Supplementary FIGURE 1

kdrl kdrl:cre

R
R

C C

12-14ss 12-14ss

24 hpf 24 hpf

48 hpf 48 hpf

Supplementary Figure 1. kdrl:Cre transgenic animals display an expression pattern

similar to the endogenous kdrl gene. During somitogenesis, expression of kdrl:cre appears
similar to the endogenous kdrl transcript (upper panels). Expression of both transcripts is
observed in rostral tissues (R), corresponding to the region that gives rise to primitive mac-
rophages, and caudal tissues (C), corresponding to the regions giving rise to primitive erythro-
cytes, HSCs, and EMPs. At later timepoints, kdrl:cre expression appears similar to endoge-
nous kdrl, appearing in cells of the developing vasculature (middle and bottom panels).
Supplementary FIGURE 2

a H 24 hpf, analyzed at 48 hpf

hsp70:Cre; switch +H hsp70:Cre; switch +H

switch + H switch + H

b hsp70:Cre; switch H 24 hpf, analyzed at 1 year of age

WKM Erythroid Lymphoid Precursors Myeloid

4
10

400 52% 80% 93% 98%

3
10

300
39% 18%
2
10
200

1
10
100

0 0
2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5
0 200 400 600 800 1000 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
FSC DsRed express DsRed express DsRed express DsRed express

Supplementary Figure 2. Characterization of actin2:loxP-STOP-loxP-DsRedexpress

reporter animals. a, Progeny of Tg(Hsp70l:Cre)zf36 and switch heterozygote matings were
heat-shocked (38C for 30) at 24 hpf and analyzed for DsRed fluorescence at 48 hpf.
Embryos were subsequently genotyped for the presence of the switch reporter transgene.
Induced double transgenic animals showed robust and uniform DsRed expression (upper
embryo). DsRed expression was never observed in the absence of Cre expression (lower
embryo). b, Similarly induced double transgenic embryos were raised to adulthood, and
whole kidney marrow (WKM) was analyzed by flow cytometry. Cre induction at 24 hpf led to
labeling of 98% of myeloid (green gate, left panel, and green box, right panel) and lymphoid
(blue gate, left panel, and blue box, right panel) cells within WKM at one year of age.
Approximately half of the erythroid cells (red gate, left panel, and red box, right panel)
expressed low levels of DsRed, whereas the other half was DsRed negative, consistent with
loss of actin expression in mature erythrocytes, as previously shown20. These results
demonstrate that the switch lineage-tracing reporter gene remains stably expressed over
time following a single induction of Cre recombinase.
Supplementary FIGURE 3

cmyb:GFP; kdrl:RFP at 8dpf

0.009%

cmyb:eGFP

Supplementary Figure 3. Transitional intermediates between hemogenic endothelium

and HSCs are not observed in larval or adult stages. Double transgenic kdrl:RFP;
cmyb:eGFP animals were raised to larval or adult stages and analyzed by flow cytometry.
Compared to double transgenic animals at 36 hpf (Figure 2), relatively few cells were
observed within the kdrl+ cmyblo (orange gate) or kdrl+ cmyb+ (yellow gate) regions in larvae
at 8 dpf or within adult WKM (not shown). Cells along the diagonal in between the orange
and yellow regions are autofluorescent pigment cells. These results suggest that the genera-
tion of HSCs from hemogenic endothelium is rare or absent past embryonic stages.