|
Graphical Abstract
|
|
Fig. 1.Schematic of Homo sapiens (H. sapiens) and X. laevis RECQL4 proteins and CRISPR sgRNAs. a) Protein domain structure for human RECQL4 and X. laevis Recql4; down arrows indicate sgRNA target positions. Asterisk indicates premature stop codon. sgRNA 8 generates predominantly 8-bp deletions that will introduce a downstream stop codon in the helicase domain, resulting in a truncated protein. sgRNA 10 generates a dominant 9-bp deletion that removes bases “I,L and S” in a conserved region of the helicase. Protein alignments are shown for human, mouse, Xenopus, and zebrafish with shading highlighting conserved residues. b) Example of sgRNA8 editing, Sanger sequence traces from control wild type, and sgRNA8 CRISPant. Guide sequence is indicated by line on top of the Sanger trace.
|
|
Fig. 2.Unilateral knockdown of recql4 results in smaller head and eye size on the edited side. a) Example of bright field and a′) fluorescence view of neurula embryo, dorsal view, and anterior uppermost. The embryo was injected on the right side at the 2-cell stage, and GFP can be seen only on the right side of the midline. b to d) Examples of tadpoles at 3 wk; outlines show the regions of interest (ROIs) used to measure area of the head and eye on the left and right sides using Fiji. Scale bar in b) applies to all panels. b) Control, injected with GFP mRNA only on the left side. c) Tadpole from the group injected with 2-ng recql4 sgRNA 8 (g8) and nGFP mRNA on the right and d) on the left. e and f) Scatter plots showing the proportions injected vs uninjected head side e) or eye f) area. Line indicates mean ± SEM. Dotted line denotes no difference between the 2 sides. Recql4-injected groups are compared to the control (only injected with GFP mRNA) using Kruskal–Wallis tests with Dunn's post hoc comparisons. Sample sizes are GFP-only controls: N = 28, 2-ng g8; N = 21, 3-ng g8; N = 24, 2-ng g10; and N = 39. Significantly different groups are indicated as ****P < 0.0001. Raw data can be found in Supplementary Tables 1 to 6 in Supplementary File 1.
|
|
Fig. 3.Forelimb development requires Recql4. a) Stage NF 58 tadpoles commonly lack a forelimb on the CRISPR-edited side. Examples of left- and right-sided Recql4 CRISPANT tadpoles, dorsal and ventral views. Missing forelimbs are indicated by an asterisk; scale bar is 5 mm. Tails have been removed to aid visualization. b) Whole skeletal preparations of example stage NF 58 tadpoles where the edited side forelimb was not missing but instead was severely disrupted or duplicated, with ectopic structures present. Blue stain is the cartilage (Alcian blue), and red stain shows the bone (alizarin red). The whole tadpole is shown in the left image and a zoomed view of the ectopic limb on the right, viewed from the side that best shows the limb elements. c) Stacked bar graph showing the observed frequency of forelimb phenotypes on the uninjected (gray) side vs injected (teal) side. Fisher’s exact test, ns = nonsignificant, **** = P < 0.0001. Raw count data can be found in Supplementary Table 7 in Supplementary File 1 and images in Supplementary Fig. 2.
|
|
Fig. 4.Hindlimbs are more resistant to loss of Recql4 than forelimbs. a) Example of NF 58 stage tadpole where CRISPR knockdown of recql4 on the left side has led to failure of the left hindlimb to develop as well as the forelimbs. Missing limbs are indicated by an asterisk. b) Skeletal preparation of NF58 tadpole showing example of a smaller hindlimb on the recql4-edited left side (white arrowhead). Bone and cartilage staining show that this limb is not missing any elements. This individual is also missing the left forelimb as indicated with an asterisk. c) A single example of an ectopic hindlimb found in the study is shown. This tadpole is stage NF55 and editing is on the right. Two limbs appear as a mirror image duplication (preaxial polydactyly) on the right side and a single limb (partially obscured by the tail) on the left. Both forelimbs are present but cannot be seen due to the prehatching stage. d) Stacked bar graph showing observed frequency of hindlimb phenotypes on the injected vs uninjected side of half-CRISPant tadpoles. Fisher's exact tests, ns = nonsignificant, *** = P < 0.001, **P < 0.01. Count data is in Supplementary Table 7 in Supplementary File 1.
|
|
Fig. 5.Recql4 unilateral CRISPants have craniofacial defects on the edited side, but this does not arise from altered migration of cranial neural crest cells. a) Tadpole stage NF58 skeletal preparations, showing a ventral view of the jaw with the cartilage stained blue and the ossified bone stained red. The left image shows a control tadpole, the center shows a tadpole where recql4 was knocked down on the left side (no ossification of Meckel's cartilage on the injected side), and the right shows a tadpole with recql4 knockdown on the right side (reduced ossification of Meckel's on the injected side. b) Diagram of control and left-sided CRISPant tadpole ventral craniofacial structures to show the positions of the cartilages. c) Stacked bar graph comparing the frequency of the reduced or absent ossification of Meckel's cartilage on the uninjected vs injected side of unilateral recql4 CRISPants. N = 52, Fisher's exact test, ****P < 0.0001. d) Stacked bar graph comparing the frequency of smaller-sized hyoid cartilage on the uninjected vs injected unilateral CRISPants. N = 52, Fisher's exact test, ****P < 0.0001. e) In situ hybridization of early cranial neural crest markers in stage 19 unilateral recql4 CRISPants (left side edited). The cranial neural crest cells migrate from the dorsal neural tube in 3 streams, the mandibular (m), hyoid (h), and branchial (b). The scale bar in the bottom right panel relates to all panels. Further examples can be found in Supplementary Fig. 3.
|
|
Fig. 6.The forelimb bud fails to develop, and the vasculature of the head is reduced in the absence of Recql4. a) Stacked bar graphs showing the observed frequency of forelimb absence or delayed development on uninjected vs injected sides of half-CRISPant tadpoles at 4 wk old (stages NF50–54). Fisher's exact test, **P < 0.01, ***P < 0.001, ****P < 0.0001. b) Dorsal and ventral views of the head of typical unilateral CRISPant tadpoles at 4 wk of development. The heart and vascular system can be clearly seen due to the natural transparency of the tadpole head. flb, forelimb bud; h, heart; ra, right aorta; la, left aorta; sc, subclavian artery. Raw data can be found in Supplementary Tables 8 to 11 in Supplementary File 1.
|
|
Supplementary Figure 1: Examples of recql4 CRISPR half tadpoles at 3 weeks of development (Figure 2E,F) Top: first tadpole batch, controls are injected with just GFP mRNA, CRISPants using sgRNA8, scale bars 2 mm. Dorsal view of head and trunk with anterior uppermost. Bottom second tadpole batch using sgRNA10, dorsal view of head and trunk with head to left, scale bars 1 mm. L, injected on Left side, R, injected on right side.
|
|
Supplementary Figure 2 Stage 58 half CRISPR recql4 sgRNA8 tadpoles (Figures 3, 4). Dorsal and ventral views for each animal are boxed, tails have been removed. Top, Left side CRISPants, Bottom, right side CRISPants, red arrowheads indicate ectopic limbs, black asterisk indicates a missing forelimb, white asterisk indicates a missing hindlimb. Scale bars 5 mm.
|
|
Supplementary Figure 3: Cartilage (blue) and bone (red) staining of half recql4 CRISPants (Figures 3, 4). Individual tadpoles are boxed, dorsal (left) and ventral (centre) showing whole stage 58 tadpole, right image is a zoom of the head cartilages from the ventral side. Top: left CRISPants, Bottom: right CRISPants.
|
|
Supplementary Figure 4: Ectopic hindlimb skeletal staining. Individual from Figure 4C, stage NF55). There is a single femur, two fibulae and one central tibia, four tarsal bones and two complete hindfeet, with the two copies of digit 1 (hallux) adjacent. Viewed from the dorsal side
|
