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 OH-TAM solution to a 2 mL microcentrifuge tube with a truncated tip and vortex at maximum speed for 10 min.</li>
		<li>Sonicate 30 min in a sonicator bath.</li>
		<li>Add 250 &#181;L of PEG-35 castor oil under the fumehood to obtain a 50 mg/mL stock solution. 
		NOTE: PEG-35 castor oil is a solvent with amphiphilic properties that binds hydrophobic molecules and solubilizes them in aqueous solvents.</li>
		<li>Vortex for approximately 5 min at maximum speed and sonicate 30 min in a sonicator bath.</li>
		<li>Aliquot 90 &#181;L (4.5 mg) per microcentrifuge tube (1 aliquot per injection).</li>
		<li>Store at 4 &#176;C for 1 week or at -20 &#176;C for long term as previously described 13.</li>
	</ol>
	</li>
	<li>Prepare the stock solution of Progesterone (10 mg/mL)
	<ol>
		<li>Add 250 &#181;L of 100% Ethanol to 25 mg of progesterone to prepare a 10 mg/100 &#181;L suspension under the fumehood. Vortex gently.</li>
		<li>Add 2250 &#181;L autoclaved sunflower oil to make 10 mg/ml progesterone solution under the hood.</li>
		<li>Vortex for approximately 5 min at maximum speed, aliquot and store at 4 &#176;C. Note that the solution is clear when stored.</li>
	</ol>
	</li>
	<li>Tamoxifen administration 
	NOTE: Embryonic development was estimated considering the day of vaginal plug formation as Embryonic day (E) 0.5. Recombination is induced by single injection at E8.5 into pregnant Csf1rMeriCreMerfemales 12. Supplement OH-TAM with 37.5 &#181;g per g of Progesterone to reduce abortion rates after tamoxifen administration. Inject at 1 pm (for vaginal plug observed in the morning).
	<ol>
		<li>On the morning of injection, sonicate an aliquot of the OH-TAM solution for 10 min (or until completely resuspended).</li>
		<li>Add 360 &#181;L NaCl 0.9% under the fumehood to obtain a 10 mg/mL OH-TAM solution and vortex thoroughly. Sonicate until the solution is clear and completely resuspended (at least 30 min).</li>
		<li>Pre-warm progesterone to room temperature.</li>
		<li>Mix 450 &#181;L of OH-TAM and 225 &#181;L of progesterone in a microcentrifuge tube. Vortex.</li>
		<li>Sonicate at least 10 min and load on to a 1 mL syringe with a 25G needle.</li>
		<li>In the animal facility, weigh the pregnant female (Csf1rMeriCreMer females are in an inbred FVB genetic background and typical weigh between 25 and 33 g).</li>
		<li>Perform an intra-peritoneal injection by slowly injecting the calculated volume into the mouse. After withdrawing the needle, gently press on the puncture wound and massage the abdomen to distribute the OH-TAM. 
		NOTE: The OH-TAM injection dose is 75 mg/kg and the Progesterone dose is 37.5 mg/kg. Table 1 provides the volume to inject into pregnant females.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mouse weight (g)</td>
			<td>Total volume to inject from the mix (&#181;L)</td>
		</tr>
		<tr>
			<td>25</td>
			<td>281</td>
		</tr>
		<tr>
			<td>26</td>
			<td>292</td>
		</tr>
		<tr>
			<td>27</td>
			<td>303</td>
		</tr>
		<tr>
			<td>28</td>
			<td>315</td>
		</tr>
		<tr>
			<td>29</td>
			<td>326</td>
		</tr>
		<tr>
			<td>30</td>
			<td>338</td>
		</tr>
		<tr>
			<td>31</td>
			<td>348</td>
		</tr>
		<tr>
			<td>32</td>
			<td>360</td>
		</tr>
		<tr>
			<td>33</td>
			<td>371</td>
		</tr>
		<tr>
			<td>34</td>
			<td>382</td>
		</tr>
		<tr>
			<td>35</td>
			<td>394</td>
		</tr>
	</tbody>
</table>
Table 1: Injection volume of 4-OH-tamoxifen (OH-TAM). Volume required for a single injection at E8.5 of 75 &#181;g per g (body weight) of OH-TAM supplemented with 37.5 &#181;g per g of progesterone.
2. Dissection of the Yolk Sac (YS) and Fetal Liver (FL)
NOTE: Rigorous sterile techniques are not necessary when manipulating embryos unless if they are going to be used for long-term culture. Nevertheless, the working area must be clean and covered in foil under absorbent paper.
<ol>
	<li>Prepare ice-cold phosphate buffered saline (PBS) and digestion mix (PBS containing 1 mg/mL collagenase D, 100 U/mL Deoxyribonuclease I (DNase I) and 3% fetal bovine serum).</li>
	<li>Sacrifice pregnant females by cervical dislocation at the required gestation day (e.g. embryonic stage E10.5).</li>
	<li>Pinch the skin just over the genitals and make a small incision at the midline with scissors. Pull the skin toward the head to expose completely the body wall without fur. Cut the abdominal muscles to expose the internal organs and push the gut to expose the two uterine horns.</li>
	<li>With middle-sized blunt forceps, grab the fat-pad attached to the ovary and gently pull the uterus. Cut at the cervical level of the uterine horns and lift the horns from the peritoneal cavity. Remove the fat-pad to completely free the uterine horns and cut the horns from the ovary on each side.</li>
	<li>Put the horns into ice-cold PBS in a 10 mm Petri dish. Rapidly grip the uterus muscle layers at one extremity (cervical end) and slide fine scissors between the muscle layer and the decidual tissue to release the embryos with the surrounding decidual tissue. 
	NOTE: Muscle tend to rapidly contract after the extraction, so this step must be as quick as possible.</li>
	<li>Use one pair of fine forceps to cut off the Reichert&#39;s membrane and the placenta.</li>
	<li>Gently remove the yolk sac and place it in a 24-well tissue culture plate with 0.5 mL of digestion mix. 
	NOTE: At this step, embryonic blood can be collected.
	<ol>
		<li>Immediately after severing the umbilical and vitelline vessels, transfer the embryo into a 12-well tissue culture plate containing 10 mM ice-cold ethylenediaminetetraacetic acid (EDTA). Decapitate the embryo using sharp fine scissors, trying to limit as much as possible tissue dilaceration. Incubate on ice for 10-15 min and collect the EDTA containing the blood.</li>
	</ol>
	</li>
	<li>Remove the amnion surrounding the embryo.</li>
	<li>For stages &#60;E11.5, count the number of somite pairs for better staging of embryos and a better time resolution (each somite pair develops in ~1 h 30 min).</li>
	<li>Cut the head of the embryo using forceps or fine scissors. Transfer the head to a 24-well plate with 0.5 mL digestion mix. 
	NOTE: The neuroectoderm and brain at later stages are further dissected for flow cytometry analysis; carefully remove the surrounding vascular plexus.</li>
	<li>Cut the embryo above the hindlimb and remove the forelimbs.</li>
	<li>To isolate the liver, open the thorax using a pair of fine forceps. Pinch anteriorly to the heart and gently pull while using the second forceps to free the organs from the body.
	<ol>
		<li>Carefully separate the fetal liver from the heart and gut. Transfer the fetal liver to a 24-well plate with 0.5 mL digestion mix. Carefully monitor the transfer under the dissecting microscope.</li>
	</ol>
	</li>
	<li>Collect the tail region (or any other embryo part) for genotyping by polymerase chain reaction assay (PCR). 
	NOTE: Other tissues and organs can be harvested from E10.5 embryos for a similar analysis using flow cytometry. Blood, head skin, aorta-gonad-mesonephros region (AGM), heart and neuroectoderm can be collected at E10.5. At later stages, lung, kidney, spleen and pancreas can also be collected. A detailed description of the dissection of the AGM at different developmental stages has been previously described 14.</li>
</ol>
3. Processing of Embryonic Tissues for Flow Cytometry
<ol>
	<li>Incubate the organs (placed in the digestion mix) for 30 min at 37 &#176;C.</li>
	<li>Transfer the tissue and enzymatic solution onto a 100 &#956;m strainer placed in a 6-well tissue culture plate filled with 6 ml of FACS buffer (0.5% Bovine Serum Albumin (BSA) and 2 mM EDTA in 1x PBS). Mechanically dissociate by gently mashing with the black rubber piston of a 2 mL syringe to obtain a single-cell suspension. 
	NOTE: From now on, all steps should be performed at 4 &#176;C.</li>
	<li>Collect the cell suspension with a Pasteur pipette and transfer into a 15 mL tube.</li>
	<li>Spin for 7 min at 320 x g at 4 &#176;C. Discard the supernatant by aspiration.</li>
	<li>Resuspend the pellet in 60 &#181;L Fc-blocking buffer (CD16/CD32 blocking antibody diluted 1/50 in FACS buffer).
	<ol>
		<li>Transfer 50 &#181;L of the single-cell suspension per well in a round bottom 96 multi-well plate. Incubate for at least 15 min on ice. 
		NOTE: The staining can also be performed in 5 mL polystyrene FACS tubes when handling a small number of samples.</li>
		<li>Transfer the remaining 10 &#181;L into a 5 mL polystyrene FACS tube to obtain a pool of the samples from each tissue. This pool will serve as the controls (i.e. unstained samples and fluorescent minus one (FMO) controls for each fluorochrome). Transfer 50 &#181;L of the pool for each fluorescent minus one (FMO) control (one per fluorochrome) to the 96 multi-well plate. 
		NOTE: Each FMO control contains all the fluorochrome-coupled antibodies from the antibody panel, except for the one that is being measured. FMOs are used to identify gate boundaries and to control for the spectral overlap in multicolor panels. To correctly address the variations in background and autofluorescence between different tissues, it is important to prepare these controls from the pool of samples of each tissue. The appropriate control for YFP expression will be the samples from Cre-negative embryos, which are confirmed by PCR genotyping.</li>
	</ol>
	</li>
</ol>
4. Surface Antigen Staining
<ol>
	<li>Prepare the antibody mix in FACS buffer (Table 2). Prepare 50 &#181;L of antibody mix per sample.
	<ol>
		<li>Use the following fluorochrome-coupled antibodies: anti-CD45.2 (clone 104); anti-CD11b (clone M1/70); anti-F4/80 (clone BM8); anti-AA4.1 (clone AA4.1); anti-Kit (clone 2B8); and anti-Ter119 (clone Ter119).</li>
		<li>Prepare six antibody mixes for the FMO controls in FACS buffer. Prepare 50 &#181;L of antibody mix per control sample. 
		NOTE: The antibody dilution in the antibody mix is two times more concentrated than the final concentration indicated in the materials table. For a panel with six fluorochrome-coupled antibodies, 6 FMO controls are prepared. Table 2 provides the composition of the antibody mix and FMO controls for one tube.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td colspan="7">Volume (&#181;L) of antibody&#160; (Final Volume 50 &#181;L)</td>
		</tr>
		<tr>
			<td>Antibody</td>
			<td>Clone</td>
			<td>Antibody Mix</td>
			<td>FMO CD45.2</td>
			<td>FMO CD11b</td>
			<td>FMO F4/80</td>
			<td>FMO AA4.1</td>
			<td>FMO&#160;&#160;&#160;&#160;&#160; Kit</td>
			<td>FMO Ter119</td>
		</tr>
		<tr>
			<td>anti-CD45.2</td>
			<td>104</td>
			<td>1</td>
			<td>0</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>anti-CD11b</td>
			<td>&#160;M1/70</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>anti-F4/80</td>
			<td>BM8</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>0</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>anti-AA4.1</td>
			<td>AA4.1</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>0</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>anti-Kit</td>
			<td>2B8</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>anti-Ter119</td>
			<td>Ter119</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0</td>
		</tr>
		<tr>
			<td colspan="2">FACS buffer</td>
			<td>45.5</td>
			<td>46.5</td>
			<td>46</td>
			<td>46.5</td>
			<td>46.5</td>
			<td>46</td>
			<td>46</td>
		</tr>
	</tbody>
</table>
Table 2: Volume of antibodies using for staining and Fluorescent minus one (FMO) controls. Volume (&#181;L) of antibody required for a final volume of 50 &#181;L.
<ol start="2">
	<li>Add 50 &#181;L of the antibody mix to the samples in the 96-multiwell plate (final volume during staining is 100 &#181;L). Gently pipette up and down twice and incubate on ice for 30 min.</li>
	<li>Spin the 96-multiwell plate for 7 min at 320 x g at 4 &#176;C and discard the supernatant. Resuspend in 200 &#181;L of FACS buffer.</li>
	<li>Repeat wash procedure (step 4.3) twice with 150 &#181;L FACS buffer.</li>
	<li>Filter the samples and the controls (FMOs and unstained pool samples) into 5 ml polystyrene tubes through a 70 &#956;m strainer. Store on ice until acquisition.</li>
</ol>
5. Identification of EMPs and YS-derived Macrophages by Flow Cytometry
NOTE: This protocol was optimized using a 4 laser flow cytometer equipped with a 405 nm Violet laser, a 488 nm Blue laser, a 562 nm Yellow laser and a 638 nm Red laser.
<ol>
	<li>Prepare compensation beads for each coupled antibody following the manufacturer&#39;s instructions. Use unstained samples and compensation beads to optimize laser intensities.</li>
	<li>To identify gate boundaries, use FMO (Fluorescence minus one) controls for each antibody.</li>
	<li>In order to exclude dead cells, add 1 &#181;L of DAPI (1 mg/ml) to the tube (containing 200 &#181;L of stained cell suspension) 1 min before acquisition. Use the strong DAPI signal and the forward-scattered parameter (FSC) to perform exclusion of dead cells and debris. 
	NOTE: Use software from the flow cytometer to draw gates during acquisition. Compensation matrix and analysis can be performed after sample acquisition using other commercially available software.</li>
	<li>Use forward and side scatter (FSC vs SSC) to perform live cell and doublet discrimination from total events.</li>
	<li>Create fluorescence dot plots in log-scale axis and draw daughter gates to, first, exclude erythrocytes (Ter119+) and, then, to identify progenitor cells (Kit+) and hematopoietic cells (CD45+ Kitneg) (See Figure 1).</li>
	<li>Create fluorescence histograms to quantify the labeling efficiency of YFP among the different identified populations in the YS (Figure 2), fetal liver (Figure 3) and brain (Figure 4).</li>
</ol>

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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-inducible Cre strains, a fusion gene is created between a bacterial Cre recombinase and a mutant form of the ligand binding domain of the human estrogen receptor (CRE-ER) or between an improved single-point mutant Cre and two mouse estrogen receptors (Mer-iCre-Mer). Fusion of Cre with estrogen receptors leads to the cytoplasmic sequestration of Cre by Hsp90, thereby preventing nuclear Cre-mediated recombination. Tamoxifen (TAM) is metabolized by the liver into 4-hydroxytamoxifen (OH-TAM), its active metabolite with high binding affinity for estrogen receptor. OH-TAM binding to the fusion Cre leads to the disruption of the interaction with Hsp90, permitting its translocation to the nucleus and initiation of Cre-mediated recombination. Injection of TAM or OH-TAM in utero into pregnant females has been shown to activate recombination in the developing embryo. Tamoxifen administration during pregnancy can lead to abortion and thus reduces litter size but it also prevents birth if delivered after mid-gestation, in which case pup delivery through a caesarean operation is required. To counterbalance tamoxifen effects during pregnancy, we supplement OH-TAM administration with a half-dose of progesterone, in order to improve survival of embryos and reduce the risk of abortions 17.
Several routes can be employed to deliver TAM or OH-TAM into pregnant females. Currently oral gavage or intra peritoneal (ip) injection is used for tamoxifen. OH-TAM has the advantage to be immediately available, as it does not require to be metabolized by the liver of the pregnant dam. However, while tamoxifen is very easy to dissolve in corn oil, OH-TAM is very difficult to prepare using the same technique and results in an emulsion. This has been a limiting step in employing OH-TAM for in utero pulse labeling. We have adapted the protocol for preparation of OH-TAM developed by the group of J.F. Nicolas 13, where they use PEG-35 castor oil, a solvent with amphiphilic properties to solubilize OH-TAM, as tamoxifen dissolution is one of the most critical steps in the procedure. This allows for reproducible and optimal preparation of OH-TAM and considerably shortens the time of tamoxifen preparation. Further, it is necessary to adapt the concentration of OH-TAM to inject when using different inducible Cre strains. The combination of a viability dye (DAPI) and size (FSC) is critical to remove dead cells and cellular debris, respectively. Dead cells and debris are highly autofluorescent in most of the violet, blue and red laser detectors, and can hamper flow cytometric analysis. Further, we do not recommend fixing the cells after staining prior to analysis with the flow cytometer. Fixation can lead to changes in cell morphology and thus renders difficult size discrimination based on Forward and Side Scatter (FSC vs. SSC). Further, fixation of samples with PFA leads to a significant loss of YFP fluorescent intensity.
The main limitation within this protocol is that the populations are not tested on their functionality and differentiation potential. In order to overcome this, cells can be sorted following a similar protocol using a Fluorescently-activated cell sorter (FACS) to perform colony-forming assay. In this case, the procedure must be performed under sterile conditions and use of fetal bovine serum (FBS) in the FACS buffer, instead of BSA, is highly recommended. The low labeling efficiency achieved with this technique and the consequently low number of YFP+ cells of interest per embryonic tissue is a major challenge in the study of EMP phenotype. This protocol uses a 4-laser flow cytometer. A flow cytometer with 3 lasers can also be used but it is important to adapt the antibody panel to take into account the increased spectral overlap between dyes. Additionally, titration of antibodies is required to find the appropriate concentration when changing the antibody panel and we recommend performing titration of antibodies and validation of the new antibody panel in a pilot experiment where all embryos per tissue are pooled, in order to increase the number of cells analyzed per tissue.
The field of developmental biology has been revolutionized by the Cre/Lox method, which allows permanent labeling of cells in situ. This approach achieves tissue- or cell type -specificity through expression of the Cre recombinase under the control of a specific promoter. In our case, we chose to study the expression of the Cre recombinase under the control of the promoter of Csf1r (Colony Stimulating Factor 1 Receptor), an important mitogenic and growth factor receptor for macrophages and their progenitors, using a strain developed by the group of J. Pollard 18. The advantage of a fate mapping strategy based on Csf1r expression compared to other published strategies is that it allows the labeling of yolk sac-derived cells (EMP and macrophages) without labeling any fetal HSCs 12. The Cx3cr1CreERT2 strain can also label yolk sac cells without labeling HSCs 19, however it can only label macrophages and macrophage precursors but it does not label EMP progenitors 1. The Cx3cr1CreERT2 strain has the same caveat as the Csf1rMerCreMer line, since it cannot distinguish between macrophages derived from unipotent primitive macrophage progenitors or from EMP. When tamoxifen is administered as early as E7.5 in both Runx1MerCreMer16 and KitMerCreMer20, peripheral blood cells are always labeled in adults, albeit at a very low efficiency, thus demonstrating labeling of HSCs during development. Further, KitMerCreMer fate mapping labels yolk sac Kit+ Sca1+ progenitors while EMP are Kit+ Sca1 negative 11. The Csf1rMeriCreMer strain is not a knock-in but it was generated by classical additive transgenesis, thus expression of the Cre may not fully recapitulate endogenous expression of Csf1r. However, this has the advantage to avoid any potential developmental defects due heterozygous expression of Csf1r. This is an important factor to take to account when analyzing other fate mapping strategies used for developmental hematopoiesis studies, such as Runx1MerCreMer, KitMerCreMer and Cx3cr1CreERT2, where the inducible Cre is inserted-in the endogenous locus. For both Runx1MerCreMer and Kit MerCreMer, developmental hematopoietic defects have been described in heterozygous embryos. Collectively, the method presented here allows to investigate yolk sac EMP biology during development and also yolk sac-derived macrophages found in adult tissues and distinct from HSC-derived macrophages. This will improve our current understanding of this lineage of resident macrophages and their specific functions in adulthood. The immunophenotypic characterization of EMPs has been improved recently using cell sorting and colony forming assays, demonstrating that yolk sac EMP specifically express CD41 and CD16/32 in early stages of development 11. However, the specificity of expression of CD41 and CD16/32 needs further characterization from E10.5 onwards, especially in the fetal liver niche, using fate-mapping models. Indeed, whether CD41 and CD16/32 expression is still specific to EMP when compared to fetal HSCs and HSC-derived progenitors in the E10.5 to E14.5 fetal liver need to be elucidated. The presented method allows to label EMP generated in the yolk sac and to follow them during embryonic development and it will contribute to a more detailed characterization of EMP phenotype when they seed the fetal liver.
This method could be important in the near future to study EMP differentiation pathways. While EMPs emerge from the yolk sac endothelium from E8.5 to E10.5 4, this method only allows to label a fraction of the progenitors emerging between E8.5-E9.5. Therefore, a limitation of this method is that only a small fraction of EMP is labeled, thus rendering difficult the analysis in classical loss-of-function studies with floxed alleles for candidate genes. Alternatively, sparse labeling could become an advantage in clonal studies of EMP differentiation in vivo, using clonal reporter. Nevertheless, the labeling efficiency needs to be characterized for each floxed reporter or floxed allele used, as recombination efficiency of floxed constructs depends on genomic locus (floxed alleles) or the Rosa26 reporter construct. Indeed, different Rosa26 reporter lines are available with different promoters and it is reported that Rosa26tdTomato and Rosa26mTmG strain have higher recombination efficiency than the Rosa26LSL-eYFP line. The reporter mouse strain used in this protocol is Rosa26LSL-eYFP line, which cannot provide information regarding the dynamic process of progenitor maturation mentioned in the results section. The question of maturation process can be partially addressed using different reporter strains like the Rosa26mTmG. In such strain, cells can be distinguished based on the time elapsed since the recombination event. All cells in the Rosa26mTmG strain express membrane-bound tdTomato fluorescent protein and Cre recombination excises the tdTomato cassette and allows for expression of membrane-bound GFP. Since tdTomato has a long half-life, cells that still contain tdTomato but have started to express the GFP (shortly after recombination) are tdTomato+ GFPlow/int and can be distinguished from tdTomatoneg GFP+ cells that do not express tdTomato anymore and express higher levels of GFP (later after recombination).
As discussed above, OH-TAM preparation is a critical step to consistently deliver similar doses of OH-TAM between experiments and to reduce tamoxifen side effects. Progesterone co-administration is also useful to limit deleterious effect of tamoxifen on pregnancy. Another critical element of the protocol is the viability of the single-cell suspension obtained from different embryonic tissues. We usually obtain &#62;90% viable cells for flow cytometric analysis. To achieve this, it is critical to follow all the steps rapidly and to maintain all samples on ice after tissue collection. Appropriate controls such as unstained samples, single stains and fluorescent minus one (FMO) controls are required to identify gate boundaries and to control for the spectral overlap in multicolor panels. Changes in the antibody need to be validated in terms of antibody titration and spectral overlap. Finally, the choice of the Rosa26 reporter strain needs to be adapted to the scientific question that is investigated.

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>
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			<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 enzymatical and mechanical dissociation and samples were analyzed by flow cytometry after staining with fluorescent-coupled antibodies. After exclusion of dead cells and doublets, single cell suspensions were analyzed (Figure 1). All gates were defined using Fluorescence Minus One (FMO) controls. It is recommended to also perform an erythrocyte exclusion (Ter119+ cells) to improve clarity of the analysis (Figure 1B). Using the cell surface markers Kit (progenitor marker) and CD45 (hematopoietic cells marker), we were able to distinguish three populations of cells of interest: Kit+ CD45neg; Kit+ CD45low and Kitneg CD45+ (Figure 1). Progenitors were further analyzed based on their expression of AA4.1 (Figures 2-4). Indeed, AA4.1 is a surface marker that allows the enrichment of the progenitor population within erythromyeloid progenitors in the yolk sac, as demonstrated in colony forming assays 15. Once the populations of interest were identified, we quantified the YFP labeling efficiency in each population using histograms. Among the yolk sac Kit+ progenitors, both CD45neg (Figure 2A) and CD45low (Figure 2B) cell populations contain AA4.1+ YFP+ cells. However, AA4.1+ YFP+ cells in the liver and brain are only found in the Kit+ CD45low progenitor population (Figure 3B and 4B). Since, the brain is not an active site of hematopoiesis, progenitors found in this tissue could correspond to circulating progenitors. This also suggests a process of progenitor maturation as they migrate from the yolk sac to the fetal liver and the peripheral tissues. Kit+ progenitors first express AA4.1, irrespectively of CD45 expression, but by the time they reach circulation and the fetal liver, all AA4.1+ YFP+ progenitors express detectable levels of cell surface CD45. Macrophages, defined as F4/80bright CD11b+, were found in the Kitneg CD45+ gate (Figure 2-4). Macrophages in the E10.5 yolk sac, liver and brain were efficiently labeled (60 to 80% of F4/80bright CD11b+ cells are YFP+) by a single OH-TAM administration at E8.5 (Figure 2C, 3C and 4C).
To compare labeling efficiency between litters, we recommend that an internal control for recombination efficiency is used. Variability in the level of expression of the reporter can be observed between experiments, due to variations in developmental timing between litters and mouse strains when OH-TAM is injected in utero. Hence, we recommend that embryo staging from E8.5 to E11.5 embryos is performed by somite pair counting. In this protocol, we propose to use the labeling efficiency in the brain resident macrophages (microglia) as a reference to compare the efficiency of Cre recombination between different experiments and strains (Figure 4C). Other resident macrophages could be used, however microglia were the first cells to be described as yolk sac-derived macrophages 16 and they represent the most abundant population of tissue resident macrophages at E10.5. Thus, YFP labeling in microglia can be used to assess the fate-mapping efficiency in each experiment and compare data from different litters.
<img alt="Figure 1" src="/files/ftp_upload/55305/55305fig1.jpg" /> 
Figure 1: Gating strategy to identify progenitor cells (Kit+ CD45neg and Kit+ CD45low) and differentiated hematopoietic cells (Kitneg CD45+). (A) Discrimination of live cells and singlets is performed using DAPI staining and Forward and Side scatter (FSC/SSC) parameters. (B) Following red blood cell exclusion (Ter119neg), three populations of cells of interest are identified based on cell surface expression of Kit and CD45: Kit+ CD45neg; Kit+ CD45low and Kitneg CD45+ cells. (C) Csf1r-expressing cells present when OH-TAM is injected and their progeny are identified in Cre-recombinase positive embryos as expressing YFP compared to the Cre-recombinase negative embryos (confirmed later by polymerase chain reaction assay, PCR). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55305/55305fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55305/55305fig2.jpg" /> 
Figure 2: Labeling efficiency in the yolk sac from E10.5 Csf1rMeriCreMer Rosa26LSL-eYFP embryospulsed with OH-TAM at E8.5. (A) Kit+ CD45neg cells are gated based on Kit and CD45 expression on live yolk sac cells (blue gate, left panel). AA4.1 and Kit expression on Kit+ CD45neg cells. Blue gate encloses AA4.1+ Kit+ CD45neg cells (middle panel). Comparison of YFP labeling in AA4.1+ Kit+ CD45neg cells (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). (B) Kit+ CD45low cells are gated based on Kit and CD45 expression (blue gate, left panel). AA4.1 and Kit expression on Kit+ CD45low cells. Blue gate encloses EMP, as defined as AA4.1+ Kit+ CD45low cells (middle panel). Comparison of YFP labeling in AA4.1+ Kit+ CD45low cells (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). (C) Hematopoietic cells are defined as Kitneg CD45+ and gated in blue (left panel). F4/80 and CD11b expression on Kitneg CD45+ cells. Macrophages are defined as F4/80bright CD11b+ cells (middle panel). Comparison of YFP labeling in F4/80bright CD11b+ macrophages (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55305/55305fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55305/55305fig3.jpg" /> 
Figure 3: Labeling efficiency in the liver from E10.5 Csf1rMeriCreMer Rosa26LSL-eYFP embryospulsed with OH-TAM at E8.5. (A) Kit+ CD45neg cells are gated based on Kit and CD45 expression on live liver cells (blue gate, left panel). AA4.1 and Kit expression on Kit+ CD45neg cells. Blue gate encloses AA4.1+ Kit+ CD45neg cells (middle panel). Comparison of YFP labeling in AA4.1+ Kit+ CD45neg cells (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). (B) Kit+ CD45low cells are gated based on Kit and CD45 expression (blue gate, left panel). AA4.1 and Kit expression on Kit+ CD45low cells. Blue gate encloses EMP, as defined as AA4.1+ Kit+ CD45low cells (middle panel). Comparison of YFP labeling in AA4.1+ Kit+ CD45low cells (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). (C) Hematopoietic cells are defined as Kitneg CD45+ and gated in blue (left panel). F4/80 and CD11b expression on Kitneg CD45+ cells. Macrophages are defined as F4/80bright CD11b+ cells (middle panel). Comparison of YFP labeling in F4/80bright CD11b+ macrophages (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55305/55305fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55305/55305fig4.jpg" /> 
Figure 4: Labeling efficiency in the brain from E10.5 Csf1rMeriCreMer Rosa26LSL-eYFP embryospulsed with OH-TAM at E8.5. (A) Kit+ CD45neg cells are gated based on Kit and CD45 expression on live brain cells (blue gate, left panel). AA4.1 and Kit expression on Kit+ CD45neg cells. Blue gate encloses AA4.1+ Kit+ CD45neg cells (middle panel). Comparison of YFP labeling in AA4.1+ Kit+ CD45neg cells (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). (B) Kit+ CD45low cells are gated based on Kit and CD45 expression (blue gate, left panel). AA4.1 and Kit expression on Kit+ CD45low cells. Blue gate encloses EMP, as defined as AA4.1+ Kit+ CD45low cells (middle panel). Comparison of YFP labeling in AA4.1+ Kit+ CD45low cells (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). (C) Hematopoietic cells are defined as Kitneg CD45+ and gated in blue (left panel). F4/80 and CD11b expression on Kitneg CD45+ cells. Macrophages are defined as F4/80bright CD11b+ cells (middle panel). Comparison of YFP labeling in F4/80bright CD11b+ macrophages (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55305/55305fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

12.9K Views.  Institut Pasteur. 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. 

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

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis.

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be difficult to study, and can lead to confounding results. The protocol described here is a straightforward breeding paradigm to increase and decrease penetrance in lethal zebrafish skeletal mutants. Because lethal mutants cannot be selectively bred directly, the classic selective breeding strategy of progeny testing is employed. This method also includes protocols for Kompetitive Allele Specific PCR (KASP) genotyping zebrafish and staining larval zebrafish cartilage and bone. Applying the husbandry strategy described here can increase the penetrance of an interesting skeletal phenotype enabling more reproducible results in downstream applications. In addition, decreasing the mutant penetrance through this selective breeding strategy can reveal the developmental processes that most crucially require the function of the mutated gene. While the skeleton is specifically considered here, we propose that this methodology will be useful for all zebrafish mutant lines.

The goal of this protocol is to alter the penetrance of lethal skeletal mutant phenotypes in zebrafish by selective breeding. Lethal mutants cannot be grown to adulthood and bred themselves, therefore this protocol describes a method for tracking and selecting penetrance through multiple generations by progeny testing.

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...