Supplemental Information

Immune suppressive and bone inhibitory effects of prednisolone in growing and

regenerating zebrafish tissues

Karina Geurtzen, Aude Vernet, Andrew Freidin, Martina Rauner, Lorenz Hofbauer,

Jürgen Schneider, Michael Brand, Franziska Knopf

Description of Supplemental Information

Supplemental Figure 1: Relates to Figure 1 and shows that macrophage number in zebrafish

larvae is already reduced to minimum levels at 1 day of treatment. It furthermore shows that

generation of hematopoetic stem cell precursors and erythrocytes in the kidney of adult

zebrafish is not affected by prednisolone treatment.

Supplemental Figure 2: Relates to Figure 2 and shows that bone mineralization in the

uninjured fin of adult zebrafish is affected by prednisolone treatment. In contrast, bone

mineralization in regenerating fins occurs proportionate to the overall regenerate length and is

thus not specifically affected. Supplemental Figure 2 furthermore shows representative

cryosections of zebrafish skulls at 27 days post injury.

Supplemental Figure 3: Relates to Figure 3 and shows that there is no significant difference

in osteoblast number in adult zebrafish fins at 7 days post amputation and treatment. It

furthermore shows that there is a reduced number of osteoblasts in prednisolone treated larvae

which is, however, not due to increased osteoblast apoptosis. In addition, qRT-PCR data on

runx2b expression in regenerating fins is presented.

Supplemental Figure 4: Relates to Figure 4 and shows the quantification of the overall

osteoclast number in regenerating fins at 4 days post amputation and days of treatment.
Supplemental Figure 1.

(A) Quantification of macrophage numbers in transgenic mpeg1:mCherry larvae after 1, 3 and

5 dt prednisolone treatment. Mean (SD), ANOVA: * p<0.05, ** p<0.01 (n = 4 DMSO, 3

Pred.)

(B) Quantification of HSC precursors in the adult zebrafish kidney 14 dt by FACS. There is

no significant difference between prednisolone and DMSO treated groups. Mean (SD)

Student's t-test (n = 5)

(C) Quantification of erythrocytes in the adult zebrafish kidney 14 dt by FACS. There is no

significant difference between prednisolone and DMSO treated groups. Mean (SD) Student's

t-test (n = 5)
Supplemental Figure 2.

(A) Alizarin+ versus alizarin- domain lengths in homeostatic fins at 6 weeks of treatment. The

alizarin+ domain length is significantly reduced by prednisolone. Mean (SD), Student’s t-test:

* p<0.05 (0,028) (n = 5, three rays per fish)

(B) Whole body bone mineral density (BMD) of zebrafish treated for 4 weeks. There is no

significant difference in BMD between control and prednisolone treated groups. Mean (SD),

Student's t-test (n = 6)

(C) Whole mount live images of fin regenerates of osterix:nGFP transgenic fish after 14 dt.

The osterix:nGFP expressing region is shorter in prednisolone treated fins. Red dashed line =

amputation plane. Scalebar = 100 µm

(D) Quantification of experiment shown in B. Mean (SD), ANOVA: * p<0.05, ** p<0.01 ***

p<0.001 (n = 5, three rays per fish)

(E) Alizarin+ versus alizarin- domain lengths in fin regenerates at 14 dpa/dt. There is no

significant difference in the relative domain lengths between both groups. Mean (SD) (n = 5,

three rays per fish)

(F) Representative images of 27 dpi/dt transverse sections of the injured skull. The injury site

is covered by newly deposited bone in DMSO and prednisolone treated fish. Scalebar = 100

µm (n = ?)
Supplemental Figure 3.

(A) Quantification of osteoblasts in the fin regenerate of osterix:nGFP transgenic zebrafish at

7 dpa/dt. The numbers do not significantly differ between prednisolone and DMSO treated

zebrafish. Mean (SD), Student's t-test (n = ?)

(B) Quantification of TUNEL, GFP double-positive osteoblasts in osterix:nGFP transgenic

zebrafish larvae at different time points of treatment. There are no significant differences in

between groups. Mean (SD), ANOVA (n=3, at least 3 sections per larvae)

(C) Quantification of osteoblasts in larval osterix:nGFP zebrafish transverse cryosections

showing a strong reduction of osteoblasts at 5 dt. Shown are the numbers of

osteoblasts/section. Mean (SD), Student’s t-test: * p<0.05 (0,04) (n = ?)

(D) Quantification of PCNA+ osteoblasts in osterix:mCherry zebrafish transverse

cryosections of the spine. Only very few osteoblasts normally undergo proliferation in this

tissue. Note the complete absence of proliferation in the prednisolone treated group. There is

no significant difference between groups. Mean (SD), Student's t-test (n = 5, at least 3

sections per fish).

(DE) Endogenous runx2b levels determined by qRT-PCR in 2 dpa/dt fin regenerates of

prednisolone treated fish shown relative to the levels of DMSO treatment. Mean (SD of

technical error).
Supplemental Figure 4.

Quantification of overall TRAP+ osteoclasts in 4 dpa/dt regenerating fin rays (stump and

regenerate added). The overall number of osteoclasts is significantly reduced as a result of

prednisolone treatment. Mean (SD), Student’s t-test: * p<0.05 (0,031) (n = 5, three rays per

fish)