Genetic and Molecular Interactions between HΔCT, a Novel Allele of the Notch Antagonist Hairless, and the Histone Chaperone Asf1 in Drosophila melanogaster.

Cellular differentiation relies on the highly conserved Notch signaling pathway. Notch activity induces gene expression changes that are highly sensitive to chromatin landscape. We address Notch gene regulation using Drosophila as a model, focusing on the genetic and molecular interactions between the Notch antagonist Hairless and the histone chaperone Asf1. Earlier work implied that Asf1 promotes the silencing of Notch target genes via Hairless (H). Here, we generate a novel HΔCT allele by genome engineering. Phenotypically, HΔCT behaves as a Hairless gain of function allele in several developmental contexts, indicating that the conserved CT domain of H has an attenuator role under native biological contexts. Using several independent methods to assay protein-protein interactions, we define the sequences of the CT domain that are involved in Hairless-Asf1 binding. Based on previous models, where Asf1 promotes Notch repression via Hairless, a loss of Asf1 binding should reduce Hairless repressive activity. However, tissue-specific Asf1 overexpression phenotypes are increased, not rescued, in the HΔCT background. Counterintuitively, Hairless protein binding mitigates the repressive activity of Asf1 in the context of eye development. These findings highlight the complex connections of Notch repressors and chromatin modulators during Notch target-gene regulation and open the avenue for further investigations.

Comparative Morphological, Metabolic and Transcriptome Analyses in elmo1 -/- , elmo2 -/- , and elmo3 -/- Zebrafish Mutants Identified a Functional Non-Redundancy of the Elmo Proteins.

The ELMO protein family consists of the homologues ELMO1, ELMO2 and ELMO3. Several studies have shown that the individual ELMO proteins are involved in a variety of cellular and developmental processes. However, it has poorly been understood whether the Elmo proteins show similar functions and act redundantly. To address this question, elmo1 -/- , elmo2 -/- and elmo3 -/- zebrafish were generated and a comprehensive comparison of the phenotypic changes in organ morphology, transcriptome and metabolome was performed in these mutants. The results showed decreased fasting and increased postprandial blood glucose levels in adult elmo1 -/- , as well as a decreased vascular formation in the adult retina in elmo1 -/- , but an increased vascular formation in the adult elmo3 -/- retina. The phenotypical comparison provided few similarities, as increased Bowman space areas in adult elmo1 -/- and elmo2 -/- kidneys, an increased hyaloid vessel diameter in elmo1 -/- and elmo3 -/- and a transcriptional downregulation of the vascular development in elmo1 -/- , elmo2 -/- , and elmo3 -/- zebrafish larvae. Besides this, elmo1 -/- , elmo2 -/- , and elmo3 -/- zebrafish exhibited several distinct changes in the vascular and glomerular structure and in the metabolome and the transcriptome. Especially, elmo3 -/- zebrafish showed extensive differences in the larval transcriptome and an impaired survivability. Together, the data demonstrated that the three zebrafish Elmo proteins regulate not only similar but also divergent biological processes and mechanisms and show a low functional redundancy.

pdx1 Knockout Leads to a Diabetic Nephropathy- Like Phenotype in Zebrafish and Identifies Phosphatidylethanolamine as Metabolite Promoting Early Diabetic Kidney Damage.

The pdx1-/- zebrafish mutant was recently established as a novel animal model of diabetic retinopathy. In this study, we investigate whether knockout of pdx1 also leads to diabetic kidney disease (DKD). pdx1-/- larvae exhibit several signs of early DKD, such as glomerular hypertrophy, impairments in the filtration barrier corresponding to microalbuminuria, and glomerular basement membrane (GBM) thickening. Adult pdx1-/- mutants show progressive GBM thickening in comparison with the larval state. Heterozygous pdx1 knockout also leads to glomerular hypertrophy as initial establishment of DKD similar to the pdx1-/- larvae. RNA sequencing of adult pdx1+/- kidneys uncovered regulations in multiple expected diabetic pathways related to podocyte disruption and hinting at early vascular dysregulation without obvious morphological alterations. Metabolome analysis and pharmacological intervention experiments revealed the contribution of phosphatidylethanolamine in the early establishment of kidney damage. In conclusion, this study identified the pdx1 mutant as a novel model for the study of DKD, showing signs of the early disease progression already in the larval stage and several selective features of later DKD in adult mutants.

Elevated 4-hydroxynonenal induces hyperglycaemia via Aldh3a1 loss in zebrafish and associates with diabetes progression in humans.

Increased methylglyoxal (MG) formation is associated with diabetes and its complications. In zebrafish, knockout of the main MG detoxifying system Glyoxalase 1, led to limited MG elevation but significantly elevated aldehyde dehydrogenases (ALDH) activity and aldh3a1 expression, suggesting the compensatory role of Aldh3a1 in diabetes. To evaluate the function of Aldh3a1 in glucose homeostasis and diabetes, aldh3a1-/- zebrafish mutants were generated using CRISPR-Cas9. Vasculature and pancreas morphology were analysed by zebrafish transgenic reporter lines. Corresponding reactive carbonyl species (RCS), glucose, transcriptome and metabolomics screenings were performed and ALDH activity was measured for further verification. Aldh3a1-/- zebrafish larvae displayed retinal vasodilatory alterations, impaired glucose homeostasis, which can be aggravated via pdx1 silencing induced hyperglycaemia. Unexpectedly, MG was not altered, but 4-hydroxynonenal (4-HNE), another prominent lipid peroxidation RCS exhibited high affinity with Aldh3a1, was increased in aldh3a1 mutants. 4-HNE was responsible for the retinal phenotype via pancreas disruption induced hyperglycaemia and can be rescued via l-Carnosine treatment. Furthermore, in type 2 diabetic patients, serum 4-HNE was increased and correlated with disease progression. Thus, our data suggest impaired 4-HNE detoxification and elevated 4-HNE concentration as biomarkers but also the possible inducers for diabetes, from genetic susceptibility to the pathological progression.