Pax6 is a developmental control gene with an essential role in development of the eye, brain and pancreas. Pax6, as many other developmental regulators, depends on a substantial number of cis-regulatory elements in addition to its promoters for correct spatiotemporal and quantitative expression. Here we report on our analysis of a set of mice transgenic for a modified yeast artificial chromosome carrying the human PAX6 locus. In this 420 kb YAC a tauGFP-IRES-Neomycin reporter cassette has been inserted into the PAX6 translational start site in exon 4. The YAC has been further engineered to insert LoxP sites flanking a 35 kb long, distant downstream regulatory region (DRR) containing previously described DNaseI hypersensitive sites, to allow direct comparison between the presence or absence of this region in the same genomic context. Five independent transgenic lines were obtained that vary in the extent of downstream PAX6 locus that has integrated. Analysis of transgenic embryos carrying full-length and truncated versions of the YAC indicates the location and putative function of several novel tissue-specific enhancers. Absence of these distal regulatory elements abolishes expression in specific tissues despite the presence of more proximal enhancers with overlapping specificity, strongly suggesting interaction between these control elements. Using plasmid-based reporter transgenic analysis we provide detailed characterization of one of these enhancers in isolation. Furthermore, we show that overexpression of a short PAX6 isoform derived from an internal promoter in a multicopy YAC transgenic line results in a microphthalmia phenotype. Finally, direct comparison of a single-copy line with the floxed DRR before and after Cre-mediated deletion demonstrates unequivocally the essential role of these long-range control elements for PAX6 expression.
During growth, homeostasis and regeneration, stem cells are exposed to different energy demands. Here, we characterise the metabolic pathways that mediate the commitment and differentiation of mouse skeletal muscle stem cells, and how their modulation can influence the cell state. We show that quiescent satellite stem cells have low energetic demands and perturbed oxidative phosphorylation during ageing, which is also the case for cells from post-mortem tissues. We show also that myogenic fetal cells have distinct metabolic requirements compared to those proliferating during regeneration, with the former displaying a low respiration demand relying mostly on glycolysis. Furthermore, we show distinct requirements for peroxisomal and mitochondrial fatty acid oxidation (FAO) in myogenic cells. Compromising peroxisomal but not mitochondrial FAO promotes early differentiation of myogenic cells. Acute muscle injury and pharmacological block of peroxisomal and mitochondrial FAO expose differential requirements for these organelles during muscle regeneration. Taken together, these observations indicate that changes in myogenic cell state lead to significant alterations in metabolic requirements. In addition, perturbing specific metabolic pathways impacts on myogenic cell fates and the regeneration process.
Neural stem cells (NSCs) in the adult vertebrate brain are found in a quiescent state and can preserve long-lasting progenitor potential (stemness). Whether and how these two properties are linked, and to what extent they can be independently controlled by NSC maintenance pathways, is unresolved. We have previously identified Notch3 signalling as a major quiescence-promoting pathway in adult NSCs of the zebrafish pallium. We now show that Notch3 also controls NSC stemness. Using parallel transcriptomic characterizations of notch3 mutant NSCs and adult NSC physiological states, we demonstrate that a set of potentially direct Notch3 target genes distinguishes quiescence and stemness control. As a proof of principle, we focus on one ‘stemness’ target, encoding the bHLH transcription factor Hey1, that has not yet been analysed in adult NSCs. We show that abrogation of Hey1 function in adult pallial NSCs in vivo, including quiescent NSCs, leads to their differentiation without affecting their proliferation state. These results demonstrate that quiescence and stemness are molecularly distinct outputs of Notch3 signalling, and identify Hey1 as a major Notch3 effector controlling NSC stemness in the vertebrate adult brain.
The transcription factor Pax6 is a developmental regulator with a crucial role in development of the eye, brain, and olfactory system. Pax6 is also required for correct development of the endocrine pancreas and specification of hormone producing endocrine cell types. Glucagon-producing cells are almost completely lost in Pax6-null embryos, and insulin-expressing beta and somatostatin-expressing delta cells are reduced. While the developmental role of Pax6 is well-established, investigation of a further role for Pax6 in the maintenance of adult pancreatic function is normally precluded due to neonatal lethality of Pax6-null mice. Here a tamoxifen-inducible ubiquitous Cre transgene was used to inactivate Pax6 at 6 months of age in a conditional mouse model to assess the effect of losing Pax6 function in adulthood. The effect on glucose homeostasis and the expression of key islet cell markers was measured. Homozygous Pax6 deletion mice, but not controls, presented with all the symptoms of classical diabetes leading to severe weight loss requiring termination of the experiment five weeks after first tamoxifen administration. Immunohistochemical analysis of the pancreata revealed almost complete loss of Pax6 and much reduced expression of insulin, glucagon, and somatostatin. Several other markers of islet cell function were also affected. Notably, strong upregulation in the number of ghrelin-expressing endocrine cells was observed. These findings demonstrate that Pax6 is essential for adult maintenance of glucose homeostasis and function of the endocrine pancreas.
BackgroundThe increasing number of available genomic sequences makes it now possible to study the evolutionary history of specific genes or gene families. Transcription factors (TFs) involved in regulation of gene-specific expression are key players in the evolution of metazoan development. The low complexity COE (Collier/Olfactory-1/Early B-Cell Factor) family of transcription factors constitutes a well-suited paradigm for studying evolution of TF structure and function, including the specific question of protein modularity. Here, we compare the structure of coe genes within the metazoan kingdom and report on the mechanism behind a vertebrate-specific exon duplication.ResultsCOE proteins display a modular organisation, with three highly conserved domains : a COE-specific DNA-binding domain (DBD), an Immunoglobulin/Plexin/transcription (IPT) domain and an atypical Helix-Loop-Helix (HLH) motif. Comparison of the splice structure of coe genes between cnidariae and bilateriae shows that the ancestral COE DBD was built from 7 separate exons, with no evidence for exon shuffling with other metazoan gene families. It also confirms the presence of an ancestral H1LH2 motif present in all COE proteins which partly overlaps the repeated H2d-H2a motif first identified in rodent EBF. Electrophoretic Mobility Shift Assays show that formation of COE dimers is mediated by this ancestral motif. The H2d-H2a α-helical repetition appears to be a vertebrate characteristic that originated from a tandem exon duplication having taken place prior to the splitting between gnathostomes and cyclostomes. We put-forward a two-step model for the inclusion of this exon in the vertebrate transcripts.ConclusionThree main features in the history of the coe gene family can be inferred from these analyses: (i) each conserved domain of the ancestral coe gene was built from multiple exons and the same scattered structure has been maintained throughout metazoan evolution. (ii) There exists a single coe gene copy per metazoan genome except in vertebrates. The H2a-H2d duplication that is specific to vertebrate proteins provides an example of a novel vertebrate characteristic, which may have been fixed early in the gnathostome lineage. (iii) This duplication provides an interesting example of counter-selection of alternative splicing.
Synaptic loss, plaques and neurofibrillary tangles are viewed as hallmarks of Alzheimer’s disease (AD). This study investigated synaptic markers in neocortical Brodmann area 9 (BA9) samples from 171 subjects with and without AD at different levels of cognitive impairment. The expression levels of vesicular glutamate transporters (VGLUT1&2), glutamate uptake site (EAAT2), post-synaptic density protein of 95 kD (PSD95), vesicular GABA/glycine transporter (VIAAT), somatostatin (som), synaptophysin and choline acetyl transferase (ChAT) were evaluated. VGLUT2 and EAAT2 were unaffected by dementia. The VGLUT1, PSD95, VIAAT, som, ChAT and synaptophysin expression levels significantly decreased as dementia progressed. The maximal decrease varied between 12% (synaptophysin) and 42% (som). VGLUT1 was more strongly correlated with dementia than all of the other markers (polyserial correlation = −0.41). Principal component analysis using these markers was unable to differentiate the CDR groups from one another. Therefore, the status of the major synaptic markers in BA9 does not seem to be linked to the cognitive status of AD patients. The findings of this study suggest that the loss of synaptic markers in BA9 is a late event that is only weakly related to AD dementia.
Mp is an irradiation-induced mouse mutation associated with microphthalmia, micropinna and hind limb syndactyly. We show that Mp is caused by a 660 kb balanced inversion on chromosome 18 producing reciprocal 3-prime gene fusion events involving Fbn2 and Isoc1. The Isoc1-Fbn2 fusion gene (Isoc1Mp) mRNA has a frameshift and early stop codon resulting in nonsense mediated decay. Homozygous deletions of Isoc1 do not support a significant developmental role for this gene. The Fbn2-Isoc1 fusion gene (Fbn2 Mp) predicted protein consists of the N-terminal Fibrillin-2 (amino acids 1–2646, exons 1–62) lacking the C-terminal furin-cleavage site with a short out-of-frame extension encoded by the final exon of Isoc1. The Mp limb phenotype is consistent with that reported in Fbn2 null embryos. However, severe eye malformations, a defining feature of Mp, are not seen in Fbn2 null animals. Fibrillin-2Mp forms large fibrillar structures within the rough endoplasmic reticulum (rER) associated with an unfolded protein response and quantitative mass spectrometry shows a generalised defect in protein secretion in conditioned media from mutant cells. In the embryonic eye Fbn2 is expressed within the peripheral ciliary margin (CM). Mp embryos show reduced canonical Wnt-signalling in the CM – known to be essential for ciliary body development - and show subsequent aplasia of CM-derived structures. We propose that the Mp “worse-than-null” eye phenotype plausibly results from a failure in normal trafficking of proteins that are co-expressed with Fbn2 within the CM. The prediction of similar trans-acting protein effects will be an important challenge in the medical interpretation of human mutations from whole exome sequencing.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.