Biology subjects

Beck, C. W.

Publications and source records attributed to Beck, C. W..

11 recordsLinked to original sources

bioRxiv2025

Seizures, increased interhemispheric synchrony, altered brain transcriptomics and a leaky blood-brain barrier result from loss of ap3b2 in a CRISPR tadpole model of DEE48

Loss-of-function variants in AP3B2, a neuronal adaptor protein required for synaptic vesicle formation, cause a severe early-onset neurodevelopmental epilepsy known as Developmental and Epileptic Encephalopathy 48 (DEE48). To investigate how AP3B2 loss alters brain development, leading to increased seizure susceptibility, we generated a Xenopus laevis model by targeting the orthologous gene using CRISPR/Cas9. Ap3b2.S-/- (mosaic) F0 tadpoles displayed increased locomotor activity with frequent seizure-like episodes when compared to sibling controls. Visualisation of forebrain and midbrain activity using the genetically encoded Ca2+ sensor GCaMP6s detected spontaneous, large amplitude, prolonged and widespread neural activity, alongside increased interhemispheric synchrony of both regions. Comparison of whole-brain transcriptomes from ap3b2 CRISPants and unedited sibling controls detected mainly downregulation of brain expressed genes, with significant over-representation of pathways involved in ion transport, axon formation and guidance, inhibitory (GABA) neurotransmission, and transport across the blood-brain barrier (BBB). In a simple assay for BBB integrity, CRISPant tadpoles were confirmed to have faster leakage of sodium fluoresceinAcute exposure to the angiotensin receptor blocker losartan significantly reduced locomotor hyperactivity, and CRISPant cohorts treated with losartan tended to have lower neural activity, indicating incomplete rescue of the ap3b2.S CRISPant phenotype. These findings demonstrate how AP3B2 loss of function alters brain development and the establishment of the BBB, with the resulting alterations in neurotransmitter pathways predisposing the brain to spontaneous seizures. Our results suggest that traditional anti-seizure medications designed to alter ion transport and GABA metabolism could be augmented with drugs targeting neuroinflammation, as adjunct seizure control options in infants with DEE48.

developmental biology↗

bioRxiv2025

Commensal skin bacteria interact with the innate immunesystem to promote tail regeneration in Xenopus laevis tadpoles.

Xenopus laevis tadpoles regenerate their tails following partial amputation, however, for a brief developmental window, some undertake wound healing rather than regeneration. Inspired by links between microbiomes and human inflammatory disease, we asked how tadpole skin microbiomes and innate immunity influence the ability to regenerate tails. Having previously shown that lipopolysaccharide-Tlr4 signalling promotes regeneration, here, we demonstrate that the peptidoglycan-Tlr2 pathway is also involved. While tadpoles acquire their commensal skin bacteria from their mothers, analysis of mitochondrial haplotypes did not explain familial regenerative bias. Levels of endogenous lipopolysaccharides on tail tips were also not predictive of regenerative success, and shotgun sequencing indicated no difference in bacterial loads. To see if microbiome composition correlated with regenerative success, we sequenced 16S rRNA amplicons from 503 tadpole tail tips and mapped these to regenerative outcomes. While no one taxon appeared critical for regenerative success, higher proportions of Gram-positives correlated with successful regeneration. Our results suggest a previously undocumented role for Tlr2, peptidoglycan and Gram-positive commensal skin bacteria in tipping the balance from wound repair to regenerative programmes in Xenopus laevis refractory stage tadpoles.

developmental biology↗

bioRxiv2025

Unilateral loss of recql4 function in Xenopus laevis tadpoles leads to ipsilateral ablation of the forelimb, hypoplastic Meckel's cartilage and vascular defects.

RECQL4 encodes a RecQ helicase, one of a family of DNA unwinding enzymes with roles in DNA replication, double strand break repair and genomic stability. Pathogenic variants in RECQL4 are clinically associated with three rare autosomal recessive conditions: Rothmund-Thomson Syndrome type II, Baller-Gerold Syndrome and RAPADILINO syndrome. These three syndromes show overlapping growth retardation, low bone density and skeletal defects affecting the arms and hands. Here, we take advantage of the ability to generate one-sided CRISPR knockdowns of recql4 in Xenopus laevis tadpoles. Tadpoles develop normally until feeding starts, after which growth slows on the edited side leading to a curved posture, smaller eyes (micropthalmia) and reduced head size (microcephaly). Forelimb buds fail to develop, leading to complete absense of the forelimb on the edited side. Additionally, Meckels cartilage (lower jaw) ossification is absent or reduced and the hyoid cartilage is smaller, but this is not due to deficiencies in cranial neural crest migration on the edited side. Knockdown of recql4 also results in hypoplastic vasculature, with reduced branching from the aorta on the edited side. Taken together, our results clearly show the utility of unilateral CRISPR editing in Xenopus for understanding the specific phenotypic developmental effects of mutations affecting cell proliferation.

developmental biology↗

bioRxiv2023

Knockdown of NeuroD2 leads to seizure-like behaviour, brain neuronal hyperactivity and a leaky blood-brain barrier in a Xenopus laevis tadpole model of DEE75

Developmental and Epileptic Encephalopathies (DEE) are a genetically diverse group of severe, early onset seizure disorders. DEE are normally identified clinically in the first six months of life by the presence of frequent, difficult to control seizures and accompanying stalling or regression of development. DEE75 results from de novo mutations of the NEUROD2 gene that result in loss of activity of the encoded transcription factor, and the seizure phenotype was shown to be recapitulated in Xenopus tropicalis tadpoles. We used CRISPR/Cas9 to make a DEE75 model in Xenopus laevis, to further investigate the developmental aetiology. NeuroD2.S CRISPR/Cas9 edited tadpoles were more active, swam faster on average, and had more unprovoked escape responses (C-starts) than their sibling controls. Live imaging of Ca2+ signalling revealed prolongued, strong signals sweeping through the brain, indicative of neuronal hyperactivity. While the resulting tadpole brain appeared grossly normal, the blood-brain barrier was found to be leakier than that of controls. Additionally, the TGF{beta} antagonist Losartan was shown to have a short-term protective effect, reducing neuronal hyperactivity and reducing permeability of the blood- brain barrier. Severity of the behavioral phenotype correlated with increased with editing efficiency. Our results support a haploinsufficiency model of DEE75 resulting from reduced NeuroD2 activity during vertebrate brain development, and indicate that a leaky blood- brain barrier contributes to epileptogenesis.

neuroscience↗

bioRxiv2023

The role of family and environment in determining the skin microbiome of captive aquatic frogs, Xenopus laevis

BackgroundThe amphibian skin microbiome has drawn interest due to the ecological threat posed by chytridiomycosis, which drives changes in symbiotic microbial communities and may be inhibited by certain bacterial taxa. However, skin microbes also play a role in amphibian tissue regeneration. Xenopus spp. are well-established model organisms used to study development, regeneration, genetics and disease. Husbandry protocols, including use of antibiotics and other sterilising agents, may affect experimental outcomes by altering microbiomes. It is therefore essential to improve our understanding of Xenopus microbiome characteristics and inheritance. We undertook bacterial 16S rRNA based sampling of a captive, closed Xenopus laevis colony. A total of 16 female frogs, their eggs, and tadpoles were sampled, covering multiple aquarium systems and tanks, along with testes from males used for in vitro fertilisation and a range of environmental samples. ResultsTank environments supported the most complex microbial communities. Mother frogs harboured the most diverse microbial communities of the frog life stages, with tadpole skin microbiomes being relatively simple. Frog samples were dominated by Proteobacteria and Bacteroidota. Rhizobium and Chryseobacterium were dominant in tadpoles, whereas mothers supported high proportions of Vogesella and Acinetobacter as well as Chryseobacterium. While the mothers habitats contained low levels of these taxa, the tadpoles environmental microbes were very similar to those on tadpole skin. A total of 34 genera were found to be differentially abundant between the mothers and tadpoles. Analysis of Bray-Curtis distances indicated that mother and tadpole microbiomes varied according to the mothers aquarium system, the tanks within them, and the individual mother. Source tracking analyses showed that egg jelly and tadpoles received a mean of approximately two thirds of their microbiomes via vertical transmission, although a sizeable proportion came from unknown sources at all life stages. ConclusionsThe skin of mother frogs appears to select for certain taxa that are otherwise present at low abundances in the environment. While tadpoles inherit a proportion of their microbiomes from their mothers via the egg, they support a distinct and less diverse microbial community than adult frogs. The microbiome varies between individual mothers, and is also affected by the aquarium system and individual tank within that the mother occupies.

microbiology↗

bioRxiv2022

Expression of mRNA encoding two gain-of-function cyfip2 variants associated with DEE65 results in spontaneous seizures in Xenopus laevis tadpoles

Developmental and epileptic encephalopathies (DEE) are a genetically diverse group of disorders with similar early clinical presentations. DEE65 is caused by de novo, non-synonymous, gain-of-function mutations in CYFIP2. It presents in early infancy as hypotonia, epileptic spasms and global developmental delay. While modelling loss-of-function mutations can be done using knockdown or knockout techniques to reduce the amount of functional protein, modelling gain-of-function mutations requires different approaches. Here, we show that transient ectopic expression of the Arg87Cys pathogenic variant of cyfip2 mRNA in Xenopus laevis tadpoles resulted in increased seizure-related behaviours such as rapid darting and swimming in circles. In contrast, expression of a second pathological variant, Tyr108Cys, did not alter tadpole behaviour. Expression of either pathogenic variant resulted in spontaneous epileptic activity in the brain. For both variants, neuronal hyperactivity was reduced by treating the tadpole with 5 mM of the anti-seizure drug valproate (VPA). mRNA overexpression of gain-of-function variants in X. laevis tadpoles may be useful both for understanding the aetiology of DEE and for pre-clinical drug testing.

genetics↗

bioRxiv2022

Characterisation and automated quantification of induced seizure-related behaviours in Xenopus laevis tadpoles

Epilepsy, a clinical diagnosis characterized by paroxysmal episodes known as seizures, affects 1% of people worldwide. Safe and patient-specific treatment is vital and can be achieved by the development of rapid pre-clinical models of for identified epilepsy genes. Epilepsy can result from either brain injury or gene mutations, and can also be induced chemically. Xenopus laevis tadpoles could be a useful model for confirmation of variants of unknown significance found in epilepsy patients, and for drug re-purposing screens that could eventually lead to benefits for patients. Here, we characterise and quantify seizure-related behaviours in X. laevis tadpoles arrayed in 24-well plates. To provoke acute seizure behaviours, tadpoles were chemically induced with either pentylenetetrazole (PTZ) or 4-aminopyridine (4-AP). To test the capacity to adapt this method for drug testing, we also exposed induced tadpoles to the anti-seizure drug valproate (VPA). Four induced seizure-like behaviours were described and manually quantified, and two of these (darting, circling) could be accurately detected automatically, using the video analysis software TopScan. Additionally, we recorded swimming trajectories and mean swimming velocity. Automatic detection showed that either PTZ or 4-AP induced darting behaviour and increased mean swimming velocity compared to untreated controls. Both parameters were significantly reduced in the presence of VPA. In particular, darting behaviour was a shown to be a sensitive measure of epileptic seizure activity. While we could not automatically detect the full range of seizure behaviours, this method shows promise for future studies, since X. laevis is a well-characterised and genetically tractable model organism.

neuroscience↗

bioRxiv2022

Gene expression analysis of the Xenopus laevis early limb bud proximodistal axis.

BackgroundLimb buds develop as bilateral outgrowths of the lateral plate mesoderm and are patterned along three axes. Current models of proximal to distal patterning of early amniote limb buds suggest that two signals, a distal organising signal from the apical epithelial ridge (AER, Fgfs) and an opposing proximal (retinoic acid) act early on pattern this axis. ResultsTranscriptional analysis of stage 51 Xenopus laevis hindlimb buds sectioned along the proximal-distal axis, showed that the distal region is distinct from the rest of the limb. Expression of capn8.3, a novel calpain, was located in cells immediately flanking the AER. The Wnt antagonist Dkk1 was AER-specific in Xenopus limbs. Two transcription factors, sall1 and zic5, were expressed in distal mesenchyme. Zic5 has no described association with limb development. We also describe expression of two proximal genes, gata5 and tnn, not previously associated with limb development. Differentially expressed genes were associated with Fgf, Wnt and retinoic acid (RA) signalling as well as differential cell adhesion and proliferation. ConclusionsWe identify new candidate genes for early proximodistal limb patterning. Our analysis of RA-regulated genes supports a role for transient RA gradients in early limb bud in proximal-to-distal patterning in this anamniote model organism.

developmental biology↗

bioRxiv2021

Manipulating the microbiome alters regenerative outcomes in Xenopus laevis tadpoles vis lipopolysaccharide signalling

Xenopus laevis tadpoles can regenerate functional tails, containing spinal cord, notochord, muscle, fin, blood vessels and nerves, except for a brief refractory period at around one week of age. At this stage, amputation of the tadpoles tail may either result in scarless wound healing, or the activation of a regeneration programme, which replaces the lost tissues. We recently demonstrated a link between bacterial lipopolysaccharides and successful tail regeneration in refractory stage tadpoles, and proposed that this could result from lipopolysaccharides binding to Toll-like receptor 4 (TLR4). Here, we have used 16S rRNA sequencing to show that the tadpole skin microbiome is highly variable between sibships and that the community can be altered by raising embryos in the antibiotic gentamicin. Six gram-negative genera, including Delftia and Chryseobacterium, were over-represented in tadpoles that underwent tail regeneration. Lipopolysaccharides purified from a commensal Chryseobacterium spp. XDS4, an exogenous Delftia spp. or Escherichia coli could significantly increase the number of antibiotic-raised tadpoles that attempted regeneration. Conversely, the quality of regeneration was impaired in native-raised tadpoles exposed to the antagonistic lipopolysaccharide of Rhodobacter sphaeroides. Editing TLR4 using CRISPR/Cas9 also reduced regeneration quality, but not quantity, at the level of the cohort. However, we found that the editing level of individual tadpoles was a poor predictor of regenerative outcome. In conclusion, our results suggest that variable regeneration in refractory stage tadpoles depends at least in part on the skin microbiome and lipopolysaccharide signalling, but that signalling via TLR4 cannot account for all of this effect.

developmental biology↗

bioRxiv2018

The Germ Theory of Regeneration

Amphibians have remarkable regenerative capabilities, but the mechanisms they use to regenerate are largely unknown. Identifying these mechanism would be of great interest for applications in medicine. Reactive oxygen species (ROS) are continually produced and required during tadpole tail regeneration (1). Production of ROS and tadpole tail regeneration are prevented by NADPH oxidase (Nox) inhibitors, suggesting Nox complexes as the source of ROS. However, the role of ROS and the mechanism of their sustained production throughout regeneration, were not known. NF-{kappa}B is a rapid-acting transcription factor with the potential to dramatically alter the activity and function of a cell (2). NF-{kappa}B is necessary for maintaining the undifferentiated state of human embryonic stem cells (3), human induced pluripotent stem cells (4) and mesenchymal stem cells (5), so may similarly be involved in maintaining the de-differentiated state of regeneration blastema cells. In the absence of an activating signal, NF-{kappa}B is sequestered in the cytoplasm by IkB (inhibitor of NF-{kappa}B), preventing its nuclear localisation and activity. The IkB kinase (IKK) complex inhibits IkB in response to multiple extracellular stimuli, but ROS can also inhibit IkB (6). Nuclear NF-{kappa}B directly activates transcription of several genes encoding Nox proteins (7, 8), so could thereby facilitate ROS production. A positive-feedback loop was hypothesised where ROS inhibit IkB to help maintain continual NF-{kappa}B activity and, in turn, facilitate the continual production of ROS by activating the transcription of Nox-encoding genes. Here we demonstrate the involvement of microorganisms in the initiation of tadpole tail regeneration. Microorganisms offer sources of ligands for toll-like receptor (TLR) pathway activation and consequently, IKK complex activity. It was also suggested that sustained NF-{kappa}B activity allows the continual expression of the genes encoding Nox4 in blastema cells and Nox2 in professional phagocytes. These findings provide potential targets for the activation of regeneration in non-regenerative animals.

developmental biology↗

bioRxiv2018

Early lethality of embryos derived from transgenic Xenopus females is associated with reduced ovarian grem1 expression

The grem1 gene codes a protein that inhibits the action of multiple members of a growth factor family known as bone morphogenetic proteins (BMPs). Certain members of this BMP family can regulate both fecundity and fertility in mammals via their action on oocyte (egg) development, and grem1 has been identified as a marker of oocyte quality in humans. The model amphibian Xenopus laevis is far more fecund than mammals, producing thousands of eggs in a clutch. However, female transgenic frogs carrying grem1 under the control of a stress inducible hsp70 promoter (\"G\" frogs) produce very few viable offspring. Here, we show that this is not due to reduced fecundity or fertilization rate, but results from a significant reduction in subsequent survival over the first day of development. Embryos that successfully survive for the first day were found to go on to develop normally when compared to their peers. Both the morphology and stage distribution of oocytes from G females appears normal, and oocytes develop at expected rates, although stage VI oocytes were found to have a lower response to in vitro progesterone treatment. Unexpectedly, levels of grem1 mRNA were found to be consistently lower in the female ovaries from four independent G transgenic lines than in wild type ovaries. Both transgenic and wild type offspring were equally affected, confirming a maternal effect. Our study shows that transgenic females with the lowest levels of grem1 transcripts in the ovary have the lowest rates of survival past the first day of amphibian embryogenesis, equivalent to pre-implantation staged mammalian embryos. The reduced expression of grem1 in the oocytes of transgenic females suggests transgene supression of an endogenous locus may occur in the Xenopus female germline, an unexpected finding.

developmental biology↗