FIGURE SUMMARY
Title

A zebrafish model of Poikiloderma with Neutropenia recapitulates the human syndrome hallmarks and traces back neutropenia to the myeloid progenitor

Authors
Colombo, E.A., Carra, S., Fontana, L., Bresciani, E., Cotelli, F., Larizza, L.
Source
Full text @ Sci. Rep.

Expression profiling of usb1 in zebrafish.

(a) Temporal and spatial usb1 expression during embryogenesis and among adult tissues. Total RNA was extracted from embryos at the indicated time-points and from the specified adult tissues, and subjected to RT-PCR with primers specific for usb1 (upper panel) or β-actin (lower panel). The sizes of the obtained PCR fragments are indicated. (b) WISH analysis of usb1. The signal obtained with usb1 antisense probe was ubiquitous until the 5-somite stage. It showed a higher intensity in the cephalic region at the 15 somites stage and displayed decreasing expression from the head to the caudal region (20 somites and 26 hpf). At 36 hpf, the signal only marked the head and the pharyngeal arches-derived structures. hpf: hour post fertilization; dpf: day post fertilization. Images showing the results obtained with sense probe are provided at each developmental stage. Scale bars: 200 μm.

Overall phenotype of usb1 morphants.

Representative live-microscopy-4X magnification-pictures of 2 dpf (a) and 5 dpf (b) embryos injected with the same dosage (0.6 pmol/embryo) of Std (top), SMO-A (central) or SMO-B (bottom) morpholinos. The morphants display a smaller size, with defective and irregular pigmentation of the skin, as compared to the continuous stripes of the control embryos, and pericardial oedema. (c) Massive skeletal defects revealed by Alcian blue staining in usb1 morphants at 5 dpf. From left to right: diagram illustrating the normal pharyngeal arches-derived bone architecture19; ventral view of a control embryo (left panel) and representative pictures of the mild (left) and severe (right) phenotypes of SMO-A and SMO-B morphants. The Meckel’s (yellow in the colour-coded diagram), the palatoquadrate (green) and ceratohyal (purple) structures appear misshaped in both mildly- and severely-affected embryos; moreover, the ceratobranchial structures (light blue) appear disorganized or missing in half of SMO-A and in a third of SMO-B severely-affected embryos. (d) Reduction of gata1-positive cells (red fluorescence) (middle panels) and a regular vasculature pattern (green fluorescence) (lower panels) in 2 dpf tg(gata1:dsRED;flk1:GFP) embryos injected with SMO-A (0.7 pmol/embryo) (right) as compared to control (left). (e) Myeloid lineage defects evinced by a reduction in mpx-positive cells (green fluorescence) (lower panels) in 2 dpf tg(mpx:GFP;lyzC:dsRED) embryos injected with SMO-A (0.7 pmol/embryo) (right) as compared to control (left). Scale bars: (a–e) 200 μm.

Effect of usb1 knockdown on haematopoiesis.

(a) Real-time PCR analysis of pu.1, gata1 and mpx expression at the indicated developmental stages of embryos injected with SMO-A or Std-MO (each at 0.6 pmol/embryo). Samples were run in triplicate and data are expressed as the mean ± standard deviation. Asterisks (*) indicate statistically significant differences (t- test; p < 0.05). (b) WISH of gata1 and mpx in SMO-A and control embryos at the indicated developmental stages. (c) O-dianisidine staining for haemoglobin in embryos injected with Std-MO (top), SMO-A (central) and SMO-B (bottom panels) (each at 0.6 pmol/embryo). The left and the right images are representative of the slight and severe erythropoiesis defects observed at the indicated percentages in SMO-A- and SMO-B-injected embryos. The # points to the caudal region of the morphant where the accumulation of blood is observed. Scale bars: (b,c) 200 μm.

EXPRESSION / LABELING:
Genes:
Fish:
Knockdown Reagent:
Anatomical Terms:
Stage Range: 26+ somites to Prim-15
PHENOTYPE:
Fish:
Knockdown Reagents:
Observed In:
Stage Range: 26+ somites to Long-pec

Phenotypes of morphants co-injected with SMO-A and SMO-B at subphenotypic dosage and rescue of morpholino-induced phenotypes with human USB1 RNA.

(a) Representative pictures of zebrafish embryos injected with Std-MO (0.6 pmol/embryo), SMO-A (0.3 pmol/embryo), SMO-B (0.3 pmol/embryo) and co-injected with SMO-A and SMO-B (each at 0.3 pmol/embryo). (b) Alcian blue staining at 5 dpf highlights the regular morphologic architecture of the pharyngeal arch cartilages in embryos injected with Std-MO, SMO-A and SMO-B at sub-phenotypic dosages and the aberrant cartilaginous structures in embryos co-injected with SMO-A and SMO-B (each at 0.3 pmol/embryo). (c) In the left panels, lateral views of 2 dpf embryos injected with Std-MO, SMO-A (0.6 pmol/embryo) and SMO-A and human USB1 RNA (300 pg/embryo). In the right panels, fluorescent images of tg(mpx:GFP;lyzC:dsRED) embryos at 2 dpf. The green signals, representing mpx-expressing cells, are reduced in embryos injected with SMO-A (0.6 pmol/embryo) as compared to the controls, but are enhanced in embryos co-injected with human USB1 RNA (300 pg/embryo). The fractions of embryos exhibiting the investigated phenotypes out of the total number of those examined are indicated. All images are at the same magnification. Scale bars: 200 μm.

EXPRESSION / LABELING:
Gene:
Fish:
Knockdown Reagent:
Anatomical Term:
Stage: Long-pec

Heart morphology in usb1-depleted embryos.

WISH analysis of cmlc2 expression showing that 2 dpf SMO-A-injected embryos display heart signals similar to those of Std-MO embryos. Scale bar: 200 µm.

Investigation of off-target effects due to usb1 knockdown.

The overall phenotype of co-injected p53-MO and SMO-A embryos is similar to that of SMO-A morphants. The number of embryos which phenotype is shown is indicated in each panel. Scale bar: 200 µm.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Sci. Rep.