Complementary and Inducible creERT2 Mouse Models for Functional Evaluation of Endothelial Cell Subtypes in the Bone Marrow

In the adult bone marrow (BM), endothelial cells (ECs) are an integral component of the hematopoietic stem cell (HSC)-supportive niche, which modulates HSC activity by producing secreted and membrane-bound paracrine signals. Within the BM, distinct vascular arteriole, transitional, and sinusoidal EC subtypes display unique paracrine expression profiles and create anatomically-discrete microenvironments. However, the relative contributions of vascular endothelial subtypes in supporting hematopoiesis is unclear. Moreover, constitutive expression and off-target activity of currently available endothelial-specific and endothelial-subtype-specific murine cre lines potentially confound data analysis and interpretation. To address this, we describe two tamoxifen-inducible cre-expressing lines, Vegfr3-creERT2 and Cx40-creERT2, that efficiently label sinusoidal/transitional and arteriole endothelium respectively in adult marrow, without off-target activity in hematopoietic or perivascular cells. Utilizing an established mouse model in which cre-dependent recombination constitutively-activates MAPK signaling within adult endothelium, we identify arteriole ECs as the driver of MAPK-mediated hematopoietic dysfunction. These results define complementary tamoxifen-inducible creERT2-expressing mouse lines that label functionally-discrete and non-overlapping sinusoidal/transitional and arteriole EC populations in the adult BM, providing a robust toolset to investigate the differential contributions of vascular subtypes in maintaining hematopoietic homeostasis. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s12015-024-10703-9.


Introduction
Hematopoietic stem cells (HSCs) are multipotent precursors that sit atop a hierarchy of hematopoietic progenitor cells (HPCs) responsible for maintaining balanced blood production throughout life [1,2].In adults, hematopoietic stem and progenitor cells (HSPCs) are localized to specialized vascularized niches within the bone marrow (BM) that direct stem cell-fate decisions, including quiescence, self-renewal, and restricted progenitor differentiation [3,4].Endothelial cells (ECs) are a critical component of the HSC-supportive BM niche, nucleating perivascular stromal and hematopoietic cells to create an instructive multicellular microenvironment through the production of extrinsic cues that maintain hematopoietic homeostasis and regeneration.Within the BM, the vasculature can be subclassified into high-pressure arterioles, branching into transitional vessels located adjacent to trabecular bone in the metaphysis and near cortical bone, before emptying into a low-pressure sinusoidal capillary network in the central marrow [5].While arteriole, transitional, and sinusoidal vascular microenvironments are anatomically distinct and can be classified by vessel morphology [6,7], accompanying perivascular stromal and hematopoietic cell association [4], and endothelial immunophenotypic labeling and gene expression signatures [8,9], the lack of high-fidelity inducible cre systems that allow for targeted genetic manipulations in niche-specific endothelial subtypes have hampered the functional characterization of these vascular subsets.
A diverse array of cre-expressing mouse lines have been successfully used to target pan-endothelial and endothelial subtypes [9][10][11][12][13][14].However, existing cre lines have two limitations: (1) Most pan-endothelial cre lines are constitutively expressed and consequently exhibit recombination in HSCs due to their shared developmental ontogeny, and (2) existing cre lines targeting vascular subsets exhibit off-target recombination within BM stromal and hematopoietic subsets.These limitations preclude the investigation of the role of vascular-subtype-specific niches in regulating HSPC activity within the adult BM.In this manuscript, we characterize two inducible cre-expressing murine lines that faithfully identify adult BM endothelial subpopulations to accurately interrogate the mechanisms of endothelial-HSPC instructive function.Herein, we describe inducible and vascular subtype-specific Vegfr3-creER T2 and Cx40-creER T2 mice that respectively target non-overlapping sinusoidal/transitional and arteriole endothelial populations within the adult BM, with no observable off-target stromal or hematopoietic activity.Using a previously described genetic model of MAPKactivation in the BM vascular niche [15], we demonstrate that Vegfr3-creER T2 and Cx40-creER T2 mice are able to faithfully segregate individual sinusoid/transitional and arteriole MAPK-dependent contributions to hematopoietic dysfunction.Taken together, these model systems provide a platform to discriminate endothelial-borne sinusoidal/ transitional and arteriole paracrine signals in the adult BM microenvironment.

Vegfr3-creER T2 Transgene Mapping
The Vegfr3-creER T2 BAC genomic insertion site was determined by long-read sequencing of high molecular weight (HMW) leukocyte DNA.In short, HMW DNA was purified from 1 mL of red blood cell (RBC)-lysed peripheral blood from a male Vegfr3-creER T2 heterozygous animal using the Monarch HMW DNA Extraction Kit for Cells & Blood (New England BioLabs) according to the manufacturer's recommendation.The resulting HMW genomic DNA was sheared to a size of ~ 20 kb using a g-TUBE (Covaris) according to the manufacturer's suggestions.The sequencing library was prepared using the Ligation Sequencing Kit V14 (Oxford Nanopore Technologies).Briefly, 1 μg of DNA was repaired and end-prepped using the NEBNext Ultra II End Repair/dA-tailing Module (New England Biolabs).Sequencing adapters were ligated to the DNA ends and the adapted library was cleaned to remove fragments shorter than 3 kb.Twenty-two fmol of library DNA was loaded onto an R10.4.1 flow cell (FLO-PRO114M) and sequencing was carried out on a PromethION (Oxford Nanopore Technologies) instrument.Base calling was carried out directly on the device with the MinKNOW software using the high-accuracy setting and a minimum quality score of 8. Long DNA sequencing reads (5.9 M reads, N50 read length = 12,705 bp, average genome coverage = 20X) were split into non-overlapping contiguous 500 bp fragments and independently mapped to the mouse genome (C57BL/6 J; reference version GRCm39) and transgene sequence using minimap2 [24].The identified 923 long reads containing at least two 500 bp fragments mapping to both the mouse genome and the transgene were mapped back to the mouse genome and custom scripts were used to identify the region with the highest number of aligned reads.The integration site was identified on unplaced contig chrUn_JH584304, representing two contiguous peaks of high coverage.BAM files produced by the alignment of the fragments to the mouse genome were visualized using Integrative Genome Viewer (IGV) [25] to verify the location of the integration site and to generate Fig. 1b.

Microscopy
Endothelium were labeled in situ in tamoxifen-induced adult Vegfr3-creER T2+ , Cx40-creER T2+ , and Bmx-creER T2+ reporter animals (ZsGreen fl/wt or tdTomato fl/wt ) by retroorbital sinus injections with an αCDH5 antibody (Supplementary Table 1).Mice were euthanized 10 min post-injection and femurs, liver, and spleen samples were collected and fixed overnight with 4% paraformaldehyde (in PBS; pH 7.2) at 4 °C.Femurs were washed three times (15 min/ wash) with PBS (pH 7.2) at room temperature, decalcified in 10% EDTA in PBS (pH 7.2) for 72 h at room temperature, and normalized to 30% sucrose (in PBS; pH 7.2) for 72 h at 4 °C.Liver and spleen samples were washed three times (15 min/wash) with PBS (pH 7.2) at room temperature and normalized to 30% sucrose (in PBS; pH 7.2) for 72 h at 4 °C.Tissues were then embedded in 1:1 mixture of Tissue-Tek O.C.T. (Sakura) and 30% sucrose (in PBS; pH 7.2) and snap frozen in N 2 (l).To expose the marrow cavity for whole mount analysis, femurs were shaved longitudinally using a cryostat (Leica 3050S) and washed three times (5 min/wash) in PBS (pH 7.2) to remove excess O.C.T. Exposed marrow was then permeabilized in PBS (pH 7.2) with 20% (v/v) Normal Goat Serum (Jackson ImmunoResearch) and 0.5% (v/v) Triton X-100 (Sigma-Aldrich) for 2 h at room temperature and stained with an αSCA1 antibody for 48 h at 4 °C (Supplementary Table 1).For liver and spleen, sections were cut (12 μm) using a cryostat (Leica 3050S) and washed three times (5 min/wash) in PBS (pH 7.2) to remove excess O.C.T., and permeabilized in PBS (pH 7.2) with 20% (v/v) Normal Goat Serum (Jackson Immu-noResearch) and 0.5% (v/v) Triton X-100 (Sigma-Aldrich) for 30 min at room temperature.Tissues were washed three times (15 min/wash) in PBS (pH 7.2) and stained with DAPI (Biolegend) at 1 μg/mL in PBS (pH 7.2) for 15 min at room temperature (where applicable).Tissues were mounted with ProLong Gold (ThermoFisher Scientific) and imaged using a Nikon C2 confocal LASER-scanning microscope; 40 μm Z-stack images were acquired and denoised (Denoise.ai)and rendered into a maximum intensity projection using NIS Elements software (Nikon).

Whole Bone Marrow Isolation
Individual femurs were disassociated using a mortar and pestle in PBS (pH 7.2) + 0.5% BSA (w/v) + 2 mM EDTA and filtered (40 μm; Corning) to ensure a single cell suspension.Cell counts were determined using a Hemocytometer (Reichert Bright-Line; Hausser) and Trypan Blue (ThermoFisher Scientific) according to the manufacturer's recommendations.For HSPC analysis by flow cytometry, whole bone marrow (WBM) suspensions were depleted of terminally-differentiated hematopoietic cells using the murine-specific Lineage Cell-Depletion Kit (MiltenyiBiotec) according to the manufacturer's recommendations.

Liver/Spleen Digestion
Individual liver or spleen samples were minced to ~ 1mm 3 using sterile scalpels and enzymatically disassociated under gentle agitation in Hanks Balanced Salt Solution (Corning) + 10 mM HEPES (pH 7.2) with 2.5 mg/ mL Collagenase A (Roche) and 1 Unit/mL Dispase II (Roche) for 30 min at 37 °C.Resulting cell suspensions were filtered (40 μm; Corning) and washed using ten times digestion volume with PBS (pH 7.2) + 0.5% BSA (w/v) + 2 mM EDTA.Cell counts were determined using a Hemocytometer (Reichert Bright-Line; Hausser) and Trypan Blue (ThermoFisher Scientific) according to the manufacturer's recommendations.

HSPC Analysis
HSPC populations were quantified by flow cytometry from WBM stained with antibodies described in Supplementary Table 1.

Colony Forming Assays
Based on Hemocytometer counts, 7.5 × 10 4 total WBM cells in 300 μL Low-Glucose DMEM (ThermoFisher Scientific) were added to 3 mL MethoCult GF M3434 (StemCell Technologies) and plated in duplicate (2.5 × 10 4 cells/well) on low-adherent 6-well plates.Cells were incubated at 37 °C 5% CO 2 and scored for hematopoietic progenitor colony-forming units ten days post-plating using an SZX16 Stereo Microscope (Olympus) according to the manufacturer's guidelines.

Peripheral Blood
To examine multilineage donor engraftment or complete blood counts, mice were bled via the retro-orbital sinus using 75 mm heparinized capillary tubes (Kimble-Chase) into microfuge tubes with PBS (pH 7.2) + 10 mM EDTA and analyzed as described.

Complete Blood Counts
Complete blood counts were quantified using an Element HT5 (Heska) veterinary hematological analyzer according to the manufacturer's recommendations.

Hematopoietic Recovery
For myelosuppression, control (creER T2− ; Mapk fl/fl ) and experimental (creER T2+ ; Mapk fl/fl ) mice were subjected to single-dose total body irradiation (450 cGy; RadSource RS2000 Small Animal X-Ray Irradiator) and bled weekly to determine complete blood count recovery kinetics.Nonirradiated baseline counts were determined two weeks prior to myelosuppression.

Statistical Analysis
Experimental significance was determined using Prism 9.5.1 Software (Graphpad).Statistical analysis and parameters are indicated in individual figure legends.

Vegfr3-creER T2 Targets Sinusoidal and Transitional Endothelium in the Bone Marrow
To generate an inducible sinusoid-specific cre-expressing mouse model, we utilized a C57BL/6 J-derived bacterial artificial chromosome (BAC) containing the Vegfr3 gene with approximately 130 kb upstream and 60 kb downstream genomic DNA sequence.This BAC was previously used to generate Vegfr3-Yfp reporter mice [21] that discriminate sinusoidal endothelium from arterioles in adult BM [27].Using bacterial recombineering, a cre-ER T2 cassette was introduced in-frame downstream of the Vegfr3 exon 1 start codon (Fig. 1a).The residual Kanamycin selection cassette was removed via FLP-mediated recombination prior to targeted-BAC linearization and pronuclear injection into fertilized C57BL/6 J zygotes.Resulting Vegfr3-creER T2+ offspring were maintained on a C57BL/6 J background.The transgene insertion site was mapped to unplaced scaffold chromosome chrUN_ JH584304 approximately 40 kb upstream of phosphatidylserine decarboxylase -pseudogene 3 (Pisd-ps3) (Fig. 1b), avoiding the disruption of any known protein-coding genes.We next sought to evaluate the fidelity of Vegfr3-creER T2+ mice to mark sinusoidal endothelium in the adult BM microenvironment.

Discussion
Vascular-directed cre mice are a vital tool to unravel the complex BM EC-instructive mechanisms that modulate HSPC function in vivo.However, model-specific differences in cre activity can have a profound impact on the interpretation of experimental phenotypes [10,51].In this study, we set out to characterize congenic C57BL/6 J cre-expressing mice models that (1) allow for inducible cre-mediated recombination in adult BM EC subsets, bypassing the potential pitfalls of embryonic HSC involvement, (2) avoid off-target cre activity in HSPC-supportive BM perivascular niche cells, and (3) define non-overlapping arteriole, transitional, and sinusoidal BM EC cre activity.In reporter mice, tamoxifen-treated Vegfr3-creER T2 and Cx40-creER T2 efficiently labeled discrete adult BM sinusoidal/transitional and arteriole EC populations, respectively, while avoiding hematopoietic and stromal involvement.Vegfr3-creER T2 and Cx40-creER T2 also specifically label discrete sinusoidal and arteriole endothelium in secondary hematopoietic tissues, including the liver and spleen, with no detectable activity in non-endothelial populations.Functionally, we demonstrated that Vegfr3-creER T2 and Cx40-creER T2 mice were able to phenotypically identify and segregate arteriole-specific activation of MAPK signaling as the source of hematopoietic dysfunction previously reported using a vascular-specific pan-endothelial Cdh5-creER T2 driver in adult animals [15].
While the identified Vegfr3-creER T2 BAC-transgene insertion point was mapped to an unplaced genomic segment and does not appear to directly disrupt any protein-coding genes, Cx40-creER T2 knock-in animals [26] disrupt the endogenous Cx40 allele.However, the cardiovascular system in Cx40 +/-knockout mice display no reported gross differences when compared with wild type littermates [52][53][54].Nonetheless, Cx40-creER T2 and Vegfr3-creER T2 mice should be maintained as heterozygotes to avoid potential complications due to excessive cre activity or loss of gene function at the transgene insertion loci.Because cre recombinase activity in murine model systems have been implicated in loxPindependent cellular cytotoxicity [55,56], we also examined the potential effect of BM EC subset-specific creER T2 induction on HSC activity.Competitive transplantation of WBM from Vegfr3-creER T2+ or Cx40-creER T2+ tamoxifen-inducted animals demonstrated that creER T2 activity in these models does not impair hematopoietic reconstitution.