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

Hernández, José Ángel; Castro, Victoria L.; Reyes-Nava, Nayeli G; Montes, Laura P.; Quintana, Anita M.

doi:10.1182/bloodadvances.2018024539

Cited by 15 publications

(24 citation statements)

References 81 publications

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“…For each sh, a minimum of 20 to 30 z-stacks were collected. Maximum intensity projection images were processed and saved (32). For cell counts, the number of cells per zstack (20-30/ sh) at each jaw joint region (3 rows of chondrocytes on the left side of the joint and 5 rows on the right) were counted using ImageJ.…”

Section: Confocal Imaging and Transgenic Cell Countsmentioning

confidence: 99%

Knockdown of hspg2 is associated with mandibular jaw joint fusion and neural crest cell dysfunction in zebrafish

Castellanos¹,

Reyes-Nava²,

Quintana³

2020

Preprint

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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 and Dyssegmental Dysplasia Silverman-Handmaker Type, 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 in zebrafish. Results: Knockdown of hspg2 resulted in a fusion 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 NK3 homeobox 2 (nkx3.2), a jaw joint molecular marker at 4 dpf. Conclusions: Collectively, these data suggest a complex role for hspg2 in jaw joint formation and late stage NCC differentiation.

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Section: Confocal Imaging and Transgenic Cell Countsmentioning

confidence: 99%

Knockdown of hspg2 is associated with mandibular jaw joint fusion and neural crest cell dysfunction in zebrafish

Castellanos¹,

Reyes-Nava²,

Quintana³

2020

Preprint

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“…Polymerase chain reaction (PCR) was used to amplify the target sequence of the hmgcs1 gene. PCR and restriction digest was performed as previously described in (Quintana, Hernandez, & Gonzalez, 2017) (Hernandez et al, 2019).…”

Section: Methodsmentioning

confidence: 99%

“…Primers for qPCR are as follows: 1) Axin2 fwd 5’-CAACCAAGCACATCCATCAC-3’ and rev 5’-TCCATGTTCACCTCCTCTCC-3’, 2) rpl fwd 5’-TCCCAGCTGCTCTCAAGATT-3’ and rev 5’-TTCTTGGAATAGCGCAGCTT-3’, and 3) Sox10 fwd 5’-ACGCTACAGGTCAGAGTCAC-3’ and rev 5’-ATGTTGGCCATCACGTCATG-3’. Whole mount in situ hybridization was performed as previously described (Hernandez et al, 2019; Quintana et al, 2017; Thisse & Thisse, 2008). The axin2 riboprobe was produced with forward primer 5’-TGCACTGCTCCTTACATTCG-3’ and reverse primer 5’-GCTCCAGGACAAGGCTACTG-3’.…”

Section: Methodsmentioning

confidence: 99%

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

Castro

Reyes-Nava

Sanchez

et al. 2020

Preprint

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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.

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“…This region was selected because these cells were consistently visible across zstacks. Statistical signi cance was obtained by using a Student t-test with random control and morphant data (63). dlx2a expression is normal in morphants.…”

Section: Confocal Imaging and Transgenic Cell Countsmentioning

confidence: 99%

Knockdown of hspg2 is associated with mandibular jaw joint fusion and neural crest cell dysfunction in zebrafish

Castellanos

Reyes-Nava

Quintana

2020

Preprint

View full text Add to dashboard Cite

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 by 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. Concurrently, morphants showed a decreased expression of NK3 homeobox 2 (nkx3.2), a jaw joint molecular marker at 4 DPF. Conclusions: Collectively, these data suggest a complex role for hspg2 in jaw joint formation and late stage NCC differentiation.

show abstract

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

Cited by 15 publications

References 81 publications

Knockdown of hspg2 is associated with mandibular jaw joint fusion and neural crest cell dysfunction in zebrafish

Knockdown of hspg2 is associated with mandibular jaw joint fusion and neural crest cell dysfunction in zebrafish

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

Knockdown of hspg2 is associated with mandibular jaw joint fusion and neural crest cell dysfunction in zebrafish

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