Dataset DOI: 10.5061/dryad.b5mkkwhss

Description of the data and file structure

Data are stored in comma-separated value (.csv) format. A total of twenty-six (26) files are included.

Each .csv file is named according to the corresponding figure number and panel in the published study associated with the data. For example, the file 1C.csv corresponds with raw data for Figure 1C.

Description: The data were generated by several methods for analysis of gene expression, hematopoietic and endothelial cell type classification, mitochondrial and bioenergetic features, protein synthesis and regulatory sequences that dictate translation of specific mRNA transcripts, hematopoietic progenitor and stem cell function, intracellular signaling pathways, and tissue concentration of drugs in the embryo following in utero administration to mothers. The raw or normalized numbers are the source for the graphs presented in the published article (https://doi.org/10.1084/jem.20250607).

Files and variables

File: 1C.csv

Description: RT-qPCR from independent experiments shows that 6-hr WSS increases ETC and mitobiogenesis mRNAs in E10.5 and E11.5 AGM.

Variables

Groups include force as a stimulus at two embryo ages

• Static
• WSS

Embryo age includes two developmental times for mice

• E10.5
• E11.5

File: 1F.csv

Description: ATP5I puncta per single mitochondrion were counted in 5 μm volumetric STED nanoscopy images. Independent samples were prepared from separate embryos.

Variables

Groups include force as a stimulus at two embryo ages

• Static
• WSS

File: 1G.csv

Description: Flow cytometric quantification of TOM20, as a measure of mitochondrial mass, shows increase in endothelial cell populations both expressing and not expressing the pan-hematopoietic surface marker CD45. Three independent experiments were conducted on different days.

Variables

Groups include force as a stimulus at two embryo ages

• Static
• WSS

Populations include cell types defined by flow cytometry

• VEC+ CD45-
• VEC+ CD45+

File: 1H.csv

Description: Seahorse assays from E10.5 and E11.5 developmental ages show elevated OXPHOS (OCR: oxygen consumption rate) with force. Various measurements were calculated from OCR readings after injection of drugs, as part of the Agilent MitoStress Test Assay. Independent experiments were conducted on different days.

Variables

Groups include force as a stimulus at two embryo ages

• Static
• WSS

Measurements include bioenergetic metrics associated with mitochondrial function or glycolysis

• Basal
• Maximal
• ATP
• Spare
• H+ Leak

File: 2E.csv

Description: Transmission electron microscopy (TEM) reveals that mitochondrial cristae are poorly developed in endothelial cells lining the Ncx1 mutant dorsal aorta. Specifically, Ncx1 mutant aortas have reduced mitochondrial numbers and fewer cristae as determined by scores generated by a pathologist blinded to genotype. Average counts of mitochondria per cell are provided for individual embryos sorted by genotype and identified as Replicate 1, Replicate 2, Replicate 3, and Replicate 4. Other scored features of morphology include metrics of ultrastructure of the mitochondria.

Variables

Genotypes of mouse embryos were analyzed for differences in drug transport into tissues after in utero delivery to the pregnant dam

• ko (Ncx1 mutant)
• wt (Wildtype)

Mitochondrial numbers per cell and morphology ultrastructure characteristics include counts and scored features

• Counts (average mitochondria per cell)
• Organized cristae
• Irregular cristae
• Vacuoles
• Few cristae

File: 3A.csv

Description: Cell type identity was determined by flow cytometry and each population assessed for mitochondrial membrane potential (MMP). The MMP-sensitive dye TMRE increases as cells acquire hematopoietic identity and as embryos develop from E8.5 to E11.5. Individual embryos (E10.5, E11.5) or groups of 2-3 embryos (E8.5, E9.5) from independent timed pregnancies were microdissected to isolate aorta-gonad-mesonephros (AGM) or para-aortic splanchnopleura (PSp), and incubated with TMRE prior to flow cytometry analysis. Each embryo or group of embryos represents a separate data point.

Variables

Populations include cell types defined by flow cytometry

• VEC-
• EC
• AE
• HE
• proHSC
• preHSC I
• preHSC II

Embryo age includes a range of developmental times for mice

• E8.5
• E9.5
• E10.5
• E11.5

File: 3D.csv

Description: The top or bottom 15% of live cells were sorted based exclusively on mitochondrial membrane potential (MMP). The sorted populations were incubated in Methocult M3434 and analyzed by scoring hematopoietic colonies after 11 days. Colonies were classified by "Type" as granulocytes (G), macrophages (M), erythroid cells (Ery), megakaryocytes (Mk), or mixed groups comprised of two (GM) or more (GEMM) myeloid lineages. Sorting of MMP high and MMP low populations permitted an assessment of hematopoietic activity, which is exclusively found in the high-MMP population, as measured by colony formation assays. Independent experiments are recorded in separate columns for each "Type" of hematopoietic colony.

Variables

Groups include mitochondrial membrane potential level

• MMP low
• MMP high

Type includes a classification system for discriminating myeloid cells that develop in vitro

• Granulocytes (G)
• Macrophages (M)
• Erythroid cells (Ery)
• Megakaryocytes (Mk)
• Granulocytes-macrophages (GM)
• Granulocytes-erythroid cells-macrophages-megakaryocytes (GEMM)

File: 3F.csv

Description: Transplantation was used to assess hematopoietic engraftment and repopulating potential in peripheral blood. The top or bottom 30% from 10 embryo equivalents (e.e.) of CD45.2 C57BL/6 AGM were sorted based on MMP using TMRM and viability dye, and irradiated CD45.1 recipient mice each received either the MMP high (MMP Hi) or low (MMP Lo) fraction of the 10 e.e. Transplants were conducted on nine independent dates, and mice were bled to sample peripheral blood chimerism (the percentage of donor cells divided by the total CD45 of both host and donor) and hematopoietic lineages in the peripheral blood. Separate data points from bleed data collected on different days are provided as replicates across columns. Time points for blood collection are listed in rows. Values for chimerism and hematopoietic lineages in the blood are provided in separate tables. Reconstitution of the peripheral blood was significantly greater from MMP-high donor cells.

Variables

Groups include mitochondrial membrane potential level

• MMP Lo
• MMP Hi

Timepoints for analysis of hematopoietic reconstitution of transplant recipients includes analysis of peripheral blood

• 4wk
• 6wk
• 8wk
• 10wk
• 12wk

Hematopoietic lineages determined by cytometry myeloid and lymphoid lineages, as characterized by expression of surface markers on the cells

• Granulocytes/Neutrophils (G)
• Macrophages (M)
• B cells (B)
• T cells (T)

File: 3G.csv

Description: Bone marrow chimerism and lineages were assessed in transplanted, irradiated CD45.1 recipient mice that received either the MMP high (MMP Hi) or low (MMP Lo) fraction of the 10 e.e. Analysis was conducted at the 13-week timepoint and data points are provided as replicates across columns. Reconstitution of the bone marrow was significantly greater from MMP-high donor cells. Values for chimerism (the percentage of donor cells divided by the total CD45 of both host and donor) and hematopoietic lineages in the bone marrow are provided in separate tables.

Variables

Groups include mitochondrial membrane potential level

• MMP Lo
• MMP Hi

The timepoints for analysis of hematopoietic reconstitution of transplant recipients includes analysis of bone marrow at 13 weeks

• 13wk

Hematopoietic lineages determined by cytometry myeloid and lymphoid lineages, as characterized by expression of surface markers on the cells

• Granulocytes/Neutrophils (G)
• Macrophages (M)
• B cells (B)
• T cells (T)

File: 4D.csv

Description: Protein synthesis was measured in aorta-gonad-mesonephros (AGM) cells cultured under force. Methionine-free media containing HPG was applied for the last 3 hr of an 18 hr static or WSS culture period, following an initial 8-hr seeding period of E10.5 AGM. Cells were labeled with surface markers, fixed, and permeabilized, then fluorescent azide was added via click labeling to prepare the samples for flow cytometry. Gates for HPG+ were established in comparison to samples that were not cultured with HPG.

Variables

Groups include force as a stimulus

• Static
• WSS

Populations include cell types defined by flow cytometry

• VEC+ CD45-
• VEC+ CD45+

File: 4H.csv

Description: Seahorse assays of cultured E11.5 aorta-gonad-mesonephros (AGM) show that inhibiting protein translation significantly diminishes the bioenergetic adaptations stimulated by 18 hr WSS. AGM were seeded for 8 hr prior to treatment with WSS. Oxidative phosphorylation (OXPHOS, with OCR as oxygen consumption rate) was collected from the MitoStress Test Assay. Separate cultures were prepared to measure glycolysis (GlycoPER, which is derived from ECAR (extracellular acidification rate)) as part of the Glycolytic Rate Assay. Mean, standard error of the mean, and N replicates are shown for each group.

Variables

Groups include force as a stimulus and various drug treatments

• Veh Static (vehicle control under static culture)
• CAP Static (chloramphenicol under static culture)
• ANS Static (anisomycin under static culture)
• Veh WSS (vehicle control under WSS culture)
• CAP WSS (chloramphenicol under WSS culture)
• ANS WSS (anisomycin under WSS culture)

Measurements include bioenergetic metrics associated with mitochondrial function or glycolysis

• Basal OCR
• Maximal OCR
• Basal GlycoPER
• Compensatory GlycoPER

File: 4K.csv

Description: Luciferase assays from murine pluripotent stem cell derived endothelium provided measurement of translation efficiency based on 5' untranslated region (UTR) sequence of mRNA transcripts. Two reporters were used to determine the influence of the 5’ UTR of mRNAs on rates of protein synthesis. Translation of Renilla luciferase is under the control of the Eef2 5’ UTR, and translation of Firefly luciferase is independent of the 5’ UTR, instead relying on IRES-dependent initiation of translation, thereby serving as an internal control for efficiency of transfection and transcription. Rotational rheometer-style flow devices with smooth cones were used to produce laminar flow (WSS), while grooved cones generated disturbed flow (DF). The mTOR inhibitor PP242 was applied to block mTOR-responsive translation. Renilla luciferase luminescence was first normalized to Firefly luciferase, then relative luminescence was calculated as the ratio of normalized Renilla activity from the TOP reporter to the TOPm reporter. Replicates were generated across three different days. Data points are the ratio of Renilla to Firefly luminescence intensity, and replicates are recorded in individual columns.

Variables

Groups include force as a stimulus and treatment with the mTOR drug PP242

• Static
• WSS
• DF
• WSS PP242

File: 5B.csv

Description: Western blots from independent experiments were used to determine effects of force on mTOR signaling in dissociated, cultured E11.5 AGM. Immunoblotting reveals the rapid activation of Akt and mTOR by WSS. Within 5 min of WSS, phosphorylation of S6K and inactivating hyper-phosphorylation of 4E-BP1 by mTORC1 are consistent with positive regulation of mTOR signaling. GAPDH was detected on all membranes for normalization for each protein. Briefly, protein bands were quantified, with normalization of intensities to GAPDH and static controls on corresponding membranes. Experiments were performed on different days, and those days are assigned names corresponding to Replicate 1, Replicate 2, etc. Replicates for 4E-BP1 phosphorylation are entered across different columns.

Variables

Groups include force as a stimulus at various timepoints

• Static
• 5 min (WSS)
• 30 min (WSS)
• 2 hr (WSS)
• 6 hr (WSS)
• 18 hr (WSS)

Proteins detected by immunoblotting include several in the mTOR pathway

• p-mTOR
• mTOR
• PI3K
• PGC1a
• p-S6K
• S6K
• p-Akt (T308)
• p-Akt (S473)
• Pan-Akt
• Hypo p-4E-BP1
• Hyper p-4E-BP1

File: 5C.csv

Description: Intracellular flow cytometry was used to detect post-translational modifications, including phosphorylation of Akt and 4E-BP1, in cells labeled with surface markers for endothelial-to-hematopoietic transition (EHT) subsets from E9.5 to E10.5 PSp/AGM. E10.5 AGM cells were treated with either vehicle or PP242 in utero. Replicates were collected on four different days and are entered across different columns.

Variables

Groups include embryo age and in utero administered drug treatment

• E9 Veh (vehicle treated embryos collected at E9.5)
• E10 Veh (vehicle treated embryos collected at E10.5)
• E10 PP242 (PP242 treated embryos collected at E10.5)

Proteins detected by intracellular flow cytometry include two as part of the mTOR pathway

• p-Akt (S473)
• p-4E-BP1 (Thr37/46)

Populations include cell types defined by surface markers detected by flow cytometry

• VEC-
• VEC+
• EC
• AE
• HE
• proHSC
• preHSC I
• preHSC II

File: 5D.csv

Description: Flow cytometry was used to detect surface markers for endothelial-to-hematopoietic transition (EHT) subsets in E10.5 AGM. E10.5 AGM cells were treated with either vehicle or PP242 in utero. Replicates were collected on four different days and are entered across different columns.

Variables

Groups include in utero administered drug treatment

• Vehicle (vehicle treated embryos collected at E10.5)
• PP242 (PP242 treated embryos collected at E10.5)

Populations include cell types defined by surface markers detected by flow cytometry

• AE
• HE
• proHSC
• preHSC I
• preHSC II

File: 5E.csv

Description: Intracellular flow cytometry was used to detect post-translational modifications, including phosphorylation of Akt and 4E-BP1, in E10.5 AGM. Cells were seeded for 18 hr, and then exposed to 15 min laminar wall shear stress (WSS) or disturbed flow (DF). PP242 drug was applied 30 min before WSS began to inhibit mTOR. Replicates were prepared on different days and are entered across different columns.

Variables

Groups include force as a stimulus and treatment with the mTOR drug PP242

• Static
• WSS
• DF
• WSS PP242

Proteins detected by intracellular flow cytometry include two as part of the mTOR pathway

• p-Akt (S473)
• p-4E-BP1 (Thr37/46)

Populations include cell types defined by surface markers detected by flow cytometry

• VEC-
• EC
• AE
• HE
• proHSC
• preHSC I
• preHSC II

File: 5F.csv

Description: Seahorse assays of cultured E10.5 aorta-gonad-mesonephros (AGM) show that inhibiting PI3K or mTOR significantly diminishes the bioenergetic adaptations stimulated by WSS. Specifically, inhibition of PI3K or mTOR by Pictilisib or PP242, respectively, significantly alters bioenergetics in response to WSS. Various groups were compared to understand impact of PI3K and mTOR signaling on force-regulated metabolism, including vehicle static, static pictilisib, static PP242, vehicle WSS, WSS pictilisib, and WSS PP242. Each replicate was prepared on different days. Oxidative phosphorylation (OXPHOS, with OCR as oxygen consumption rate) was collected from the MitoStress Test Assay. Separate cultures were prepared to measure glycolysis (GlycoPER, which is derived from ECAR (extracellular acidification rate)) as part of the Glycolytic Rate Assay. Mean, standard error of the mean, and N replicates are shown for each group.

Variables

Groups include force as a stimulus and various drug treatments

• Veh Static (vehicle control under static culture)
• Pict Static (pictilisib under static culture)
• PP242 Static (PP242 under static culture)
• Veh WSS (vehicle control under WSS culture)
• Pict WSS (pictilisib under WSS culture)
• PP242 WSS (PP242 under WSS culture)

Measurements include bioenergetic metrics associated with mitochondrial function or glycolysis

• Basal OCR
• Maximal OCR
• Basal GlycoPER
• Compensatory GlycoPER

File: 6A.csv

Description: The Seahorse MitoStress Test Assay was used to measure the effects of the mTOR activator, MHY1485, on mitochondrial capacity in E10.5 AGM cells cultured in vitro. Various doses were applied immediately after seeding cells and were left for 24 hr prior to measurement of OCR. Seahorse assays from E10.5 AGM cultures show elevated maximal and spare OXPHOS (OCR: oxygen consumption rate) with application of the mTOR drug MHY1485. Three different groups of embryos were collected for analysis of the dose-response. Various measurements were calculated from OCR readings after injection of drugs. Independent experiments were conducted on different days and replicates are entered across columns.

Variables

Groups include drug dose at a single embryo age

• 0 μM MHY1485
• 1 μM MHY1485
• 5 μM MHY1485
• 10 μM MHY1485
• 30 μM MHY1485
• 50 μM MHY1485

Measurements include bioenergetic metrics associated with mitochondrial function

• Basal
• Maximal
• Spare

File: 6C.csv

Description: Pregnant dams received MHY1485 (10 mg/kg) once daily by oral gavage beginning at E7.5, and embryos were recovered for analysis by mass spectrometry. AGM from embryos collected at E10.0 was analyzed by mass spectrometry for MHY1485 after receiving in utero exposure with four doses (one per day), with the last dose administered 3 hours before harvest. Tissue concentration was compared to weight. The AGM region was weighed and subsequently flash frozen for mass spectrometry, while the remaining embryo tissue was genotyped.

Variables

Genotypes of mouse embryos were analyzed for differences in drug transport into tissues after in utero delivery to the pregnant dam

• m (Ncx1 mutant)
• H (Ncx1 heterozygous)
• W (Wildtype)

File: 6D.csv

Description: Intracellular flow cytometry was used to detect post-translational modifications, including phosphorylation of Akt and 4E-BP1, in E10.5 AGM treated in utero with MHY1485 (10 mg/kg) once daily by oral gavage beginning at E7.5. E10.0 AGM were collected from Ncx1 heterozygous intercrosses, and all embryos were processed independently for genotyping and analysis. Replicates were prepared on different days and are entered across different columns.

Variables

Genotypes of mouse embryos were analyzed for differences in mTOR signaling after in utero delivery of the mTOR activator drug to the pregnant dam

• Control Veh (wildtype and heterozygous Ncx1 embryos treated with vehicle)
• Control MHY (wildtype and heterozygous Ncx1 embryos treated with MHY1485)
• Mutant Veh (mutant/ko Ncx1 embryos treated with vehicle)
• Mutant MHY (mutant/ko Ncx1 embryos treated with MHY1485)

Proteins detected by intracellular flow cytometry include two as part of the mTOR pathway

• p-Akt (S473)
• p-4E-BP1 (Thr37/46)

Populations include cell types defined by surface markers detected by flow cytometry

• VEC-
• EC
• AE
• HE
• proHSC
• preHSC I
• preHSC II

File: 6E.csv

Description: Flow cytometry was used to detect surface markers for endothelial-to-hematopoietic transition (EHT) subsets in E10.0 AGM treated with the MHY1485 mTOR activator in utero. Pregnant dams from Ncx1 heterozygous intercrosses received MHY1485 (10 mg/kg) once daily by oral gavage beginning at E7.5, and embryos were recovered for analysis by flow cytometry. Frequency of EHT subsets is shown. Replicates are entered across different columns.

Variables

Genotypes of mouse embryos were analyzed for differences in frequency of EHT subsets produced in response to chemical mTOR activation

• Ncx1 (Ncx1 mutant)
• Wild Type (Ncx1 wildtype or heterozygous)

Groups include in utero administered drug treatment

• Vehicle (vehicle treated embryos collected at E10.0)
• MHY1485 (MHY1485 treated embryos collected at E10.0)

Populations include cell types defined by surface markers detected by flow cytometry

• EC
• AE
• HE
• proHSC
• preHSC I
• preHSC II

File: 6G.csv

Description: ATP5I puncta per single mitochondrion were counted in 5 μm volumetric STED nanoscopy images of AGM treated with MHY1485 mTOR activator. STED nanoscopy shows an increase in ATP5I within mitochondria of VEC+ cells from E11.5 wild-type embryos treated in utero with MHY1485. ATP5I puncta per mitochondrion is provided for VEC+ cells. Four groups of embryos treated in utero with vehicle or MHY1485 were analyzed across different days.

Variables

Groups include drug treatment at a single embryo age

• Vehicle
• MHY1485

File: 6H.csv

Description: Transplantation was used to assess hematopoietic engraftment and repopulating potential in peripheral blood using AGM cells treated in utero with MHY1485. Engraftment from E11.5 wild-type donor AGM treated with MHY1485 in utero enabled greater reconstitution of peripheral blood. Briefly, mice were bled to sample peripheral blood chimerism (the percentage of donor cells divided by the total CD45 of both host and donor) and hematopoietic lineages in the peripheral blood. Separate data points from bleed data collected on different days are provided as replicates across columns. Time points for blood collection are listed in rows.

Variables

Groups include drug treatment at a single embryo age

• Vehicle
• MHY1485

Timepoints for analysis of hematopoietic reconstitution of transplant recipients includes analysis of peripheral blood

• 4wk
• 10wk
• 16wk

Hematopoietic lineages determined by cytometry myeloid and lymphoid lineages, as characterized by expression of surface markers on the cells

• Granulocytes/Neutrophils (G)
• Macrophages (M)
• B cells (B)
• T cells (T)

File: 6I.csv

Description: Bone marrow chimerism and lineages were assessed in transplanted, irradiated CD45.1 recipient mice that received either E11.5 wild-type donor AGM treated with MHY1485 in utero or vehicle control. Analysis was conducted at the 17-week timepoint and data points are provided as replicates across columns. Bone marrow analyzed at 17 weeks showed an increase in donor chimerism with MHY1485. Values for chimerism (the percentage of donor cells divided by the total CD45 of both host and donor) and hematopoietic lineages in the bone marrow are provided in separate tables.

Variables

Groups include drug treatment at a single embryo age

• Vehicle
• MHY1485

The timepoints for analysis of hematopoietic reconstitution of transplant recipients includes analysis of bone marrow at 17 weeks

• 17wk

Hematopoietic lineages determined by cytometry myeloid and lymphoid lineages, as characterized by expression of surface markers on the cells

• Granulocytes/Neutrophils (G)
• Macrophages (M)
• B cells (B)
• T cells (T)

File: S3C.csv

Description: Cell type identity was determined by flow cytometry, and each population was assessed for reactive oxygen species (ROS) with MitoSOX dye. Superoxide level, as detected by mean fluorescence intensity (MFI) of MitoSOX in E11.5 AGM, is elevated upon commitment to a hematopoietic fate. Samples were prepared on four independent days. Embryos from independent timed pregnancies were microdissected to isolate aorta-gonad-mesonephros (AGM) and incubated with MitoSOX prior to flow cytometry analysis. Each embryo or group of embryos represents a separate data point and is labeled as Replicate 1, Replicate 2, etc.

Variables

Populations include cell types defined by flow cytometry

• EC
• AE
• HE
• preHSC I
• preHSC II

File: S4E.csv

Description: Western blots from independent experiments were used to determine effects of force and PI3K activity on mTOR signaling in dissociated, cultured E11.5 AGM. Immunoblotting reveals the rapid activation of Akt and mTOR by WSS, while the PI3K-specific inhibitor pictilisib blocks WSS-dependent activation of signaling downstream of PI3K. GAPDH was detected on all membranes for normalization for each protein. Briefly, protein bands were quantified, with normalization of intensities to GAPDH and static controls on corresponding membranes. Experiments were performed on different days, and those days are entered across columns.

Variables

Groups include force as a stimulus at various timepoints

• Static
• 5 min WSS
• 18 hr WSS

Groups also include PI3K drug as a stimulus

• Vehicle
• Pictilisib

Proteins detected by immunoblotting include several in the mTOR pathway

• p-mTOR
• mTOR
• PI3K
• PGC1a
• p-S6K
• S6K
• p-Akt (T308)
• p-Akt (S473)
• Pan-Akt
• p-4E-BP1

Code/software

Microsoft Excel or any text editor can be used to open the files.

Access information

Graphical depictions of the data are available in the associated published article (https://doi.org/10.1084/jem.20250607).