Biology subjects

Mullins, J. J.

Publications and source records attributed to Mullins, J. J..

7 recordsLinked to original sources

bioRxiv2022

Glucocorticoid involvement in reproductive biology

Oestrogen and progesterone play essential roles in the release of mature oocytes, the priming and cycling of the uterine lining, and the maintenance of mammalian pregnancy. Progesterone is synthesized de novo at the embryo implantation site in the mouse, during decidualization of the endometrium. During early stages of pregnancy, the locally produced progesterone is thought to act as an immunosuppressant, preventing rejection of the fetal allograft at the fetal-maternal interface. However, both uterine natural killer cells and dendritic cells express glucocorticoid receptor rather than progesterone receptor. The importance of glucocorticoids in early pregnancy is inferred from the presence of steroid receptors and the 11{beta}-hydroxysteroid dehydrogenase enzymes, which modulate corticosterone action in the decidua, the trophoblast, the placenta, and the fetus. 11{beta}-hydroxylase is the last enzyme in the metabolism of cholesterol to corticosterone and, in a mouse model of 11{beta}-hydroxylase deficiency, complications of reproduction suggested its requirement for normal ovulation and uterine cell turnover. We present evidence that, in this model, folliculogenesis occurs normally but ovulation is inhibited, and abnormal uterine cell turnover ultimately leads to adenomyosis. Ovaries respond to a superovulation protocol by releasing oocytes and forming corpora lutea, and homozygous null blastocysts are capable of implantation, but the pregnancy is not maintained. We show that glucocorticoid is produced locally at the implantation site in control animals, revealing wide involvement of glucocorticoids in reproductive biology.

physiology↗

bioRxiv2022

Dynamic Compartmentalisation of Intracellular Sodium in collecting duct cells

In Principal cells (PC) of the cortical collecting duct (CCD), the highly regulated and coordinated reabsorption of sodium occurs through the epithelial sodium channel (ENaC) at the apical membrane and Na/K ATPase at the basolateral membrane. However, it is not known how sodium ions (Na+) are transported across the cell. We investigated intracellular transport in mCCDcl1 cells using a fluorescent sodium dye, CoroNa Green AM. Dye uptake was stimulated by aldosterone, blocked by amiloride (an ENaC inhibiter), and basolateral transport was prevented by ouabain (an Na/K ATPase blocker) thus validating the dyes apparently faithful replication of sodium transport. Cells exhibited a consistent pattern of sodium-containing vesicles, of various sizes, surrounded by cytoskeleton and lipid membrane. While the smallest vesicles ([~]0.5m) co-stained with lysotracker, larger vesicles (up to 6.4m) did not co-stain with either lysosomal- or mitochondrial-specific dyes and appeared to have internal structure, suggesting that they were multivesicular bodies. Time-lapse imaging showed a subset of these multivesicular bodies release or take up sodium dye in a controlled manner. Our novel data suggest that intracellular sodium compartmentalisation is highly regulated and offer new insights into intracellular sodium dynamics in the collecting duct, revealing potential new targets for control of sodium homeostasis. New and NoteworthyThe article shows for the first time, to our knowledge, intracellular sodium transport mechanism in mCCDcl1 cells in the form of dynamic vesicular bodies. These structures offer new targets for the regulation of sodium homeostasis and transport in the kidney collecting duct, with wider implications for blood pressure regulation.

cell biology↗

bioRxiv2022

Late Fetal and Newborn Granulopoiesis but not Active Renin is Increased by Maternal Captopril Treatment During Perinatal Kidney Development

Renin expression follows vascular development through the mouse kidney, regressing to glomerular poles by about P10, where renin is stored in dense core granules in juxtaglomerular cells. Homeostatic challenge to blood pressure causes release of active renin from the granules and recruitment of the renin lineage cells. We investigated the response to homeostatic challenge during late fetal development and following birth in a transgenic line expressing GFP under the renin promotor. Pregnant females were treated with water or captopril (30mg/kg/day), which inhibits angiotensin converting enzyme, from E15.5. We found an increase in renin transcription and expression by P1 following captopril treatment, with granulation increased at the glomerular poles and major arteries from E18.5. At P1, the granules showed a wide variation in electron density. Notably, rough endoplasmic reticulum was expanded in vascular smooth muscle cells (VSMCs) of captopril-treated pups at both time-points suggesting increased transcriptional activity. Paracrystalline material was observed in granules of captopril treated fetuses at E18.5 and in both treated and untreated pups at P1. Renin expression and some granules were confirmed in the kidney VSMCs by immuno-gold staining against GFP at E18.5. Importantly, we found no difference in active renin content between kidneys from treated and untreated pups at either age group. We therefore demonstrate a disconnect between granulation and active renin production in newborns when exposed to homeostatic challenge in utero.

developmental biology↗

bioRxiv2022

Vagal involvement in non-dipping phenotype of Hsd11b2 knockout rats

The Syndrome of Apparent Mineralocorticoid Excess (SAME) is a hypertensive disorder caused by deficiency of 11b-hydroxysteroid dehydrogenase type 2. Blood pressure is directly influenced by dietary salt intake, but the causes of salt-sensitivity are not fully resolved. We modelled SAME in Fischer 344 rats, using zinc finger nuclease targeting of the Hsd11b2 gene. The F344 genetic background showed modest salt sensitivity: blood pressure increased by [~]6mmHg when diet was switched from control (0.3% Na) to high salt (3% Na) diet. Homozygous knockout (Hsd2-/-) rats exhibited severe hypertension on control diet (mean arterial blood pressure of [~]180 mmHg compared to [~]115 mmHg in wild-types) and displayed no dipping in blood pressure in the inactive/sleep phase. They also displayed reduced heart rate (339 bpm versus 384 bpm in F344 controls). Low salt diet (0.03% Na) caused a dramatic fall in Hsd2-/- blood pressure (to [~]141mmHg), restoration of robust circadian variation in blood pressure, and an increase in heart rate (to 364bpm). This was mirrored by a restoration of circadian variation in the Poincare plot descriptor, SD1, suggesting involvement of parasympathetic dysfunction in the non-dipping phenotype. Alpha adrenoceptor blockade with prazosin treatment resulted in a further decrease in blood pressure (to [~]124mmHg), which blunted circadian rhythm, together with an increase in heart rate (to [~]394bpm). This rat model of human hypertension reveals clear links between dietary salt, autonomic nervous system dysfunction, and the non-dipping blood pressure phenotype.

physiology↗

bioRxiv2021

Macrophages stimulate epicardial VEGFaa to trigger cardiomyocyte proliferation in larval zebrafish heart regeneration

Cardiac injury induces a sustained macrophage response in both zebrafish and mammals. Macrophages perform a range of both beneficial and detrimental functions during mammalian cardiac repair, yet their precise roles in zebrafish cardiac regeneration are not fully understood. Here we characterise cardiac regeneration in the rapidly regenerating larval zebrafish laser injury model and use macrophage ablation and macrophage-null irf8 mutants to define the role of macrophages in key stages of regeneration. Live heartbeat-synchronised imaging and RNA sequencing revealed an early proinflammatory phase, marked by tnfa+ macrophages, which then resolved to an anti-inflammatory, profibrotic phase. Macrophages were required for cardiomyocyte proliferation but not for functional or structural recovery following injury. Importantly, we found that macrophages are specifically recruited to the epicardial-myocardial niche, triggering the expansion of the epicardium which upregulates VEGFaa expression to induce cardiomyocyte proliferation. Hence, revealing a novel mechanism by which macrophages facilitate cardiac regeneration.

immunology↗

bioRxiv2019

A detailed graphical and computational model of the mammalian renal circadian clock

Here we describe the construction of a detailed graphical and computational model of the mammalian circadian clock. We use it to simulate the clock activity within the kidney, where it plays a pivotal role in regulating blood pressure. First, we assembled a network-based process diagram, which includes all known components of the clock and the interactions between them. Parameterisation of the model for Petri net-based simulation experiments used mRNA levels in the kidney to define initial conditions. With empirical testing, model parameterisation was further refined such that the simulated activity of core genes closely matched their measured activity. Furthermore, virtual knockout experiments performed on the model were shown to reflect experimental gene knockout data. It also identified points at which canonical clock genes may integrate with downstream genes likely to affect blood pressure and other aspects of kidney function. We believe that the model provides new insights into the complexity and function of this most central of physiological pathways and provides a valuable resource for the research community.

systems biology↗

bioRxiv2019

Hybrid optical gating for long-term 3D time-lapse imaging of the beating embryonic zebrafish heart

Three-dimensional fluorescence time-lapse imaging of structural, cellular and sub-cellular processes in the beating heart is an increasingly achievable goal using the latest imaging and computational techniques. However, previous approaches have had significant limitations. Temporarily arresting the heart using drugs disrupts the hearts physiological state, and the use of ultra-high frame-rates for fluorescence image acquisition causes phototoxic cell damage. Real-time triggered imaging, synchronized to a specific phase in the cardiac-cycle, can computationally "freeze" the heart to acquire the minimal number of fluorescence images required for 3D time-lapse imaging. However, until now no solution has been able to maintain phase-lock to the same point in the cardiac cycle for more than about one hour. Our new hybrid optical gating system maintains phase-lock for up to 24 h, acquiring synchronized 3D+time video stacks of the unperturbed heart in vivo. This approach has enabled us to observe detailed developmental, structural, cellular and subcellular processes, including live cell division and cell fate tracking, in the embryonic zebrafish heart using transgenic fish lines expressing cell-specific fluorophores. We show that our approach not only provides high spatial and temporal resolution 3D-imaging, but also avoids phototoxic injury, where alternative approaches induce measurable harm. This provides superb cellular and subcellular imaging of the heart while it is beating in its normal physiological state, and opens up new and exciting opportunities for further study in the heart and other moving cellular and subcellular structures in vivo.

bioinformatics↗