10 research outputs found

    Additional file 1: Table S1. of Partial uniparental isodisomy of chromosome 16 unmasks a deleterious biallelic mutation in IFT140 that causes Mainzer-Saldino syndrome

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    Clinical genetic testing summary. Table S2. Whole exome sequencing capture statistics. Table S3. Final variant list following trio-based exome analysis (<1% minor allele frequency (MAF), homozygous, compound heterozygous, de novo, or X-linked). Figure S1. Fundus imaging and full field electroretinograms indicate retinal dystrophy. a. Fundus imaging of the proband at 5 years 6 months in the right (top) and left (bottom) eyes show mottling of the retinal pigmentary epithelium. b. Photopic (light adapted) testing of cone function demonstrated reduced a-wave implicit time and amplitudes with slowed implicit times for the b-wave. c. Scotopic (dark adapted) testing of rod function demonstrated slower implicit time and diminished amplitudes for both a- and b-waves. The a-wave was substantially delayed with reduced incremental increases in a-wave and b-wave. Figure S2. Validation of the ift140 splice-blocking (sb) morpholino (MO). a. Schematic of the D. rerio ift140 locus at chr24:37,937,737-38,013,596 (GRCz10; top) and translated protein (bottom). Exons are depicted as green boxes; untranslated regions are shown as white boxes (ENSDART00000129889.4). The sb-MO targets the splice donor of exon (Ex) 2 (red box). Protein schematic (blue) indicates predicted WD40 repeat (WD40) and tetratricopeptide-like helical domains (TPR). b. ift140 sb-MO induces aberrant splicing of endogenous transcript. ift140 transcript was evaluated by RT-PCR; embryos were injected with 9 ng MO at the one-to-four-cell stage and harvested for RNA extraction at the mid-somitic stage. Resulting cDNA was amplified with primers flanking the MO target site (arrows in panel a), and migrated on a 2.5% agarose gel. β–actin was used as a control to ensure RNA integrity. c. Chromatograms indicate splicing defects in ift140 mRNA in morphants. Sequence analysis of purified PCR product indicates that the majority of ift140 message is missing exon 2 (bottom), which contains the translation initiation codon. Modest amounts of wild type (wt; center) and mRNA containing a 33 nucleotide in-frame intronic insertion (top) are also detectable. d. Representative dose curve of the ift140 sb-MO. Embryo batches were injected at the one-to-four-cell stage with increasing amounts of ift140 sb-MO and scored for gastrulation defects at the mid-somitic stage (eight to ten somites). Embryos were scored live according to previously established phenotypic criteria: class I, modest shortening of the body axis and reduction in size of anterior structures; class II, severe shortening of the body axis and decreased anterior structures accompanied by broadening and/or kinking of the notochord and thinning of the somites (See Figure 3; n = 59–70 embryos/injection; repeated twice). Figure S3. Read depth and variant calls of the homozygous variants called on 16p13. Plot of the 76 maternally inherited homozygous variants identified in the 16p13 region of the proband. Red indicates alternate (alt) reads; blue indicates reference (ref) reads; 49/76 (64%) are comprised only of alternate reads; 27/76 (36%) had one to three reference reads at positions annotated as homozygous. (PDF 20018 kb

    Transient <i>laminin beta</i> 1a Induction Defines the Wound Epidermis during Zebrafish Fin Regeneration

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    <div><p>The first critical stage in salamander or teleost appendage regeneration is creation of a specialized epidermis that instructs growth from underlying stump tissue. Here, we performed a forward genetic screen for mutations that impair this process in amputated zebrafish fins. Positional cloning and complementation assays identified a temperature-sensitive allele of the ECM component <i>laminin beta 1a</i> (<i>lamb1a</i>) that blocks fin regeneration. <i>lamb1a</i>, but not its paralog <i>lamb1b</i>, is sharply induced in a subset of epithelial cells after fin amputation, where it is required to establish and maintain a polarized basal epithelial cell layer. These events facilitate expression of the morphogenetic factors <i>shha</i> and <i>lef1</i>, basolateral positioning of phosphorylated Igf1r, patterning of new osteoblasts, and regeneration of bone. By contrast, <i>lamb1a</i> function is dispensable for juvenile body growth, homeostatic adult tissue maintenance, repair of split fins, or renewal of genetically ablated osteoblasts. <i>fgf20a</i> mutations or transgenic Fgf receptor inhibition disrupt <i>lamb1a</i> expression, linking a central growth factor to epithelial maturation during regeneration. Our findings reveal transient induction of <i>lamb1a</i> in epithelial cells as a key, growth factor-guided step in formation of a signaling-competent regeneration epidermis.</p></div

    <i>lamb1a</i>, not <i>lamb1b</i>, is induced during regeneration.

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    <p>(A) RT-qPCR analysis indicates that <i>lamb1a</i>, but not <i>lamb1b</i>, is induced during regeneration. qPCR results were normalized to <i>rpl13a</i> and to the basal expression of <i>lamb1a</i>/<i>lamb1b</i> at 0 hours post-amputation (hpa). (n = 3; mean ± SEM). (B) Section ISH indicating that <i>lamba1</i> becomes visually detectable in the basal epithelial layer between 6 and 24 hpa. (C) Left: cartoon depicting basic cellular makeup of the fin regenerate. Right: <i>lamb1a</i> is expressed in basal epithelial cells and some mesenchymal cells at 3 dpa. (D) Antibody staining for Laminin expression in regenerating fins. Laminin protein is restricted to the basal side of the basal epithelial cells layer by 2 dpa in wild-type or <i>sde1/+</i> regenerates, but mislocalized to all regions of basal cells in <i>sde1</i> mutants. (E) Laminin expression at 5 dpa and 60 dpa, indicating that Laminin presence is transient during regenerative outgrowth. i: distal, newly regenerated tissue; ii: proximal regenerated tissue. Laminin, red; DAPI, blue. Scale bars, 50 μm. White dashed boxes indicate areas of enlarged view. Red arrows indicate plane of amputation.</p

    Association of <i>lamb1a</i> expression and function with key regeneration effector pathways.

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    <p>(A) Longitudinal sections of 2 dpa fin regenerates stained for <i>lamb1a</i> by ISH, indicating sparse expression in <i>fgf20a</i> mutants (<i>dob</i>). Scale bars, 50 μm. (B) RT-qPCR analysis indicating depleted levels of <i>lamb1a</i> RNA in <i>fgf20a</i> mutants (left). When Fgf signaling is blocked by induced expression of a dominant–negative Fgf receptor for just 6 hours at 4 dpa, <i>lamb1a</i> levels drop by nearly 50%. qPCR results were normalized to <i>rpl13a</i> and to the basal expression of <i>lamb1a</i> at 0 hpa. (n = 4; mean ± SEM; Student’s <i>t</i> -test, ***<i>P</i> < 0.001, **<i>P</i> < 0.01). (C) Whole-mount images of fin regenerates stained by alizarin red staining for calcium deposition, after treatment of <i>sde1</i> animals with DMSO or FK506. Scale bars, 0.5 mm. (D) Measurement of the length of alizarin red-positive domains at 7 dpa (n = 9 and 10; mean ± SEM; Student’s <i>t</i> -test, ***<i>P</i> < 0.001). (E) Antibody staining for Laminin protein in vehicle- or FK506-treated <i>sde1</i> fin regenerates at 7 dpa. Laminin, red; DAPI, blue. (F) Antibody co-staining for aPKC (red) and P63 (green) expression in vehicle or FK506-treated <i>sde1</i> fin regenerates at 7 dpa. FK506 treatment partially rescued bone regeneration in <i>sde1</i> mutants, with no detectable impact on Laminin localization or basal epithelial cell polarity. Scale bars, 50 μm. White dashed boxes indicate areas of enlarged view. White arrows indicate basal cell nuclei. Red arrows indicate plane of amputation.</p

    <i>sde1</i> requirements for tissue regeneration depend on injury context.

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    <p>(A) Measurement of <i>sde1</i> fin regenerates at 7 dpa. After amputation, animals were shifted from the permissive temperature (25°C) to the restrictive temperature (33°C) at 0, 1 or 2 dpa. TS, temperature shift. (n = 15, 16, and 21; Student’s <i>t</i> -test, ***<i>P</i> < 0.001; NS, non-significant). (B) Adult <i>sde1/+</i> and <i>sde1</i> animals were incubated at 33°C for two months (n = 11 and 9). Red arrows point to a damaged fin edge in <i>sde1</i>. The most severe example of damage in <i>sde1</i> animals is displayed here (3 of 9 showed damage in the experiment). (C) (Top) Cartoon depicting the model of incision injuries. (Bottom) Whole-mount images were acquired at 0 and 2 days post incision injury (dpi). Images from the same animal are shown before (top) and after repair (bottom). White arrows indicate sites of injury (n = 12 and 16). (D) (Top) Cartoon depicting the model of osteoblast ablation. (Bottom) Fluorescence intensity, indicating recovery of genetically labeled osteoblasts after ablation, was recorded at 4, 7, 10, and 14 days post Mtz treatment (dpt) and quantified using ImageJ software. White dashed lines indicate fin boundaries. Images from the same animal are shown throughout recovery. (E) Quantification of relative fluorescence intensity from individual animals after osteoblasts ablation (n = 9 and 12; mean ± SEM; Student’s <i>t</i>-test; NS, non-significant). Scale bars, 1 mm.</p

    Model for morphogenesis of the regeneration epidermis.

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    <p>After initial wound closure, <i>fgf20a</i> is induced at the epithelial-mesenchymal boundary by 6 hours post-amputation. <i>fgf20a</i> signaling then contributes to induction of expression of the <i>lamb1a</i> paralog in the adjacent epithelial cell layer to establish a basement membrane and to polarize the basal cell layer. This polarization is essential for positioning signaling receptors and induction of morphogenetic factors that guide osteoblast patterning and bone formation.</p

    <i>lamb1a</i> is required for juvenile fin growth but not body growth.

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    <p><b>(A)</b> Juvenile <i>sde1</i> animals, after incubation from 4 to 6 weeks post-fertilization (wpf) at 33°C, acquire degraded fins. Scale bars, 2 mm. (B) <i>sde1</i> mutations have minimal impact on juvenile outgrowth. Body length was measured from the tip of the snout to the base of caudal fin. (C) RT-qPCR analysis indicates that both <i>lamb1a</i> and <i>lamb1b</i> are induced in fin tissue during juvenile outgrowth. qPCR results were normalized to <i>rpl13a</i> and to the basal expression of <i>lamb1a/lamb1b</i> in adult uninjured fins (n = 4; mean ± SEM). (D) Antibody staining for Laminin expression in juvenile fins. Laminin protein in longitudinal sections of fins is localized to the basal side of the epitheilum in <i>sde1/+</i> animals, but becomes mislocalized in <i>sde1</i> mutants.</p

    Forward genetic screen for signaling defects in the fin regeneration epidermis.

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    <p>(A) Whole-mount images of wild-type and (<i>chc1-9</i>) mutant regenerates at 7 days post amputation (dpa). Red dashed lines indicate plane of amputation. Scale bars, 1 mm. (B) Whole-mount RNA ISH of <i>lef1</i> expression in wild-type and mutant regenerates at 1 dpa. <i>chc1</i>, <i>chc3</i>, and <i>chc4</i> mutant families show reduced or undetectable <i>lef1</i> expression when compared to their respective heterozygous siblings. (C, D) Longitudinal sections of 2 dpa fin regenerates assessed by RNA ISH, showing reduced <i>lef1</i> and <i>shha</i> in <i>chc1</i> mutant regenerates. (E) Whole-mount images of <i>sde1</i> (formerly, <i>chc1</i>) fin regenerates at 4 dpa. Scale bars, 0.5 mm. (F) Longitudinal sections of 3 dpa fin regenerates show impaired patterning of osteoblasts in <i>sde1</i> (<i>chc1</i>) mutants, assessed by Zns-5 antibody staining (green). DAPI, blue. Scale bars, 50 μm (unless otherwise indicated). Red arrows indicate plane of amputation.</p

    <i>sde1</i> encodes a <i>laminin beta1</i> paralog.

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    <p>(A) Analysis of genomic homozygosity in <i>sde1</i> mutants. (B) Log likelihood analysis using SNPtrack. (C) High-resolution mapping using linked SNP markers. Three and five recombinants were found for SNP32559436 and SNP32681121, respectively, on each side of a 121 kb region. After genotyping 453 adult animals, no recombinants were identified for a novel SNP at position 32605161. (D) Sanger sequencing readouts from wild-type and <i>sde1</i> cDNA. SNP32605161 is within the coding region of the gene <i>laminin beta 1a</i> (<i>lamb1a</i>), causing a leucine to proline change. (E) Amino acid alignment across distant species. Red star marks the location of the leucine. Differential gray scale indicates level of conservation across listed species. (F) Cartoon depicting major structural domains in Lamb1a. Blue and red arrows indicate the locations of the <i>sde1</i> mutation, along with two previously identified alleles <i>gup</i><sup><i>m189</i></sup> and <i>s804</i>. Lam NT, Laminin N-terminal domain; Blue hexagons, Laminin-type epidermal growth factor-like domain; CC, uncharacterized coiled-coil domain. (G) <i>sde1</i> embryos incubated at 31°C have shortened trunks. Representative embryos from an <i>sde1</i> x <i>sde1/+</i> cross. Embryos were transferred to 31°C at 3 hours post-fertilization (hpf). Images were acquired at 28 hpf. Approximately 48% of embryos (32 out of 67) showed phenotypes representative of the <i>gup</i><sup><i>m189</i></sup> mutation after the temperature shift, consistent with expected Mendelian ratio. The phenotype and the ratio were consistent across three independent crosses. (H) Complementation tests showing both <i>gup</i><sup><i>m189</i></sup> and <i>s804</i> alleles fail to complement the 7 dpa regeneration defects of the <i>sde1</i> mutation in adult animals, yielding expected ratios (~50%; n = 65 and 30). Red dashed lines indicate plane of amputation. Scale bars, 1 mm.</p

    <i>lamb1a</i> induction defines cell polarity and signaling competence in basal cells of the regeneration epidermis.

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    <p>(A) Antibody co-staining for aPKC (red) and P63 (green; an epithelial maker for all basal and some suprabasal epithelial cells) in longitudinal sections of <i>sde1/+</i> and <i>sde1</i> fin regenerates at 2 dpa. (B) Antibody staining for Laminin in fin regenerates at 4 dpa after a temperature shift from 25°C to 33°C at 3 dpa, indicating induced mislocalization. Laminin, red; DAPI, blue. White dashed boxes indicate areas of enlarged view. (C) Antibody co-staining for aPKC (red) and P63 (green) in longitudinal sections of <i>sde1/+</i> and <i>sde1</i> fin regenerates at 4 dpa after a temperature shift from 25°C to 33°C at 3 dpa, indicating loss of basal cell polarity. iii: distal regenerated tissue (<i>sde1/+</i>); iv: distal regenerated tissue (<i>sde1</i>). White arrows indicate basal cell nuclei. (D) Antibody co-staining for phosphorylated Igf1r (red) and P63 (green) in longitudinal sections of 5 dpa <i>sde1/+</i> and <i>sde1</i> fin regenerates, after a temperature shift from 25°C to 33°C at 4 dpa. The basal localization of p-Igf1r is enriched in basal epithelial cells in <i>sde1</i> mutants. (E) <i>shha</i> RNA expression is reduced in <i>sde1</i> fin regenerates at 4 dpa after a temperature shift from 25°C to 33°C at 3 dpa. Scale bars, 50 μm. White dashed boxes indicate areas of enlarged view. Red arrows indicate plane of amputation.</p
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