American Society of Hematology

Figure 3

$Figure 3. Defining the onset of progenitor-derived megakaryopoiesis in vivo. (A) Representative flow cytometry plots of E9.5 and E10.5 YS CD45+CD41low HPC populations showing the acquisition of CD42D expression by a subpopulation of cells at E10.5 (n = 9). (B) CFU-MK and CFU-myeloid/erythroid frequency (per 1000 cells plated) in E10.5 YS CD42D− and CD42D+ HPC populations. Bars, SEM (n = 3). *P < .05. (C) Purified E10.5 YS HPCs (CD45+CD41low cells), segregated according to CD42D expression, were cultured in proplatelet-forming medium for 72 hours. (i) Ploidy analysis by flow cytometry revealed that although highly polyploid MKs were generated by both fractions, only CD42D+ HPCs were capable of acute proplatelet formation (ii); n = 3. Arrow indicates proplatelet. (D) Representative flow cytometry plots of E9.5 and E10.5 peripheral blood (PB) demonstrating that CD42D+ HPCs entered the circulation from E10.5; n = 3. Values represent the number of CD45+CD41lowCD42D+ cells per embryo (±standard deviation). (E) Quantification of CD41highCD42D+ MKs in individual livers from E10.5 to E18.5 embryos, demonstrating that numbers dramatically increase until E14.5, from which point they remain relatively stable. n = 7 to 15 embryos per developmental stage. Bars, standard deviation. (F) Quantification of circulating platelet numbers in the peripheral blood of individual E10.5 to E18.5 embryos, revealing a gradual increase in numbers until E14.5, and a rapid spike by E16.5. n = 7 to 15 embryos per developmental stage. Bars, standard deviation. (G) Ploidy analysis of CD41highCD42D+ liver MKs from E10.5 to E13.5 reveals that the increase in circulating platelet numbers coincides with an increase ploidy of the liver MK population. Highly polyploid MKs are first seen at E11.5 (achieving an 8n state), from which point the population successively achieves higher ploidy states. n = 4 to 18 individual embryos per developmental stage. (H) Representative 3-dimensional projection of a confocal z-stack of CD41+ (red) and CD42D+ (green) MKs in the E13.5 liver. Inset is an optical section of boxed region showing a highly polyploid MK. Arrows indicated examples of platelets, which often display filopodia extensions. Bar, 20 μm. n = 11.$

Defining the onset of progenitor-derived megakaryopoiesis in vivo. (A) Representative flow cytometry plots of E9.5 and E10.5 YS CD45⁺CD41^low HPC populations showing the acquisition of CD42D expression by a subpopulation of cells at E10.5 (n = 9). (B) CFU-MK and CFU-myeloid/erythroid frequency (per 1000 cells plated) in E10.5 YS CD42D⁻ and CD42D⁺ HPC populations. Bars, SEM (n = 3). *P < .05. (C) Purified E10.5 YS HPCs (CD45⁺CD41^low cells), segregated according to CD42D expression, were cultured in proplatelet-forming medium for 72 hours. (i) Ploidy analysis by flow cytometry revealed that although highly polyploid MKs were generated by both fractions, only CD42D⁺ HPCs were capable of acute proplatelet formation (ii); n = 3. Arrow indicates proplatelet. (D) Representative flow cytometry plots of E9.5 and E10.5 peripheral blood (PB) demonstrating that CD42D⁺ HPCs entered the circulation from E10.5; n = 3. Values represent the number of CD45⁺CD41^lowCD42D⁺ cells per embryo (±standard deviation). (E) Quantification of CD41^highCD42D⁺ MKs in individual livers from E10.5 to E18.5 embryos, demonstrating that numbers dramatically increase until E14.5, from which point they remain relatively stable. n = 7 to 15 embryos per developmental stage. Bars, standard deviation. (F) Quantification of circulating platelet numbers in the peripheral blood of individual E10.5 to E18.5 embryos, revealing a gradual increase in numbers until E14.5, and a rapid spike by E16.5. n = 7 to 15 embryos per developmental stage. Bars, standard deviation. (G) Ploidy analysis of CD41^highCD42D⁺ liver MKs from E10.5 to E13.5 reveals that the increase in circulating platelet numbers coincides with an increase ploidy of the liver MK population. Highly polyploid MKs are first seen at E11.5 (achieving an 8n state), from which point the population successively achieves higher ploidy states. n = 4 to 18 individual embryos per developmental stage. (H) Representative 3-dimensional projection of a confocal z-stack of CD41⁺ (red) and CD42D⁺ (green) MKs in the E13.5 liver. Inset is an optical section of boxed region showing a highly polyploid MK. Arrows indicated examples of platelets, which often display filopodia extensions. Bar, 20 μm. n = 11.

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