Characterization of the zebrafish gabra1sa43718/sa43718 germline loss of function allele confirms a function for Gabra1 in motility and nervous system development.

Mutation of the GABRA1 gene is associated with neurodevelopmental defects and epilepsy. GABRA1 encodes for the α1 subunit of the γ-aminobutyric acid type A receptor (GABAAR), which regulates the fast inhibitory impulses of the nervous system. Multiple model systems have been developed to understand the function of GABRA1, but these models have produced complex and, at times, incongruent data. Thus, additional model systems are required to validate and substantiate previous results. We sought to provide initial phenotypic analysis of a novel germline mutant allele. Our analysis provides a solid foundation for the future use of this allele to characterize gabra1 functionally and pharmacologically using zebrafish. We investigated the behavioral swim patterns associated with a nonsense mutation of the zebrafish gabra1 (sa43718 allele) gene. The sa43718 allele causes a decrease in gabra1 mRNA expression, which is associated with light induced hypermotility, one phenotype previously associated with seizure like behavior in zebrafish. Mutation of gabra1 was accompanied by decreased mRNA expression of gabra2, gabra3, and gabra5, indicating a reduction in the expression of additional α sub-units of the GABAAR. Although multiple sub-units were decreased, larvae continued to respond to pentylenetetrazole (PTZ), indicating that a residual GABAAR exists in the sa43718 allele. Proteomics analysis demonstrated that mutation of gabra1 is associated with abnormal expression of proteins that regulate synaptic vesicle fusion, vesicle transport, synapse development, and mitochondrial protein complexes. These data support previous studies performed in a zebrafish nonsense allele created by CRISPR/Cas9 and validate that loss of function mutations in the gabra1 gene result in seizure-like phenotypes with abnormal development of the GABA synapse. Our results add to the existing body of knowledge as to the function of GABRA1 during development and validate that zebrafish can be used to provide complete functional characterization of the gene.

ATF4-dependent increase in mitochondrial-endoplasmic reticulum tethering following OPA1 deletion in skeletal muscle.

Mitochondria and endoplasmic reticulum (ER) contact sites (MERCs) are protein- and lipid-enriched hubs that mediate interorganellar communication by contributing to the dynamic transfer of Ca2+, lipid, and other metabolites between these organelles. Defective MERCs are associated with cellular oxidative stress, neurodegenerative disease, and cardiac and skeletal muscle pathology via mechanisms that are poorly understood. We previously demonstrated that skeletal muscle-specific knockdown (KD) of the mitochondrial fusion mediator optic atrophy 1 (OPA1) induced ER stress and correlated with an induction of Mitofusin-2, a known MERC protein. In the present study, we tested the hypothesis that Opa1 downregulation in skeletal muscle cells alters MERC formation by evaluating multiple myocyte systems, including from mice and Drosophila, and in primary myotubes. Our results revealed that OPA1 deficiency induced tighter and more frequent MERCs in concert with a greater abundance of MERC proteins involved in calcium exchange. Additionally, loss of OPA1 increased the expression of activating transcription factor 4 (ATF4), an integrated stress response (ISR) pathway effector. Reducing Atf4 expression prevented the OPA1-loss-induced tightening of MERC structures. OPA1 reduction was associated with decreased mitochondrial and sarcoplasmic reticulum, a specialized form of ER, calcium, which was reversed following ATF4 repression. These data suggest that mitochondrial stress, induced by OPA1 deficiency, regulates skeletal muscle MERC formation in an ATF4-dependent manner.

Abnormal chondrocyte development 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 mutations in MMACHC cause craniofacial defects are yet to be completely elucidated. Here we have characterized the craniofacial phenotypes in a zebrafish model of cblC (hg13) and performed restoration experiments with either a wildtype or a cobalamin binding deficient MMACHC protein. Homozygous mutants did not display gross morphological defects in facial development but did have abnormal chondrocyte nuclear organization and an increase in the average number of neighboring cell contacts, both phenotypes were fully penetrant. Abnormal chondrocyte nuclear organization was not associated with defects in the localization of neural crest specific markers, sox10 (RFP transgene) or barx1. Both nuclear angles and the number of neighboring cell contacts were fully restored by wildtype MMACHC and a cobalamin binding deficient variant of the MMACHC protein. Collectively, these data suggest that mutation of MMACHC causes mild to moderate craniofacial phenotypes that are independent of cobalamin binding.

Knockdown of hspg2 is associated with abnormal mandibular joint formation and neural crest cell dysfunction in zebrafish.

BACKGROUND: Heparan sulfate proteoglycan 2 (HSPG2) encodes for perlecan, a large proteoglycan that plays an important role in cartilage formation, cell adhesion, and basement membrane stability. Mutations in HSPG2 have been associated with Schwartz-Jampel Syndrome (SJS) and Dyssegmental Dysplasia Silverman-Handmaker Type (DDSH), two disorders characterized by skeletal abnormalities. These data indicate a function for HSPG2 in cartilage development/maintenance. However, the mechanisms in which HSPG2 regulates cartilage development are not completely understood. Here, we explored the relationship between this gene and craniofacial development through morpholino-mediated knockdown of hspg2 using zebrafish. RESULTS: Knockdown of hspg2 resulted in abnormal development of the mandibular jaw joint at 5 days post fertilization (DPF). We surmised that defects in mandible development were a consequence of neural crest cell (NCC) dysfunction, as these multipotent progenitors produce the cartilage of the head. Early NCC development was normal in morphant animals as measured by distal-less homeobox 2a (dlx2a) and SRY-box transcription factor 10 (sox10) expression at 1 DPF. However, subsequent analysis at later stages of development (4 DPF) revealed a decrease in the number of Sox10 + and Collagen, type II, alpha 1a (Col2a1a)+ cells within the mandibular jaw joint region of morphants relative to random control injected embryos. Concurrently, morphants showed a decreased expression of nkx3.2, a marker of jaw joint formation, at 4 DPF. CONCLUSIONS: Collectively, these data suggest a complex role for hspg2 in jaw joint formation and late stage NCC differentiation.

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

Inborn 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 are associated with reduced WNT signaling, consequently resulting in abnormal craniofacial development. To test our hypothesis we performed a combination of pharmaceutical inhibition, gene expression assays, and targeted rescue experiments to understand the function of the CSP and WNT signaling during craniofacial development. We demonstrate reduced expression of four canonical WNT downstream target genes in homozygous carriers of the Vu57 allele and reduced axin2 expression, a known WNT target gene, in larvae treated with Ro-48-8071, an inhibitor of cholesterol synthesis. Moreover, activation of WNT signaling via treatment with WNT agonist I completely restored the craniofacial defects present in a subset of animals carrying the Vu57 allele. Collectively, these data suggest interplay between the CSP and WNT signaling during craniofacial development.

Hcfc1a regulates neural precursor proliferation and asxl1 expression in the developing brain.

BACKGROUND: Precise regulation of neural precursor cell (NPC) proliferation and differentiation is essential to ensure proper brain development and function. The HCFC1 gene encodes a transcriptional co-factor that regulates cell proliferation, and previous studies suggest that HCFC1 regulates NPC number and differentiation. However, the molecular mechanism underlying these cellular deficits has not been completely characterized. METHODS: Here we created a zebrafish harboring mutations in the hcfc1a gene (the hcfc1aco60/+ allele), one ortholog of HCFC1, and utilized immunohistochemistry and RNA-sequencing technology to understand the function of hcfc1a during neural development. RESULTS: The hcfc1aco60/+ allele results in an increased number of NPCs and increased expression of neuronal and glial markers. These neural developmental deficits are associated with larval hypomotility and the abnormal expression of asxl1, a polycomb transcription factor, which we identified as a downstream effector of hcfc1a. Inhibition of asxl1 activity and/or expression in larvae harboring the hcfc1aco60/+ allele completely restored the number of NPCs to normal levels. CONCLUSION: Collectively, our data demonstrate that hcfc1a regulates NPC number, NPC proliferation, motor behavior, and brain development.

Mutations in THAP11 cause an inborn error of cobalamin metabolism and developmental abnormalities.

CblX (MIM309541) is an X-linked recessive disorder characterized by defects in cobalamin (vitamin B12) metabolism and other developmental defects. Mutations in HCFC1, a transcriptional co-regulator which interacts with multiple transcription factors, have been associated with cblX. HCFC1 regulates cobalamin metabolism via the regulation of MMACHC expression through its interaction with THAP11, a THAP domain-containing transcription factor. The HCFC1/THAP11 complex potentially regulates genes involved in diverse cellular functions including cell cycle, proliferation, and transcription. Thus, it is likely that mutation of THAP11 also results in biochemical and other phenotypes similar to those observed in patients with cblX. We report a patient who presented with clinical and biochemical phenotypic features that overlap cblX, but who does not have any mutations in either MMACHC or HCFC1. We sequenced THAP11 by Sanger sequencing and discovered a potentially pathogenic, homozygous variant, c.240C > G (p.Phe80Leu). Functional analysis in the developing zebrafish embryo demonstrated that both THAP11 and HCFC1 regulate the proliferation and differentiation of neural precursors, suggesting important roles in normal brain development. The loss of THAP11 in zebrafish embryos results in craniofacial abnormalities including the complete loss of Meckel's cartilage, the ceratohyal, and all of the ceratobranchial cartilages. These data are consistent with our previous work that demonstrated a role for HCFC1 in vertebrate craniofacial development. High throughput RNA-sequencing analysis reveals several overlapping gene targets of HCFC1 and THAP11. Thus, both HCFC1 and THAP11 play important roles in the regulation of cobalamin metabolism as well as other pathways involved in early vertebrate development.

Kabuki syndrome genes KMT2D and KDM6A: functional analyses demonstrate critical roles in craniofacial, heart and brain development.

Kabuki syndrome (KS) is a rare multiple congenital anomaly syndrome characterized by distinctive facial features, global developmental delay, intellectual disability and cardiovascular and musculoskeletal abnormalities. While mutations in KMT2D have been identified in a majority of KS patients, a few patients have mutations in KDM6A. We analyzed 40 individuals clinically diagnosed with KS for mutations in KMT2D and KDM6A. Mutations were detected in KMT2D in 12 and KDM6A in 4 cases, respectively. Observed mutations included single-nucleotide variations and indels leading to frame shifts, nonsense, missense or splice-site alterations. In two cases, we discovered overlapping chromosome X microdeletions containing KDM6A. To further elucidate the functional roles of KMT2D and KDM6A, we knocked down the expression of their orthologs in zebrafish. Following knockdown of kmt2d and the two zebrafish paralogs kdm6a and kdm6al, we analyzed morphants for developmental abnormalities in tissues that are affected in individuals with KS, including craniofacial structures, heart and brain. The kmt2d morphants exhibited severe abnormalities in all tissues examined. Although the kdm6a and kdm6al morphants had similar brain abnormalities, kdm6a morphants exhibited craniofacial phenotypes, whereas kdm6al morphants had prominent defects in heart development. Our results provide further support for the similar roles of KMT2D and KDM6A in the etiology of KS by using a vertebrate model organism to provide direct evidence of their roles in the development of organs and tissues affected in KS patients.

An X-linked cobalamin disorder caused by mutations in transcriptional coregulator HCFC1.

Derivatives of vitamin B12 (cobalamin) are essential cofactors for enzymes required in intermediary metabolism. Defects in cobalamin metabolism lead to disorders characterized by the accumulation of methylmalonic acid and/or homocysteine in blood and urine. The most common inborn error of cobalamin metabolism, combined methylmalonic acidemia and hyperhomocysteinemia, cblC type, is caused by mutations in MMACHC. However, several individuals with presumed cblC based on cellular and biochemical analysis do not have mutations in MMACHC. We used exome sequencing to identify the genetic basis of an X-linked form of combined methylmalonic acidemia and hyperhomocysteinemia, designated cblX. A missense mutation in a global transcriptional coregulator, HCFC1, was identified in the index case. Additional male subjects were ascertained through two international diagnostic laboratories, and 13/17 had one of five distinct missense mutations affecting three highly conserved amino acids within the HCFC1 kelch domain. A common phenotype of severe neurological symptoms including intractable epilepsy and profound neurocognitive impairment, along with variable biochemical manifestations, was observed in all affected subjects compared to individuals with early-onset cblC. The severe reduction in MMACHC mRNA and protein within subject fibroblast lines suggested a role for HCFC1 in transcriptional regulation of MMACHC, which was further supported by the identification of consensus HCFC1 binding sites in MMACHC. Furthermore, siRNA-mediated knockdown of HCFC1 expression resulted in the coordinate downregulation of MMACHC mRNA. This X-linked disorder demonstrates a distinct disease mechanism by which transcriptional dysregulation leads to an inborn error of metabolism with a complex clinical phenotype.

Hcfc1b, a zebrafish ortholog of HCFC1, regulates craniofacial development by modulating mmachc expression.

Mutations in HCFC1 (MIM300019), have been recently associated with cblX (MIM309541), an X-linked, recessive disorder characterized by multiple congenital anomalies including craniofacial abnormalities. HCFC1 is a transcriptional co-regulator that modulates the expression of numerous downstream target genes including MMACHC, but it is not clear how these HCFC1 targets play a role in the clinical manifestations of cblX. To begin to elucidate the mechanism by which HCFC1 modulates disease phenotypes, we have carried out loss of function analyses in the developing zebrafish. Of the two HCFC1 orthologs in zebrafish, hcfc1a and hcfc1b, the loss of hcfc1b specifically results in defects in craniofacial development. Subsequent analysis revealed that hcfc1b regulates cranial neural crest cell differentiation and proliferation within the posterior pharyngeal arches. Further, the hcfc1b-mediated craniofacial abnormalities were rescued by expression of human MMACHC, a downstream target of HCFC1 that is aberrantly expressed in cblX. Furthermore, we tested distinct human HCFC1 mutations for their role in craniofacial development and demonstrated variable effects on MMACHC expression in humans and craniofacial development in zebrafish. Notably, several individuals with mutations in either HCFC1 or MMACHC have been reported to have mild to moderate facial dysmorphia. Thus, our data demonstrates that HCFC1 plays a role in craniofacial development, which is in part mediated through the regulation of MMACHC expression.

3D Mitochondrial Structure in Aging Human Skeletal Muscle: Insights into MFN-2 Mediated Changes.

Age-related atrophy of skeletal muscle, is characterized by loss of mass, strength, endurance, and oxidative capacity during aging. Notably, bioenergetics and protein turnover studies have shown that mitochondria mediate this decline in function. Although exercise has been the only therapy to mitigate sarcopenia, the mechanisms that govern how exercise serves to promote healthy muscle aging are unclear. Mitochondrial aging is associated with decreased mitochondrial capacity, so we sought to investigate how aging affects mitochondrial structure and potential age-related regulators. Specifically, the three-dimensional (3D) mitochondrial structure associated with morphological changes in skeletal muscle during aging requires further elucidation. We hypothesized that aging causes structural remodeling of mitochondrial 3D architecture representative of dysfunction, and this effect is mitigated by exercise. We used serial block-face scanning electron microscopy to image human skeletal tissue samples, followed by manual contour tracing using Amira software for 3D reconstruction and subsequent analysis of mitochondria. We then applied a rigorous in vitro and in vivo exercise regimen during aging. Across 5 human cohorts, we correlate differences in magnetic resonance imaging, mitochondria 3D structure, exercise parameters, and plasma immune markers between young (under 50 years) and old (over 50 years) individuals. We found that mitochondria we less spherical and more complex, indicating age-related declines in contact site capacity. Additionally, aged samples showed a larger volume phenotype in both female and male humans, indicating potential mitochondrial swelling. Concomitantly, muscle area, exercise capacity, and mitochondrial dynamic proteins showed age-related losses. Exercise stimulation restored mitofusin 2 (MFN2), one such of these mitochondrial dynamic proteins, which we show is required for the integrity of mitochondrial structure. Furthermore, we show that this pathway is evolutionarily conserved as Marf, the MFN2 ortholog in Drosophila, knockdown alters mitochondrial morphology and leads to the downregulation of genes regulating mitochondrial processes. Our results define age-related structural changes in mitochondria and further suggest that exercise may mitigate age-related structural decline through modulation of mitofusin 2.

MICOS Complex Loss Governs Age-Associated Murine Mitochondrial Architecture and Metabolism in the Liver, While Sam50 Dictates Diet Changes.

The liver, the largest internal organ and a metabolic hub, undergoes significant declines due to aging, affecting mitochondrial function and increasing the risk of systemic liver diseases. How the mitochondrial three-dimensional (3D) structure changes in the liver across aging, and the biological mechanisms regulating such changes confers remain unclear. In this study, we employed Serial Block Face-Scanning Electron Microscopy (SBF-SEM) to achieve high-resolution 3D reconstructions of murine liver mitochondria to observe diverse phenotypes and structural alterations that occur with age, marked by a reduction in size and complexity. We also show concomitant metabolomic and lipidomic changes in aged samples. Aged human samples reflected altered disease risk. To find potential regulators of this change, we examined the Mitochondrial Contact Site and Cristae Organizing System (MICOS) complex, which plays a crucial role in maintaining mitochondrial architecture. We observe that the MICOS complex is lost during aging, but not Sam50. Sam50 is a component of the sorting and assembly machinery (SAM) complex that acts in tandem with the MICOS complex to modulate cristae morphology. In murine models subjected to a high-fat diet, there is a marked depletion of the mitochondrial protein SAM50. This reduction in Sam50 expression may heighten the susceptibility to liver disease, as our human biobank studies corroborate that Sam50 plays a genetically regulated role in the predisposition to multiple liver diseases. We further show that changes in mitochondrial calcium dysregulation and oxidative stress accompany the disruption of the MICOS complex. Together, we establish that a decrease in mitochondrial complexity and dysregulated metabolism occur with murine liver aging. While these changes are partially be regulated by age-related loss of the MICOS complex, the confluence of a murine high-fat diet can also cause loss of Sam50, which contributes to liver diseases. In summary, our study reveals potential regulators that affect age-related changes in mitochondrial structure and metabolism, which can be targeted in future therapeutic techniques.

Overexpression of MMACHC Prevents Craniofacial Phenotypes Caused by Knockdown of znf143b.

ZNF143 is a sequence-specific DNA binding protein that regulates the expression of protein-coding genes and small RNA molecules. In humans, ZNF143 interacts with HCFC1, a transcriptional cofactor, to regulate the expression of downstream target genes, including MMACHC, which encodes an enzyme involved in cobalamin (cbl) metabolism. Mutations in HCFC1 or ZNF143 cause an inborn error of cobalamin metabolism characterized by abnormal cbl metabolism, intellectual disability, seizures, and mild to moderate craniofacial abnormalities. However, the mechanisms by which ZNF143 mutations cause individual phenotypes are not completely understood. Defects in metabolism and craniofacial development are hypothesized to occur because of decreased expression of MMACHC. But recent results have called into question this mechanism as the cause for craniofacial development. Therefore, in the present study, we implemented a loss of function analysis to begin to uncover the function of ZNF143 in craniofacial development using the developing zebrafish. The knockdown of znf143b, one zebrafish ortholog of ZNF143, caused craniofacial phenotypes of varied severity, which included a shortened and cleaved Meckel's cartilage, partial loss of ceratobranchial arches, and a distorted ceratohyal. These phenotypes did not result from a defect in the number of total chondrocytes but were associated with a mild to moderate decrease in mmachc expression. Interestingly, expression of human MMACHC via endogenous transgene prevented the onset of craniofacial phenotypes associated with znf143b knockdown. Collectively, our data establishes that knockdown of znf143b causes craniofacial phenotypes that can be alleviated by increased expression of MMACHC.

Missense and nonsense mutations of the zebrafish hcfc1a gene result in contrasting mTor and radial glial phenotypes.

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, murine Hcfc1 regulates neural precursor (NPCs) proliferation and number, which has been validated in zebrafish. However, conditional deletion of mouse Hcfc1 in Nkx2.1 + cells increased cell death, reduced Gfap expression, and reduced numbers of GABAergic neurons. Thus, the role of this gene 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. 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 may be associated with contrasting cellular phenotypes.

Characterization of the zebrafish gabra1sa43718/sa43718 germline loss of function allele confirms a function for Gabra1 in motility and nervous system development.

Mutation of the GABRA1 gene is associated with neurodevelopmental defects and epilepsy. GABRA1 encodes for the α1 subunit of the gamma-aminobutyric acid type A receptor (GABAAR), which regulates the fast inhibitory impulses of the nervous system. Multiple model systems have previously been developed to understand the function of GABRA1 during development, but these models have produced complex and at times incongruent data. Thus, additional model systems are required to validate and substantiate previously published results. We investigated the behavioral swim patterns associated with a nonsense mutation of the zebrafish gabra1 (sa43718 allele) gene. The sa43718 allele causes a decrease in gabra1 mRNA expression, which is associated with light induced hypermotility, one phenotype associated with seizure like behavior in zebrafish. Mutation of gabra1 was accompanied by decreased mRNA expression of gabra2, gabra3, and gabra5, indicating a reduction in the expression of additional alpha sub-units of the GABAAR. Although multiple sub-units were decreased in total expression, larvae continued to respond to pentylenetetrazole (PTZ) indicating that a residual GABAAR exists in the sa43718 allele. Proteomics analysis demonstrated that nonsense mutation of gabra1 is associated with abnormal expression of proteins that regulate proton transport, ion homeostasis, vesicle transport, and mitochondrial protein complexes. These data support previous studies performed in a zebrafish nonsense allele created by CRISPR/Cas9 and validate that loss of function mutations in the gabra1 gene result in seizure like phenotypes with abnormal function of inhibitory synapses.

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.

3D reconstruction of murine mitochondria reveals changes in structure during aging linked to the MICOS complex.

During aging, muscle gradually undergoes sarcopenia, the loss of function associated with loss of mass, strength, endurance, and oxidative capacity. However, the 3D structural alterations of mitochondria associated with aging in skeletal muscle and cardiac tissues are not well described. Although mitochondrial aging is associated with decreased mitochondrial capacity, the genes responsible for the morphological changes in mitochondria during aging are poorly characterized. We measured changes in mitochondrial morphology in aged murine gastrocnemius, soleus, and cardiac tissues using serial block-face scanning electron microscopy and 3D reconstructions. We also used reverse transcriptase-quantitative PCR, transmission electron microscopy quantification, Seahorse analysis, and metabolomics and lipidomics to measure changes in mitochondrial morphology and function after loss of mitochondria contact site and cristae organizing system (MICOS) complex genes, Chchd3, Chchd6, and Mitofilin. We identified significant changes in mitochondrial size in aged murine gastrocnemius, soleus, and cardiac tissues. We found that both age-related loss of the MICOS complex and knockouts of MICOS genes in mice altered mitochondrial morphology. Given the critical role of mitochondria in maintaining cellular metabolism, we characterized the metabolomes and lipidomes of young and aged mouse tissues, which showed profound alterations consistent with changes in membrane integrity, supporting our observations of age-related changes in muscle tissues. We found a relationship between changes in the MICOS complex and aging. Thus, it is important to understand the mechanisms that underlie the tissue-dependent 3D mitochondrial phenotypic changes that occur in aging and the evolutionary conservation of these mechanisms between Drosophila and mammals.

Identification and regulation of c-Myb target genes in MCF-7 cells.

BACKGROUND: The c-Myb transcription factor regulates differentiation and proliferation in hematopoietic cells, stem cells and epithelial cells. Although oncogenic versions of c-Myb were first associated with leukemias, over expression or rearrangement of the c-myb gene is common in several types of solid tumors, including breast cancers. Expression of the c-myb gene in human breast cancer cells is dependent on estrogen stimulation, but little is known about the activities of the c-Myb protein or what genes it regulates in estrogen-stimulated cells. METHODS: We used chromatin immunoprecipitation coupled with whole genome promoter tiling microarrays to identify endogenous c-Myb target genes in human MCF-7 breast cancer cells and characterized the activity of c-Myb at a panel of target genes during different stages of estrogen deprivation and stimulation. RESULTS: By using different antibodies and different growth conditions, the c-Myb protein was found associated with over 10,000 promoters in MCF-7 cells, including many genes that encode cell cycle regulators or transcription factors and more than 60 genes that encode microRNAs. Several previously identified c-Myb target genes were identified, including CCNB1, MYC and CXCR4 and novel targets such as JUN, KLF4, NANOG and SND1. By studying a panel of these targets to validate the results, we found that estradiol stimulation triggered the association of c-Myb with promoters and that association correlated with increased target gene expression. We studied one target gene, CXCR4, in detail, showing that c-Myb associated with the CXCR4 gene promoter and activated a CXCR4 reporter gene in transfection assays. CONCLUSIONS: Our results show that c-Myb associates with a surprisingly large number of promoters in human cells. The results also suggest that estradiol stimulation leads to large-scale, genome-wide changes in c-Myb activity and subsequent changes in gene expression in human breast cancer cells.

Leading by example: developing mentoring strategies to promote diversity and student success.

Research has shown that individuals from diverse backgrounds and women are underrepresented in the science, technology, engineering, and mathematics (STEM) fields. A lack of identifiable role models/mentorship and poor mentoring experiences are a few cited factors that continue to limit increased diversity. As an underrepresented individual and a faculty member at a minority-serving institution, I strive to provide my students with a strong example, one that they can identify with. Part of my approach has been to develop mentoring pillars and strategies that seek to build relationships with my mentees and that aim to improve their research experience. This essay briefly describes my experiences as a mentor and the mentoring pillars I developed to promote a diverse and inclusive environment for my current and future mentees.

The role of HCFC1 in syndromic and non-syndromic intellectual disability.

Mutations in the HCFC1 gene are associated with cases of syndromic (cblX) and non-syndromic intellectual disability. Syndromic individuals present with severe neurological defects including intractable epilepsy, facial dysmorphia, and intellectual disability. Non-syndromic individuals have also been described and implicate a role for HCFC1 during brain development. The penetrance of phenotypes and the presence of an overall syndrome is associated with the location of the mutation within the HCFC1 protein. Thus, one could hypothesize that the positioning of HCFC1 mutations lead to different neurological phenotypes that include but are not restricted to intellectual disability. The HCFC1 protein is comprised of multiple domains that function in cellular proliferation/metabolism. Several reports of HCFC1 disease variants have been identified, but a comprehensive review of each variant and its associated phenotypes has not yet been compiled. Here we perform a detailed review of HCFC1 function, model systems, variant location, and accompanying phenotypes to highlight current knowledge and the future status of the field.