the mechanotransduction by the hair cells, making SMPX a potential key player in the construction of the machinery necessary for sound detection. This wealth of evidence provides the first possible explanation for hearing loss in SMPX-mutated patients. Additionally, we observed a clear muscular phenotype consisting of the defective organization and functioning of muscle fibers, strongly suggesting a potential role for the protein in the development of muscle fibers. This piece of evidence highlights the need for more in-depth analyses in search for possible correlations between SMPX mutations and muscular disorders in humans, thus potentially turning this non-syndromic hearing loss-associated gene into the genetic cause of dysfunctions characterized by more than one symptom, making SMPX a novel syndromic gene.

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Identification and validation of novel candidate risk genes in endocytic vesicular trafficking associated with esophageal atresia and tracheoesophageal fistulas

10.1101/2021.07.18.21260699 ◽

2021 ◽

Author(s):

Guojie Zhong ◽

Priyanka Ahimaz ◽

Nicole A. Edwards ◽

Jacob J. Hagen ◽

Christophe Faure ◽

...

Keyword(s):

Genetic Variants ◽

Congenital Anomalies ◽

De Novo ◽

Simulation Analysis ◽

Loss Of Function ◽

Developmental Defects ◽

Risk Genes ◽

Significant Burden ◽

Background Mutation Rate ◽

Complex Cases

Esophageal atresias/tracheoesophageal fistulas (EA/TEF) are rare congenital anomalies caused by aberrant development of the foregut. Previous studies indicate that rare or de novo genetic variants significantly contribute to EA/TEF risk, and most individuals with EA/TEF do not have pathogenic genetic variants in established risk genes. To identify novel genetic contributions to EA/TEF, we performed whole genome sequencing of 185 trios (probands and parents) with EA/TEF, including 59 isolated and 126 complex cases with additional congenital anomalies and/or neurodevelopmental disorders. There was a significant burden of protein altering de novo coding variants in complex cases (p=3.3e-4), especially in genes that are intolerant of loss of function variants in the population. We performed simulation analysis of pathway enrichment based on background mutation rate and identified a number of pathways related to endocytosis and intracellular trafficking that as a group have a significant burden of protein altering de novo variants. We assessed 18 variants for disease causality using CRISPR-Cas9 mutagenesis in Xenopus and confirmed 13 with tracheoesophageal phenotypes. Our results implicate disruption of endosome-mediated epithelial remodeling as a potential mechanism of foregut developmental defects. This research may have implications for the mechanisms of other rare congenital anomalies.

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Two homologous regulatory genes, lin-12 and glp-1, have overlapping functions

Development ◽

10.1242/dev.112.1.231 ◽

1991 ◽

Vol 112 (1) ◽

pp. 231-240 ◽

Cited By ~ 54

Author(s):

E.J. Lambie ◽

J. Kimble

Keyword(s):

Gene Expression ◽

Double Mutant ◽

Transmembrane Proteins ◽

Cell Interactions ◽

Loss Of Function ◽

Cell Fates ◽

Single Mutant ◽

C Elegans ◽

Homologous Genes ◽

Mutant Phenotypes

Two homologous genes, lin-12 and glp-1, encode transmembrane proteins required for regulatory cell interactions during C. elegans development. Based on their single mutant phenotypes, each gene has been thought to govern a distinct set of cell fates. We show here that lin-12 and glp-1 are functionally redundant during embryogenesis: Unlike either single mutant, the lin-12 glp-1 double mutant dies soon after hatching. Numerous cellular defects can be observed in these Lag (for lin-12 and glp-1) double mutants. Furthermore, we have identified two genes, lag-1 and lag-2, that appear to be required for both lin-12 and glp-1-mediated cell interactions. Strong loss-of-function lag mutants are phenotypically indistinguishable from the lin-12 glp-1 double; weak lag mutants have phenotypes typical of lin-12 and glp-1 single mutants. We speculate that the lin-12 and glp-1 proteins are biochemically interchangeable and that their divergent roles in development may rely largely on differences in gene expression.

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Age-dependent degeneration of an identified adult leg motor neuron in a Drosophila SOD1 model of ALS

Biology Open ◽

10.1242/bio.049692 ◽

2020 ◽

Vol 9 (10) ◽

pp. bio049692

Author(s):

Anthony Agudelo ◽

Victoria St. Amand ◽

Lindsey Grissom ◽

Danielle Lafond ◽

Toni Achilli ◽

...

Keyword(s):

Motor Neuron ◽

Gene Replacement ◽

Loss Of Function ◽

Developmental Defects ◽

Age Related ◽

Disease Mutations ◽

Discs Large ◽

Age Dependent ◽

Motor Neuron Loss ◽

Lateral Sclerosis

ABSTRACTMutations in superoxide dismutase 1 (SOD1) cause familial amyotrophic lateral sclerosis (ALS) in humans. ALS is a neurodegenerative disease characterized by progressive motor neuron loss leading to paralysis and inevitable death in affected individuals. Using a gene replacement strategy to introduce disease mutations into the orthologous Drosophila sod1 (dsod1) gene, here, we characterize changes at the neuromuscular junction using longer-lived dsod1 mutant adults. Homozygous dsod1H71Y/H71Y or dsod1null/null flies display progressive walking defects with paralysis of the third metathoracic leg. In dissected legs, we assessed age-dependent changes in a single identified motor neuron (MN-I2) innervating the tibia levitator muscle. At adult eclosion, MN-I2 of dsod1H71Y/H71Y or sod1null/null flies is patterned similar to wild-type flies indicating no readily apparent developmental defects. Over the course of 10 days post-eclosion, MN-I2 shows an overall reduction in arborization with bouton swelling and loss of the post-synaptic marker discs-large (dlg) in mutant dsod1 adults. In addition, increases in polyubiquitinated proteins correlate with the timing and extent of MN-I2 changes. Because similar phenotypes are observed between flies homozygous for either dsod1H71Y or dsod1null alleles, we conclude these NMJ changes are mainly associated with sod loss-of-function. Together these studies characterize age-related morphological and molecular changes associated with axonal retraction in a Drosophila model of ALS that recapitulate an important aspect of the human disease.This article has an associated First Person interview with the first author of the paper.

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Mechanistic Insights into Axenfeld–Rieger Syndrome from Zebrafish foxc1 and pitx2 Mutants

International Journal of Molecular Sciences ◽

10.3390/ijms221810001 ◽

2021 ◽

Vol 22 (18) ◽

pp. 10001

Author(s):

Curtis R. French

Keyword(s):

Transcription Factors ◽

Developmental Disorders ◽

Anterior Segment ◽

Therapeutic Interventions ◽

Loss Of Function ◽

Cardiovascular Malformations ◽

Developmental Defects ◽

Rieger Syndrome ◽

Function Models ◽

Shed Light

Axenfeld–Rieger syndrome (ARS) encompasses a group of developmental disorders that affect the anterior segment of the eye, as well as systemic developmental defects in some patients. Malformation of the ocular anterior segment often leads to secondary glaucoma, while some patients also present with cardiovascular malformations, craniofacial and dental abnormalities and additional periumbilical skin. Genes that encode two transcription factors, FOXC1 and PITX2, account for almost half of known cases, while the genetic lesions in the remaining cases remain unresolved. Given the genetic similarity between zebrafish and humans, as well as robust antisense inhibition and gene editing technologies available for use in these animals, loss of function zebrafish models for ARS have been created and shed light on the mechanism(s) whereby mutations in these two transcription factors cause such a wide array of developmental phenotypes. This review summarizes the published phenotypes in zebrafish foxc1 and pitx2 loss of function models and discusses possible mechanisms that may be used to target pharmaceutical development and therapeutic interventions.

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Transvection in the Drosophila Ultrabithorax gene: a Cbx1 mutant allele induces ectopic expression of a normal allele in trans.

Genetics ◽

10.1093/genetics/126.1.177 ◽

1990 ◽

Vol 126 (1) ◽

pp. 177-184 ◽

Cited By ~ 2

Author(s):

J E Castelli-Gair ◽

J L Micol ◽

A García-Bellido

Keyword(s):

Double Mutant ◽

Ectopic Expression ◽

Imaginal Discs ◽

Wing Disc ◽

Loss Of Function ◽

Normal Allele ◽

Single Mutant ◽

Adult Wing ◽

Disc Cells ◽

In Cis

Abstract In wild-type Drosophila melanogaster larvae, the Ultrabithorax (Ubx) gene is expressed in the haltere imaginal discs but not in the majority of cells of the wing imaginal discs. Ectopic expression of the Ubx gene in wing discs can be elicited by the presence of Contrabithorax (Cbx) gain-of-function alleles of the Ubx gene or by loss-of-function mutations in Polycomb (Pc) or in other trans-regulatory genes which behave as repressors of Ubx gene activity. Several Ubx loss-of-function alleles cause the absence of detectable Ubx proteins (UBX) or the presence of truncated UBX lacking the homeodomain. We have compared adult wing phenotypes with larval wing disc UBX patterns in genotypes involving double mutant chromosomes carrying in cis one of those Ubx mutations and the Cbx1 mutation. We show that such double mutant genes are (1) active in the same cells in which the single mutant Cbx1 is expressed, although they are unable to yield functional proteins, and (2) able to induce ectopic expression of a normal homologous Ubx allele in a part of the cells in which the single mutant Cbx1 is active. That induction is conditional upon pairing of the homologous chromosomes (the phenomenon known as transvection), and it is not mediated by UBX. Depletion of Pc gene products by Pc3 mutation strongly enhances the induction phenomenon, as shown by (1) the increase of the number of wing disc cells in which induction of the homologous allele is detectable, and (2) the induction of not only a paired normal allele but also an unpaired one.

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Isthmin1, a secreted signaling protein, acts downstream of diverse embryonic patterning centers in development

Cell and Tissue Research ◽

10.1007/s00441-020-03318-2 ◽

2020 ◽

Author(s):

Gokul Kesavan ◽

Florian Raible ◽

Mansi Gupta ◽

Anja Machate ◽

Dilara Yilmaz ◽

...

Keyword(s):

Single Molecule ◽

Gene Expression Pattern ◽

Correlation Spectroscopy ◽

Dependent Manner ◽

Embryonic Patterning ◽

Loss Of Function ◽

Concentration Dependent Manner ◽

Developmental Defects ◽

Extracellular Signals

AbstractExtracellular signals play essential roles during embryonic patterning by providing positional information in a concentration-dependent manner, and many such signals, like Wnt, fibroblast growth factor (FGF), Hedgehog (Hh), and retinoic acid, act by being secreted into the extracellular space, thereby triggering receptor-mediated responses in other cells. Isthmin1 (ism1) is a secreted protein whose gene expression pattern coincides with that of early dorsal determinants, nodal ligand genes like sqt and cyc, and with fgf8 during various phases of zebrafish development. Ism1 functions in early embryonic patterning and development are poorly understood; however, it has recently been shown to interact with nodal pathway genes to control organ asymmetry in chicken. Here, we show that misexpression of ism1 deletion constructs disrupts embryonic patterning in zebrafish and exhibits genetic interactions with both Fgf and nodal signaling. Unlike Fgf and nodal pathway mutants, CRISPR/Cas9-engineered ism1 mutants did not show obvious developmental defects. Further, in vivo single molecule fluorescence correlation spectroscopy (FCCS) showed that Ism1 diffuses freely in the extra-cellular space, with a diffusion coefficient similar to that of Fgf8a; however, our measurements do not support direct molecular interactions between Ism1 and either nodal ligands or Fgf8a in the developing zebrafish embryo. Together, data from gain- and loss-of-function experiments suggest that zebrafish Ism1 plays a complex role in regulating extracellular signals during early embryonic development.

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Efficient genome-wide first-generation phenotypic screening system in mice using thepiggyBactransposon

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1906354116 ◽

2019 ◽

Vol 116 (37) ◽

pp. 18507-18516 ◽

Cited By ~ 3

Author(s):

Hao Chang ◽

Yukun Pan ◽

Sean Landrette ◽

Sheng Ding ◽

Dong Yang ◽

...

Keyword(s):

First Generation ◽

Cranial Nerves ◽

Model Organisms ◽

Biological Traits ◽

Screening System ◽

Loss Of Function ◽

Developmental Defects ◽

Genome Wide ◽

A Genome ◽

Milk Ingestion

Genome-wide phenotypic screens provide an unbiased way to identify genes involved in particular biological traits, and have been widely used in lower model organisms. However, cost and time have limited the utility of such screens to address biological and disease questions in mammals. Here we report a highly efficientpiggyBac(PB) transposon-based first-generation (F1) dominant screening system in mice that enables an individual investigator to conduct a genome-wide phenotypic screen within a year with fewer than 300 cages. ThePBscreening system uses visually trackable transposons to induce both gain- and loss-of-function mutations and generates genome-wide distributed new insertions in more than 55% of F1 progeny. Using this system, we successfully conducted a pilot F1 screen and identified 5 growth retardation mutations. One of these mutants, a Six1/4PB/+mutant, revealed a role in milk intake behavior. The mutant animals exhibit abnormalities in nipple recognition and milk ingestion, as well as developmental defects in cranial nerves V, IX, and X. ThisPBF1 screening system offers individual laboratories unprecedented opportunities to conduct affordable genome-wide phenotypic screens for deciphering the genetic basis of mammalian biology and disease pathogenesis.

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