Biology subjects

Castro, V. L.

Publications and source records attributed to Castro, V. L..

5 recordsLinked to original sources

bioRxiv2025

A zebrafish seizure model of cblX syndrome reveals a dose-dependent refractory response to mTor inhibition.

Mutations in the transcriptional cofactor HCFC1 cause methylmalonic aciduria and homocystinemia, cblX type (cblX) (MIM#309541), non-syndromic X-linked intellectual disability (XLID), and focal epilepsy. Zebrafish studies have revealed increased activation of the Akt/mTor signaling pathway after mutation of hcfc1a, one ortholog of HCFC1. mTOR hyperactivation is linked to seizures and its inhibition alleviates epilepsy in other preclinical models. We hypothesized that mTor overactivity in hcfc1a mutant zebrafish increases seizure susceptibility and/or severity. We employed a two-concentration model of the seizure inducing agent, pentylenetetrazol (PTZ), with or without pretreatment of the mTor inhibitor, torin1. Mutation of hcfc1a did not increase seizure susceptibility at sub-optimal concentrations of PTZ and the pharmaceutical inhibition of mTor reduced seizure severity when utilized at a dose of 250nM. Higher doses of torin1 treatment exacerbated seizure response in mutant larvae but not in wildtype siblings. These data suggest that in an hcfc1a deficient background, moderate inhibition of mTor signaling may partially alleviate seizure phenotypes, however, over-inhibition of the pathway causes a refractory response to PTZ. Collectively, we present a model that can be used to test dose response and for the development of combinatorial treatment approaches in a high throughput manner.

developmental biology↗

bioRxiv2023

Abnormal chondrocyte intercalation in a zebrafish model of cblC syndrome restored by an MMACHC cobalamin binding mutant.

Variants in the MMACHC gene cause combined methylmalonic acidemia and homocystinuria cblC type, the most common inborn error of intracellular cobalamin (vitamin B12) metabolism. cblC is associated with neurodevelopmental, hematological, ocular, and biochemical abnormalities. In a subset of patients, mild craniofacial dysmorphia has also been described. Mouse models of Mmachc deletion are embryonic lethal but cause severe craniofacial phenotypes such as facial clefts. MMACHC encodes an enzyme required for cobalamin processing and variants in this gene result in the accumulation of two metabolites: methylmalonic acid (MMA) and homocysteine (HC). Interestingly, other inborn errors of cobalamin metabolism, such as cblX syndrome, are associated with mild facial phenotypes. However, the presence and severity of MMA and HC accumulation in cblX syndrome is not consistent with the presence or absence of facial phenotypes. Thus, the mechanisms by which mutation of MMACHC cause craniofacial defects have not been completely elucidated. Here we have characterized the craniofacial phenotypes in a zebrafish model of cblC (hg13) and performed restoration experiments with either wildtype or a cobalamin binding deficient MMACHC protein. Homozygous mutants did not display gross morphological defects in facial development, but did have abnormal chondrocyte intercalation, which was fully penetrant. Abnormal chondrocyte intercalation was not associated with defects in the expression/localization of neural crest specific markers, sox10 or barx1. Most importantly, chondrocyte organization was fully restored by wildtype MMACHC and a cobalamin binding deficient variant of MMACHC protein. Collectively, these data suggest that mutation of MMACHC causes mild to moderate craniofacial phenotypes that are independent of cobalamin binding.

developmental biology↗

bioRxiv2022

Differential Regulation of mTor signaling defines subtypes of cblX syndrome

Mutations in the HCFC1 transcriptional co-factor protein are the cause of cblX syndrome and X-linked intellectual disability (XLID). cblX is the more severe disorder associated with intractable epilepsy, abnormal cobalamin metabolism, facial dysmorphia, cortical gyral malformations, and intellectual disability. In vitro, Hcfc1 regulates neural precursor (NPCs) proliferation and number, which has been validated in zebrafish. However, conditional deletion of Hcfc1 in Nkx2.1+ NPCs increased cell death, reduced Gfap expression, and reduced numbers of GABAergic neurons. Thus, the role of HCFC1 in brain development is not completely understood. Recently, knock-in of both a cblX (HCFC1) and cblX-like (THAP11) allele were created in mice. Knock-in of the cblX-like allele was associated with increased expression of proteins required for ribosome biogenesis. However, the brain phenotypes were not comprehensively studied due to sub-viability and therefore, a mechanism underlying increased ribosome biogenesis was not described. We used a missense, a nonsense, and two conditional zebrafish alleles to further elucidate this mechanism during brain development. We observed contrasting phenotypes at the level of Akt/mTor activation, the number of radial glial cells, and the expression of two downstream target genes of HCFC1, asxl1 and ywhab. Despite these divergent phenotypes, each allele studied demonstrates with a high degree of face validity when compared to the phenotypes reported in the literature. Collectively, these data suggest that individual mutations in the HCFC1 protein result in differential mTor activity which is associated with contrasting cellular phenotypes.

developmental biology↗

bioRxiv2020

Activation of WNT signaling restores the facial deficits in a zebrafish with defects in cholesterol metabolism.

BackgroundInborn errors of cholesterol metabolism occur as a result of mutations in the cholesterol synthesis pathway (CSP). Although mutations in the CSP cause a multiple congenital anomaly syndrome, craniofacial abnormalities are a hallmark phenotype associated with these disorders. Previous studies have established that mutation of the zebrafish hmgcs1 gene (Vu57 allele), which encodes the first enzyme in the CSP, causes defects in craniofacial development and abnormal neural crest cell (NCC) differentiation. However, the molecular mechanisms by which the products of the CSP disrupt NCC differentiation are not completely known. Cholesterol is known to regulate the activity of WNT signaling, an established regulator of NCC differentiation. We hypothesized that defects in cholesterol synthesis reduce WNT signaling, consequently resulting in abnormal craniofacial development. MethodsTo test our hypothesis we performed a combination of pharmaceutical inhibition, gene expression assays, and targeted rescue experiments to understand the function of CSP and WNT signaling during craniofacial development. ResultsWe demonstrate reduced expression of axin2, a WNT downstream target gene in homozygous carriers of the Vu57 allele and in larvae treated with Ro-48-8071, which inhibits the synthesis of cholesterol. Moreover, activation of WNT signaling via treatment with a WNT agonist completely restored the craniofacial defects present in the Vu57 allele. ConclusionsCollectively, these data suggest interplay between the CSP and WNT signaling during craniofacial development.

developmental biology↗

bioRxiv2018

Mutations in the zebrafish hmgcs1 gene reveal a novel function for isoprenoids during red blood cell development.

Erythropoiesis is the process by which new red blood cells (RBCs) are formed and defects in this process can lead to anemia or thalassemia. The GATA1 transcription factor is an established mediator of RBC development. However, the upstream mechanisms that regulate the expression of GATA1 are not completely characterized. Cholesterol is one potential upstream mediator of GATA1 expression because previously published studies suggest that defects in cholesterol synthesis disrupt RBC differentiation. Here we characterize RBC development in a zebrafish harboring a single missense mutation in the hmgcs1 gene (Vu57 allele). hmgcs1 encodes the first enzyme in the cholesterol synthesis pathway and mutation of hmgcs1 inhibits cholesterol synthesis. We analyzed the number of RBCs in hmgcs1 mutants and their wildtype siblings. Mutation of hmgcs1 resulted in a decrease in the number of mature RBCs, which coincides with reduced gata1a expression. We combined these experiments with pharmacological inhibition and confirmed that cholesterol and isoprenoid synthesis are essential for RBC differentiation, but that gata1a expression is isoprenoid dependent. Collectively, our results reveal two novel upstream regulators of RBC development and suggest that appropriate cholesterol homeostasis is critical for primitive erythropoiesis.\n\nKey PointsO_LIThe products of the cholesterol synthesis pathway regulate red blood cell development during primitive erythropoiesis.\nC_LIO_LIIsoprenoids regulate erythropoiesis by modulating the expression of the GATA1 transcription factor.\nC_LI

developmental biology↗