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. 2014 Dec 1;396(1):94-106.
doi: 10.1016/j.ydbio.2014.09.026. Epub 2014 Oct 2.

Hcfc1b, a Zebrafish Ortholog of HCFC1, Regulates Craniofacial Development by Modulating Mmachc Expression

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Free PMC article

Hcfc1b, a Zebrafish Ortholog of HCFC1, Regulates Craniofacial Development by Modulating Mmachc Expression

Anita M Quintana et al. Dev Biol. .
Free PMC article

Abstract

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.

Keywords: Cobalamin; Craniofacial defects; Facial dysmorphia; HCFC1; MMACHC.

Figures

Figure 1
Figure 1. Knockdown of hcfc1b causes craniofacial abnormalities
A. Morpholino (MO) mediated knockdown of hcfc1a and hcfc1b. Western blot with anti-HCFC1 antibodies performed at 24 hours post fertilization (hpf) on wildtype (WT) or on embryos co-injected with morpholinos targeting hcfc1a or hcfc1b. Pan actin is shown as a loading control. The HCFC1 antibody is predicted to recognize both Hcfc1a and Hcfc1b and therefore a single band represents expression of both isoforms. B. RT-PCR demonstrating the effectiveness of a MO specifically targeting hcfc1a mRNA splicing. Injection of the morpholino causes out of frame excision of exon 2 and results in a premature stop codon (Schematic demonstrates normal and aberrant splicing).
Figure 2
Figure 2. Loss of hcfc1b causes defects in craniofacial development
A-D. Alcian:Alizarin staining was performed to visualize the developing cartilage in non-injected controls (NI), hcfc1a morphants (hcfc1a MO), hcfc1b morphants (hcfc1b MO), or embryos co-injected with hcfc1b MO and in vitro synthesized human HCFC1 mRNA. Embryos were stained at 5 days post fertilization and manual dissection of the viscerocranium and neurocranium was performed. Neurocranium is depicted in A-D and the vicserocranium is depicted in A’-D’.
Figure 3
Figure 3. NCCs are specified and migrate correctly in morphant animals
A-B. Dorsal view (anterior to posterior from left to right) of Tg(Sox10:mRFP) embryos injected with an hcfc1b targeting morpholino (hcfc1b-MO) were visualized for RFP expression at the 12 somite stage. C-D. Sagittal view of non-injected (NI) or hcfc1b MO injected Tg(Sox10:mRFP) larvae at 30 hours post fertilization (hpf). Arrows depict the formation of the pharyngeal arches in both wildtype and morphant animals. E-H. In situ hybridization (ISH) of dlx2a in non-injected (NI) and hcfc1b morphants (hcfc1b MO) at 1 day post fertilization (dpf). I-J. Lateral view of non-injected Tg(prdm1a:EGFP) (NI) or Tg(prdm1a:EGFP) embryos injected with the hcfc1b targeting morpholino (hcfc1b MO). The encircled area depicts posterior arch expression of EGFP in transgenic animals.
Figure 4
Figure 4. Knockdown of hcfc1b results in reduced expression of the molecular markers associated with chondrocyte differentiation
A-B. Ventral view of RNA in situ hybridization (ISH) of col2a1 or sox9a expression in non-injected (NI) control larvae or hcfc1b morphants (hcfc1b MO) at 3 days post fertilization (dpf). C-C’. Ventral view of non-injected (NI) control, Tg(sox10:memRFP) larvae, or transgenic larvae injected with hcfc1b morpholino (hcfc1b MO) at 3 days post fertilization.
Figure 5
Figure 5. hcfc1b regulates cell proliferation
A-B Z-stacks for whole mount immunohistochemistry with an anti-pH3 antibody in non-injected Tg(sox10:EGFP) larvae or larvae injected with an hcfc1b targeting morpholino (hcfc1b MO). C. Quantification of the number of pH3/Sox10 positive cells in non-injected controls or hcfc1b morphants (p= 2.61431E-05, n=6 fish per group). Statistical analysis was performed using a standard T-test.
Figure 6
Figure 6. Hcfc1 by modulates mmachc expression
A. Real time quantitative PCR analysis measuring mmachc expression in non-injected embryos (NI), hcfc1a morphant embryos (hcfc1a MO), hcfc1b morphant embryos (hcfc1b MO), or combined hcfc1a and hcfc1b morphant embryos (combo-MO). A total of ten individual embryos were analyzed per group on 2 independent occasions.
Figure 7
Figure 7. Craniofacial defects associated will loss of hcfc1b are mediated through mmachc expression
A-F. Staining of cartilage with Alcian blue in non-injected (NI), hcfc1b morphants (hcfc1b MO), mmachc morphants (mmachc MO), human MMACHC mRNA, hcfc1b MO co-injected MMACHC mRNA, or mmachc-MO injected with MMACHC mRNA. Numbers of animals are listed in Supplementary Tables 2 and 4. Embryos were stained at 4 days post fertilization and manual dissection of the viscerocranium and neurocranium was performed. Neurocranium is depicted in A-F and the vicserocranium is depicted in A’-F’.
Figure 8
Figure 8. Mutations in HCFC1 abrogate binding and reduce the expression of MMACHC
A. QPCR demonstrating the relative expression of MMACHC in fibroblasts obtained from a normal reference donor (Ref), subject with mutation c.344C>T (Sbj. 1 344) or subject with mutation c.218C>T, (Sbj. 2 218). QPCR was performed with two independent biological replicates. *p= 0.000593 ** p=0.000456 Statistical analysis was performed using a standard T-test. B. Chromatin Immunoprecipitation with either control IgG or anti-HCFC1 antibodies was performed on fibroblasts obtained from a normal reference donor (Ref), a subject with mutation c.344C>T (Sbj. 1 344), or a subject with mutation c.218C>T, (Sbj. 2 218). QPCR with primers flanking the presumed HCFC1 binding motif in the MMACHC regulatory region was used to address occupancy at the promoter region. Assay was performed in two independent biological replicates. Standard T-test was used to determine statistical significance. *p=9.8E-7, **p=0.0014. Dark line demonstrates normalization from IgG reference sample.
Figure 9
Figure 9. Mutations in HCFC1 differentially affect craniofacial development
A-F. Staining of cartilage with Alcian blue on non-injected (NI), hcfc1b MO, or hcfc1b MO co-injected with the indicated mutated version of HCFC1. Numbers of animals affected are presented in Supplementary Table 5. Embryos were stained at 5 days post fertilization and manual dissection of the viscerocranium and neurocranium was performed. Neurocranium is depicted in A-F and the vicserocranium is depicted in A’-F’.

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