The NOTCH pathway contributes to cell fate decision in myelopoiesis

Abstract

Background: Controversy persists regarding the role of Notch signaling in myelopoiesis. We have used genetic approaches, employing two Notch zebrafish mutants deadly seven (DES) and beamter (BEA) with disrupted function of notch1a and deltaC, respectively, and Notch1a morphants to analyze the development of leukocyte populations in embryonic and mature fish.

Design and methods: Myelomonocytes were quantified in early embryos by in situ hybridization using a myeloper-oxidase (mpx) probe. Morpholinos were used to knock down expression of Notch1a or DeltaC. Wound healing assays and/or flow cytometry were used to quantify myelomonocytes in 5-day post-fertilization (dpf) Notch mutants (BEA and DES), morphants or pu.1:GFP, mpx:GFP and fms:RFP transgenic embryos. Flow cytometry was performed on 2-3 month old mutant fish.

Results: The number of mpx(+) cells in embryos was reduced at 48 hpf (but not at 26 hpf) in DES compared to WT. At 5 dpf this was reflected by a reduction in the number of myelomonocytic cells found at the wound site in mutants and in Notch1a morphants. This was due to a reduced number of myelomonocytes developing rather than a deficit in the migratory ability since transient inhibition of Notch signaling using DAPT had no effect. The early deficit in myelopoiesis was maintained into later life, 2-3 month old BEA and DES fish having a decreased proportion of myelomonocytes in both the hematopoietic organ (kidney marrow) and the periphery (coelomic cavity).

Conclusions: Our results indicate that defects in Notch signaling affect definitive hematopoiesis, altering myelopoiesis from the early stages of development into the adult.

Figure 1.

Reduced number of neutrophils in DES mutant embryos at 48 hpf. Heterozygote DES adult fish were crossed and embryos were harvested and fixed at (A) 26 hpf or (B) 48 hpf. Following whole mount ISH with an mpx probe, the total number of stained cells was counted, genotyping by SNP analysis and real time PCR was performed on individual embryos and each dot shows the total number of mpx cells in each embryo according to their genotype for WT and DES mutants. A representative image of an mpx probed embryo is shown for each stage of development above the corresponding graph. Scale bars: (A) 250 μm, (B) 400 μm. (*P<0.05).

Figure 2.

Total number of myelomonocytes is reduced in 5 dpf Notch1a morphants. WT (A), pu.1:GFP (B), mpx:GFP (C) and fms:RFP (D) fertilized embryos were injected with Notch1a or control MO. (A) Representative images of somite organization in control and Notch1a morphants at 2 dpf (arrow shows somite disorganization), scale bars: 100 μm. (B-D) Percentage of GFP⁺ or RFP⁺ cells obtained from flow cytometry analysis of single cell suspensions prepared from individual 5 dpf whole morphant embryos (each dot represents one embryo) (*P<0.05).

Figure 3.

Decreased myelomonocyte numbers in Notch1a morphants. mpx:GFP fertilized embryos were injected with a second non-overlapping translation blocking Notch1a morpholino or control MO. Percentage of GFP⁺ cells obtained from flow cytometry analysis of single cell suspension prepared from individual 5 dpf whole morphant embryo were plotted (each dot represents one embryo) (*** P<0.001).

Figure 4.

Reduced numbers of neutrophils at a tail wound in 5 dpf embryos with Notch1a defect. (A) Representative image of MPO cells recruited to tail injury. Embryos at 5 dpf were submitted to tail transection and were fixed and stained for MPO 4 h post injury. Scale bar: 50 μm, arrows indicate examples of MPO positive cells. (B) MPO positive cells present at the wound site were counted and groups compared as follows: WT (light gray bar) and DES (black bar). (C) WT embryos were injected with Notch1a or control MO, at 5 dpf tail transection was performed on morphants and stained for MPO after 4 h. Graph shows average cell counts obtained. (D) 5 dpf WT embryos were treated with DAPT (50 μM) or with equivalent levels of DMSO control 30 min prior to tail transection and for 4 h post injury. Fish were then stained for MPO and MPO positive cells present at the wound site were counted. (E) Embryos were collected following DAPT and DMSO treatment and RNA extracted for analysis of hes1 mRNA levels by quantitative (TaqMan) RT-PCR. Results were normalized to 18S and relative expression compared to untreated controls. Graphs display mean values and error bars represent the 95% confidence intervals of the data sets. Graphs B (n>150 in each group), C (n>60 in each group) and D (n>125 in each group). (*** P<0.001).

Figure 5.

Leukocyte proportions in whole kidney marrow and coelomic cavity are affected in Notch1a mutants. Cells isolated from WKM and coelomic cavity of WT and DES mutants were obtained from 2–3 month old zebrafish and analyzed using flow cytometry based on their forward and side scatter characteristics. (A-B) Typical profiles of live cells obtained for WKM and coelomic cavity of WT fish. The gates used for the analysis of various cell populations were as follows: R2=myelomonocytes, R3=precursors and R4=lymphocytes for WKM, R5=myelomonocytes, R6=lymphocytes, R7=NCC for coelomic cavity. Percentage of gated cells were plotted for individual fish (C-E) in WKM and (F-H) in coelomic cavity. (*P<0.05, **P<0.01).

Figure 6.

Decreased myelomonocyte numbers in DeltaC morphants and BEA mutants. (A) mpx:GFP fertilized embryos were injected with DeltaC or control MO. The percentage of GFP+ cells obtained from flow cytometry analysis of single cell suspensions prepared from individual 5 dpf whole morphant embryos (each dot represents one embryo). (B) 5 dpf BEA zebrafish embryos were subjected to a tail transection and stained for MPO after 4 h. MPO positive cells present at the wound site were counted and groups compared as follows: WT (light gray bar, n>150), BEA (dark gray bar, n>150). (C-H) Cells isolated from WKM and coelomic cavity of WT and BEA mutants were obtained from 2–3 month old zebrafish and analyzed using flow cytometry based on their forward and side scatter characteristics. Percentage of gated cells were plotted for individual fish (C-E) in WKM and (F-H) in coelomic cavity. Data were produced in the same experiment as shown in Figures 4 for (A) and 5 for (C-H). (*P<0.05, **P<0.01, ***P<0.001).