The authors thank Prof Frederic Geissmann and Prof Christian Schulz for insightful discussions; Dr Hannah Garner for critical reading of the manuscript, Dr Xavier Montagutelli and Dr Jean Jaubert and the staff of the Institut Pasteur animal facility for support with mouse husbandry; and Pascal Dardenne and Vytaute Boreikaite, an Amgen Scholar, for their technical assistance. Research in the E.G.P. laboratory is funded by the Institut Pasteur, the CNRS, the Cercle FSER (FRM and a starting package from the Institut Pasteur and the REVIVE consortium. L.I. is supported by a PhD fellowship from the REVIVE consortium.

Animal procedures were performed in accordance with the approved institutional animal care and use committee of the Institut Pasteur (CETEA).
1. In Utero Pulse Labeling in Csf1r MeriCreMer Rosa LSL-YFP Embryos
<ol>
	<li>Prepare the stock solution of 4-hydroxytamoxifen (OH-TAM, 50 mg/mL).
	<ol>
		<li>Under the fumehood, open the 25 mg vial of OH-TAM and add 250 &#181;L ethanol (100%).</li>
		<li>Transfer the ...

<ol>
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Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCL2+recruits+inflammatory+monocytes+to+facilitate+breast-tumour+metastasis.">CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis.</a> Nature. 475 (7355), 222-225 (2011).</li><li>Yona, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+mapping+reveals+origins+and+dynamics+of+monocytes+and+tissue+macrophages+under+homeostasis.">Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis.</a> Immunity. 38 (1), 79-91 (2013).</li><li>Sheng, J., Ruedl, C., Karjalainen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+Tissue-Resident+Macrophages+Except+Microglia+Are+Derived+from+Fetal+Hematopoietic+Stem+Cells.">Most Tissue-Resident Macrophages Except Microglia Are Derived from Fetal Hematopoietic Stem Cells.</a> Immunity. 43 (2), 382-393 (2015).</li></ol>

The authors have nothing to disclose.

The different waves of hematopoietic precursor cells partially overlap within a short time frame, which makes the analysis of the contribution of each wave of developmental hematopoiesis to immune cells technically very challenging.
Tamoxifen-inducible Cre systems offer the opportunity to tag specific cells in a temporally inducible manner and to perform lineage analysis in embryos or adults, without the need for ex vivo or in vitro culture or transplantation. In tamoxifen-in...

There are several successive but overlapping waves of hematopoietic progenitors during development whose myeloid progeny remain into adulthood. First, unipotent &#34;primitive&#34; progenitors emerge in the mouse yolk sac 1,2 between E7.5-E8.25 and give rise to embryonic macrophages without any monocytic intermediate. Whether macrophages derived from primitive progenitors persist in the adult brain as microglia remains a subject of active investigation. Second, Erythro-Myeloid Precursors (EMPs) arise in the yolk sac at E8.5, enter the bloodstream and colonize the embryo. EMPs emerge from the yolk sac hemogenic endothelium in a Runx1-dependant endothelial-to-hematopoietic transition 3,4. While EMP can differentiate into macrophages within the yolk sac, they also colonize the fetal liver from embryonic day (E)95 and differentiate into erythrocytes, megakaryocytes, macrophages, monocytes granulocytes and mast cells 6. The macrophages that derive from EMPs exhibit proliferative capacity in developmental and adult tissues. Whether EMP-derived macrophages bypass the monocyte stage of differentiation is still controversial as very little is known about their differentiation pathway 7,8. Finally, Hematopoietic Stem Cells (HSCs) emerge at E10.5 within the embryo proper from the aorta-gonad-mesonephros region and migrate to the fetal liver. HSCs with long-term repopulation capacity are only detected after E11 (at the 42 somite pair stage)9. There, they expand and differentiate from E12.5 until definitive hematopoiesis begins to shift to the bone marrow, which becomes the predominant site of blood cell production for the duration of postnatal life 10.
The spatial and temporal overlap in emergence, as well as shared immuno-phenotypic markers has thus far hampered our ability to distinguish the specific contributions of these waves of embryonic hematopoietic progenitors. While both EMP and HSC are generated in a Runx1-dependent manner and express the transcription factor Myb and the growth factor receptor Csf1r (Colony-Stimulating Factor 1 Receptor, also known as Macrophage Colony Stimulating Factor Receptor) among others, EMPs can be distinguished from HSCs by their lack of lymphoid potential, both in vitro and in vivo, their lack of long-term repopulating potential and lack of surface expression of the lineage marker Sca-1 11. Genetic fate mapping models are required to characterize macrophage ontogeny since they allow targeting embryonic progenitors in a cell-specific and time-specific manner. Here we present the fate-mapping protocol used in our laboratory to discriminate between the two lineages of macrophages found in most adult tissues: HSC-derived infiltrating macrophages and HSC-independent resident macrophages.
Tissue resident macrophages have been traced back to Myb-independent precursor cells expressing the cytokine receptor Csf1r12 and are present in the embryo at E8.5-E10.5 using three complementary fate-mapping strategies 6. In order to study yolk sac hematopoiesis without labeling fetal HSCs, we use a transgenic strain, Csf1rMeriCreMer, expressing a tamoxifen-inducible fusion protein of &#39;improved&#39; Cre recombinase and two mouse estrogen receptors (Mer-iCre-Mer) under the control of the Csf1r promoter. Hence, the Cre recombinase will be active in Csf1r-expressing cells during a limited time-window. When used with a reporter strain containing a fluorescent protein downstream of a lox-STOP-lox cassette (Rosa26LSL-eYFP), it will lead to the permanent genetic labeling of the cells present at the time of induction but also of their progeny. Administration at E8.5 of the active form of tamoxifen, 4-hydroxytamoxifen (OH-TAM), labels EMPs and macrophages, without labeling yolk sac unipotent &#34;primitive&#34; progenitors or fetal HSCs. Thereby, we have characterized the immunophenotype of EMPs and their progeny during embryonic development, as well as assessed the contribution of yolk sac-derived macrophages to the adult macrophage pools. Further work is required to characterize whether primitive progenitor-derived macrophages are also labeled using this approach and whether they can contribute to adult macrophage pools.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#5 Straight Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>MC19/B Pascheff-Wolff Spring Scissors&#160;</td>
			<td>Fine Science Tools</td>
			<td>15371-92</td>
			<td></td>
		</tr>
		<tr>
			<td>8.5 cm straight scissors</td>
			<td>Fine Science Tools</td>
			<td>14090-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse Kit-PE (clone 2B8)</td>
			<td>BD Pharmingen</td>
			<td>553355</td>
			<td>1/200 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD45.2 APC-Cy7 (clone 104)</td>
			<td>Sony</td>
			<td>1149120</td>
			<td>1/100 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse AA4.1-APC (CD93, clone AA4.1)</td>
			<td>eBioscience</td>
			<td>17-5892</td>
			<td>1/100 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse Ter119 PerCP-Cy5.5 (clone Ter119)</td>
			<td>Biolegend</td>
			<td>560512</td>
			<td>1/200 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse F4/80-BV421 (clone BM8)</td>
			<td>BD Pharmingen</td>
			<td>123137</td>
			<td>1/100 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD11b PE-Cy7 (clone M1/70)</td>
			<td>BD Pharmingen</td>
			<td>552850</td>
			<td>1/200 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD16/CD32 (Mouse BD Fc Block, clone 2.4G2)</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>Fischer Scientific</td>
			<td>12559069</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Fischer Scientific</td>
			<td>11570506</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D&#160;</td>
			<td>Roche</td>
			<td>11088882001</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Sigma</td>
			<td>D4527-20KU</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>Dutscher Dominique</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well tissue culture plate</td>
			<td>Dutscher Dominique</td>
			<td>353224</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture plate</td>
			<td>Fischer Scientific</td>
			<td>11874235</td>
			<td></td>
		</tr>
		<tr>
			<td>96 wells U-shape bottom tissue culture plate</td>
			<td>Dutscher Dominique</td>
			<td>353227</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Syringe Plastipak 2 mL&#160;</td>
			<td>Dutscher Dominique</td>
			<td>300185</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon grid 100 &#181;m cell strainers</td>
			<td>Dutscher Dominique</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon grid 70 &#181;m cell strainers</td>
			<td>Dutscher Dominique</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml Falcon tubes</td>
			<td>Dutscher Dominique</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup GP Millipore filtration kit, 0.2 &#956;m</td>
			<td>Dutscher Dominique</td>
			<td>51246</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A7906-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>(Z)-4-HYDROXYTAMOXIFEN 25mg</td>
			<td>Sigma</td>
			<td>H7904-25MG</td>
			<td>Special care should be taken when preparing and working with tamoxifen and its derivates. Always use appropriate personal protective equipment</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P3972</td>
			<td>Always use appropriate personal protective equipment</td>
		</tr>
		<tr>
			<td>PEG-35 castor oil (Kolliphor/Cremophor EL)</td>
			<td>Sigma</td>
			<td>C5135</td>
			<td></td>
		</tr>
		<tr>
			<td>Sunflower oil</td>
			<td>Sigma</td>
			<td>S5007-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol&#160;</td>
			<td>Sigma</td>
			<td>24103-1L-R-D</td>
			<td>Always use appropriate personal protective equipment and use under the fumehood</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E9884-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI, 1 ML (1 MG/ML IN WATER)</td>
			<td>Fischer Scientific</td>
			<td>10116287</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFLEX S B2-R3-V4-Y4 flow cytometer</td>
			<td>Beckman Coulter</td>
			<td>B75408</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFLEX Daily QC Fluorospheres</td>
			<td>Beckman Coulter&#160;</td>
			<td>B53230</td>
			<td></td>
		</tr>
		<tr>
			<td>VersaComp Antibody Capture Bead kit (2x5 mL)</td>
			<td>Beckman Coulter&#160;</td>
			<td>B22804</td>
			<td></td>
		</tr>
		<tr>
			<td>FVB-Tg(Csf1r-cre/Esr1*)1Jwp/J</td>
			<td>The Jackson laboratory</td>
			<td>19098</td>
			<td></td>
		</tr>
		<tr>
			<td>B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J</td>
			<td>The Jackson laboratory</td>
			<td>6148</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of erythromyeloid progenitors and their progeny in the mouse embryo by flow cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

Genetic fate mapping was achieved by administration at E8.5 of OH-TAM into Csf1r-Mer-iCre-Mer females mated with males carrying a Rosa26-LSL-eYFP reporter. In the presence of OH-TAM, excision of the stop cassette leads to the permanent expression of YFP in the cells expressing Csf1r. We collected two hematopoietic tissues: the yolk sac and the fetal liver and a non-hematopoietic tissue, the neuroectoderm, from the embryos at E10.5. Single-cell suspensions were obtained by enzymat...

Watch this Scientific Journal Video about Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry at JoVE.com

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

The overall goal of this experiment is to characterize yolk sac hematopoietic progenitors and their progeny in vivo using a mouse tamoxifen induced fate mapping system and flow cytometry. This can help answer key questions in the immunology field concerning the development and differentiation of hematopoietic progenitors that are distinct from hematopoietic stem cells. The main advantage of this technique is that the hematopoietic progenitors labeled within a limited time window and the progeny such as resident macrophages can be monitored during the development and adulthood.Generally, individuals new to this method will struggle because of the deal of cells for embryonic tissue and the large volume of the simultaneously treated samples. To begin the experiment, first transfer dissected mouse uterine horns into a 10 millimeter petri dish filled with approximately 30 milliliters of ice cold PBS. Immediately after, grasp the uterus muscle layers at the cervical end of the horn and slide fine scissors between the muscle layer and a decidual tissue to release the embryos.Then with fine forceps remove Reichert's membrane and remove the placenta. Gently dissect the yolk sac and transfer it to a 24-weld tissue culture plate filled with 0.5 milliliters of the digestion solution. After severing the umbilical and vituline vessels immediately transfer the embryo into a 12-weld tissue culture plate filled with 2 milliliters of 10 millimolar ice cold EDTA.After removing the head from the embryo with sharp fine scissors, incubate the body and the head on ice for 10 to 15 minutes and then remove the embryo and collect the EDTA containing the blood. Next, remove the amnion surrounding the embryo. Afterward remove the hind limbs and foreign limbs from the embryo.Using a pair of fine forceps, open the thorax. Then, grasp the anterior of the heart and gently pull the tissue while releasing the internal organs from the body with a second pair of forceps. Under a dissecting microscope, separate the fetal liver from the heart and gut.And transfer the organ to a 24 well plate filled with 0.5 milliliters of the digestion solution. Anatomical connection with the cardiovascular system is critical for fetal liver identification. The fetal liver is still in this developmental stage and the younger the embryos are, the more difficult the dissection can be.To prepare primary cell suspensions, incubate the collected organs in the digestion solution at 37 degrees Celsius for 30 minutes. Load the tissue in the enzymatic solution into a 10 micrometer strainer installed over one weld of a six-weld tissue culture plate filled with 1 milliliter of FACS buffer. Use a black rubber piston from a 2 millimeter sysringe to dissociate the tissue by gently mashing to obtain a single cell suspension.Then, aspirate the cell suspension with a Pasteur pipette and transfer it into a 15 milliliter tube. Centrifuge the suspension at 320 times G at 4 degrees Celsius for 7 minutes. After centrifugation, aspirate and discard the supernatant.Next, prepare fresh Fc blocking buffer and the resuspend the pellet in 60 microliters. Then, transfer 50 microliters of the single cell suspension into a well of a 96-well round bottom plate. Incubate the plate on ice for at least 15 minutes.Next, transfer the remaining 10 microliters of the suspension into a 5 milliliter polystyrene FACS tube to obtain a pool of samples derived from different tissues. Transfer 50 microliters of the pool for FMO controls for each fluorochrome to the 96 multi well plate. To proceed with antigen surface staining, use a set of six flourochrome antibodies to prepare an antibody solution and FACS buffer.Then prepare six antibody solutions and FACS buffer used for the FMO control staining. Afterward add 50 microliters of the antibody solutions to the samples in the 96 multi well plate. Mix the samples by gentle double pipetting and then incubate the plate on ice for 30 minutes.Centrifuge the 96 multi well plate at 320 times G at 4 degrees Celsius for 7 minutes and remove the supernatant. Wash the cells in 200 microliters of FACS buffer and spin the plate one more time. Special care should be taken when removing the supernatant by plate inversion.The 96 multi-well plate must be inverted with a single sharp wrist motion to avoid loosening the cells. Finally, using a 70 micro strainer, filter the tissue and control samples into 6 millimeter polystyrene tubes. Flow cytometric analysis can now be carried out.A population of a live non-erythrocytic cytic single cells was analyzed for the expression of the progenitor marker kit and the hematopoietic cell marker CD45. Within the targeted population, three different cell sub populations were distinguished. KIT positive CD45 negative, KIT positive CD45 low and KIT negative CD45 positive.Among the yolk sac kit positive progenitors, both CD45 negative and CD45 low cell populations contained AA4.1 positive cells that express the yellow fluorescent protein or YFP, indicating that immature KIT positive progenitors first express AA4.1 irrespective of CD45 expression. AA4.1 positive, YFP positive cells in the liver and the brain were only found in the KIT positive CD45 low progenitor population and correspond most likely to circulating progenitors that originated from the yolk sac progenitors. Macrophages were found in the kit negative CD45 positive population and were identified as F480 bright CD11 B positive cells.Macrophages in the yolk sac, liver and brain were efficiently labeled by a single 4-hydroxy tamoxifen administration that may indicate their ENP origin. Once mastered, the procedure for cell isolation and staining can be done in three to four hours if it's performed properly. While attempting this procedure, it's important to remember to always keep samples on ice as soon as they are collected.Fresh FACS buffers should be prepared daily and filtered. Following this procedure, other mash reporter or flux strains like Rosa MTMG or Rosa confetti lines can be used in order to answer additional questions on the indifferentiation and proto phase potential of the post labeled cells.

identification-erythromyeloid-progenitors-their-progeny-mouse-embryo

Research

JoVE Journal

Developmental Biology

12.8K vistas.  Institut Pasteur. The overall goal of this experiment is to characterize yolk sac hematopoietic progenitors and their progeny in vivo using a mouse tamoxifen induced fate mapping system and flow cytometry. This can help answer key questions in the immunology field concerning the development and differentiation of hematopoietic progenitors that are distinct from hematopoietic stem cells. The main advantage of this technique is that the hematopoietic progenitors labeled within a limited time window and the proge...