Col Expression Research Articles

Chronic intermittent hypoxia impaired collagen synthesis in mouse genioglossus via ROS accumulation: A transcriptomic analysis

Obstructive sleep apnea (OSA) is a sleep-related breathing disorder characterized by intermittent and recurrent upper airway collapse during sleep that leads to chronic intermittent hypoxia (CIH). The genioglossus (GG) is the largest dilator muscle, which controls the upper airway and plays an important role in OSA pathology. Elucidating its genetic alterations may help identify potential targets for OSA. However, the genetic aspects of the GG in CIH mice remain unclear. Here, we have conducted an RNA sequencing (RNA-Seq) analysis to assess the differentially expressed genes (DEGs) in the GG between CIH mice and normoxia (NOR) mice. A total of 637 DEGs were identified to be dysregulated in CIH mice compared with control mice. Bioinformatics analysis showed that the DEGs were related to various physiological processes, such as the endogenous stimulus responses, cellular component organization and metabolic processes. Extracellular matrix (ECM)–receptor interaction was the top KEGG pathway in the environmental information processing category with high significance and large fold changes. From the gene weight distributions of collagen (Col)-related biological processes (BPs), we found several significant DEGs, such as Col1a1, Col1a2, Mmp2, Col3a1, Col5a1, Fmod, and Col5a2. A PPI network showed that Col1a1 was linked to ECM–receptor interactions, responses to reactive oxygen species (ROS) and Col-related BPs. It was verified in vivo and in vitro that hypoxia can induce excess ROS and reduce Col expression levels. Moreover, we found NAC can effectively scavenge ROS and restore collagen synthesis. These findings contribute to a better understanding of the mechanisms linking OSA and upper airway muscle injury and may help identify potential therapeutic targets.

The EBF transcription factor Collier directly promotes Drosophila blood cell progenitor maintenance independently of the niche

The maintenance of stem or progenitor cell fate relies on intrinsic factors as well as local cues from the cellular microenvironment and systemic signaling. In the lymph gland, an hematopoietic organ in Drosophila larva, a group of cells called the Posterior Signaling Centre (PSC), whose specification depends on the EBF transcription factor Collier (Col) and the HOX factor Antennapedia (Antp), has been proposed to form a niche required to maintain the pool of hematopoietic progenitors (prohemocytes). In contrast with this model, we show here that genetic ablation of the PSC does not cause an increase in blood cell differentiation or a loss of blood cell progenitors. Furthermore, although both col and Antp mutant larvae are devoid of PSC, the massive prohemocyte differentiation observed in col mutant is not phenocopied in Antp mutant. Interestingly, beside its expression in the PSC, Col is also expressed at low levels in prohemocytes and we show that this expression persists in PSC-ablated and Antp mutant larvae. Moreover, targeted knockdown and rescue experiments indicate that Col expression is required in the prohemocytes to prevent their differentiation. Together, our findings show that the PSC is dispensable for blood cell progenitor maintenance and reveal the key role of the conserved transcription factor Col as an intrinsic regulator of hematopoietic progenitor fate.

Tup/Islet1 integrates time and position to specify muscle identity in Drosophila

The LIM-homeodomain transcription factor Tailup/Islet1 (Tup) is a key component of cardiogenesis in Drosophila and vertebrates. We report here an additional major role for Drosophila Tup in specifying dorsal muscles. Tup is expressed in the four dorsal muscle progenitors (PCs) and tup-null embryos display a severely disorganized dorsal musculature, including a transformation of the dorsal DA2 into dorsolateral DA3 muscle. This transformation is reciprocal to the DA3 to DA2 transformation observed in collier (col) mutants. The DA2 PC, which gives rise to the DA2 muscle and to an adult muscle precursor, is selected from a cluster of myoblasts transiently expressing both Tinman (Tin) and Col. The activation of tup by Tin in the DA2 PC is required to repress col transcription and establish DA2 identity. The transient, partial overlap between Tin and Col expression provides a window of opportunity to distinguish between DA2 and DA3 muscle identities. The function of Tup in the DA2 PC illustrates how single cell precision can be reached in cell specification when temporal dynamics are combined with positional information. The contributions of Tin, Tup and Col to patterning Drosophila dorsal muscles bring novel parallels with chordate pharyngeal muscle development.

Control of multidendritic neuron differentiation in Drosophila: The role of Collier

Proper sampling of sensory inputs critically depends upon neuron morphogenesis and expression of sensory channels. The highly stereotyped organisation of the Drosophila peripheral nervous system (PNS) provides a model to study neuronal determination and morphogenesis. Here, we report that Collier/Knot (Col/Kn), the Drosophila member of the COE family of transcription factors, is transiently expressed in the subset of multidendritic arborisation (da) sensory neurons that display an highly branched dendritic arborisation, class IV neurons. When lacking Col activity, class IV da neurons are formed but display a reduced dendrite arborisation. Col control on dendrite branching is distinct from that exerted by Cut, another transcription factor expressed in class IV neurons and necessary for proper dendrite morphogenesis. Col is also required for the class IV da-specific expression of pickpocket ( ppk), which encodes a degenerin/epithelial sodium channel subunit required for larval locomotion. Characterisation of the col upstream region identified a 9-kb cis-regulatory region driving col expression in all class IV md neurons, even though these originate from two types of sensory precursor cells. Altogether, these findings indicate that col is required in at least two distinct programs that control the morphological and sensory specificity of Drosophila md neurons.

Effect of excess synthesis of extracellular matrix components by trabecular meshwork cells: Possible consequence on aqueous outflow

The extracellular matrix (ECM) of the trabecular meshwork (TM) is an important determinant of its functional properties. This study was performed to investigate whether overexpression of ECM components, laminin (LM) and collagen type IV (Col) by TM cells may play a role in the development of outflow resistance. To determine the effect of excess LM and Col expression on cell monolayer permeability, an in vitro cell culture model was used in which overexpression of the two ECM components, LM and Col, was induced by high glucose (HG) (30 mM) or 0.1 μM dexamethasone (D) in bovine and human trabecular meshwork (BTM and HTM) cells. Western blot analysis and immunofluorescence staining confirmed increased LM and Col synthesis in cells exposed to HG or D. Increased level of LM and Col protein resulted in reduced cell monolayer permeability. Transfection with antisense oligos (AS-oligos) targeted against LM or Col inhibited HG- or D-induced LM and Col gene overexpression in TM cells with concomitant increase in permeability. The AS-oligo strategy was effective in reducing LM or Col level in the TM cells in all conditions tested in this study. These findings suggest that increased LM and Col deposition in the outflow pathway may cause resistance to aqueous outflow and contribute to the development of primary open angle glaucoma (POAG).

Cellular immune response to parasitization in Drosophila requires the EBF orthologue collier.

Drosophila immune response involves three types of hemocytes (‘blood cells’). One cell type, the lamellocyte, is induced to differentiate only under particular conditions, such as parasitization by wasps. Here, we have investigated the mechanisms underlying the specification of lamellocytes. We first show that collier (col), the Drosophila orthologue of the vertebrate gene encoding early B-cell factor (EBF), is expressed very early during ontogeny of the lymph gland, the larval hematopoietic organ. In this organ, Col expression prefigures a specific posterior region recently proposed to act as a signalling centre, the posterior signalling centre (PSC). The complete lack of lamellocytes in parasitized col mutant larvae revealed the critical requirement for Col activity in specification of this cell type. In wild-type larvae, Col expression remains restricted to the PSC following parasitization, despite the massive production of lamellocytes. We therefore propose that Col endows PSC cells with the capacity to relay an instructive signal that orients hematopoietic precursors towards the lamellocyte fate in response to parasitization. Considered together with the role of EBF in lymphopoiesis, these findings suggest new parallels in cellular immunity between Drosophila and vertebrates. Further investigations on Col/EBF expression and function in other phyla should provide fresh insight into the evolutionary origin of lymphoid cells.

Vein-positioning in the Drosophila wing in response to Hh; new roles of Notch signaling

The Drosophila wing is a classical model for studying the generation of developmental patterns. Previous studies have suggested that vein primordia form at boundaries between discrete sectors of gene expression along the antero-posterior (A/P) axis in the larval wing imaginal disc. Observation that the vein marker rhomboid ( rho) is expressed at the centre of wider vein-competent domains led to propose that narrow vein primordia form first, and produce secondary short-range signals activating provein genes in neighbouring cells (see Curr. Opin. Genet. Dev. 10 (2000) 393). Here, we examined how the central L3 and L4 veins are positioned relative to the limits of expression of Collier (Col), a dose-dependent Hedgehog (Hh) target activated in the wing A/P organiser. We found that rho expression is first activated in broad domains adjacent to Col-expressing cells and secondarily restricted to the centre of these domains. This restriction which depends upon Notch (N) signaling sets the L3 and L4 vein primordia off the boundaries of Col expression. N activity is also required to fix the anterior limit of Col expression by locally antagonising Hh activation, thus precisely positioning the L3 vein primordium relative to the A/P compartment boundary. Experiments using Nts mutants further indicated that these two activities of N could be temporally uncoupled. Together, these observations highlight new roles of N in topologically linking the position of veins to prepattern gene expression.

The COE transcription factor Collier is a mediator of short-range Hedgehog-induced patterning of the Drosophila wing

Background: The secreted Hedgehog (Hh) proteins have been implicated as mediators of positional information in vertebrates and invertebrates. A gradient of Hh activity contributes to antero-posterior (A/P) patterning of the fly wing. In addition to inducing localised expression of Decapentaplegic (Dpp), which in turn relays patterning cues at long range, Hh directly patterns the central region of the wing.Results: We show that short-range, dose-dependent Hh activity is mediated by activation of the transcription factor Collier (Col). In the absence of col activity,longitudinal veins 3 and 4 (L3 and L4) are apposed and the central intervein is missing. Hh expression induces col expression in a narrow stripe of cells along the A/P boundary through a dual-input mechanism: inhibition of proteolysis of Cubitus-interruptus (Ci) and activation of the Fused (Fu) kinase. Col, in cooperation with Ci, controls the formation of the central intervein by activating the expression of blistered (bs), which encodes the Drosophila serum response factor (D-SRF), the activity of which is required for the adoption and maintenance of the intervein cell fate. Furthermore, col is allelic to knot, a gene involved in the formation of the central part of the wing. This finding completes our understanding of the sectorial organisation of the Drosophila wing.Conclusions: Col, the Drosophila member of the COE family (Col/Olf-1/EBF) of non-basic, helix–loop–helix (HLH)-containing transcription factors, is a mediator of the short-range organising activity of Hh in the Drosophila wing. Our results support the idea that Hh controls target gene expression in a concentration-dependent manner and highlight the importance of the Fu kinase in this differential regulation. The high degree of evolutionary conservation of the COE proteins and the diversity of developmental processes controlled by Hh signalling raises the possibility that the specific genetic interactions depicted here may also operate in vertebrates.

Requirement for the Drosophila COE transcription factor Collier in formation of an embryonic muscle: transcriptional response to notch signalling.

During Drosophila embryogenesis, mesodermal cells are recruited to form a stereotyped pattern of about 30 different larval muscles per hemisegment. The formation of this pattern is initiated by the specification of a special class of myoblasts, called founder cells, that are uniquely able to fuse with neighbouring myoblasts. We report here the role of the COE transcription factor Collier in the formation of a single muscle, muscle DA3([A])(DA4([T])). Col expression is first observed in two promuscular clusters (in segments A1-A7), the two corresponding progenitors and their progeny founder cells, but its transcription is maintained in only one of these four founder cells, the founder of muscle DA3([A]). This lineage-specific restriction depends on the asymmetric segregation of Numb during the progenitor cell division and involves the repression of col transcription by Notch signalling. In col mutant embryos, the DA3([A]) founder cells form but do not maintain col transcription and are unable to fuse with neighbouring myoblasts, leading to a loss-of-muscle DA3([A]) phenotype. In wild-type embryos, each of the DA3([A])-recruited myoblasts turns on col transcription, indicating that the conversion, by the DA3([A]) founder cell, of 'naive' myoblasts to express its distinctive pattern of gene expression involves activation of col itself. We find that muscles DA3([A]) and DO5([A]) (DA4([T]) and DO5([T])) derive from a common progenitor cell. Ectopic expression of Col is not sufficient, however, to switch the DO5([A]) to a DA3([A]) fate. Together these results lead us to propose that specification of the DA3([A]) muscle lineage requires both Col and at least one other transcription factor, supporting the hypothesis of a combinatorial code of muscle-specific gene regulation controlling the formation and diversification of individual somatic muscles.