Supported by the Pasteur Institute, INSERM, Agence Nationale de Recherche ANR (Grant &#8216;Lymphopoiesis&#8217;), the REVIVE Future Investment Program and &#8220;La Ligue contre le Cancer&#8221;.

Ethics statement: all experiments were performed according to the Pasteur Institute Ethic Charter, approved by the French Agriculture Ministry, and to the EU guidelines. A manipulator with training on small rodent surgery, certified by the French Ministry of Agriculture, performs all surgical interventions. 
NOTE: See in annex Table 1 showing the 5-step plan procedure.
1. Selection of the Embryos
<ol>
	<li>Use 36 C57BL/6 CD45.2 females, 12 females CD45.1, 12 C57BL/6 CD45.1 males and 12 C57BL/6 CD45.2 males. Crossing the females with either male genotype will produce embryos CD45.1/2 and CD45.1 used as a source of TSP or CD45.2 used as a source of recipient thymic lobes. House all males individually.</li>
	<li>To obtain embryos for E18 TSP isolation, place 2 females in a cage with one male CD45.1 at 6 pm, 21 days before the grafting experiment. Check the presence of a vaginal plug the next day, before noon. Separate the plugged females.</li>
	<li>To obtain E14 embryos to be colonized by TSP, repeat the procedure above (1.2), using males CD45.2, 17 days before the grafting experiment.</li>
	<li>To obtain embryos to isolate E13 TSP, repeat the procedure above (1.2), using males C57BL/6 CD45.1, 16 days before the grafting experiment.</li>
</ol>
2. Dissection of the Embryos Under a Horizontal Laminar Flow Hood
NOTE: Two days before the grafting experiment.
<ol>
	<li>Sacrifice the pregnant females by cervical dislocation or by progressive administration of CO2 gas during at least 5 min, in a saturating closed chamber. Ensure death by definitive arrest of cardio-respiratory movements. Wet the abdomen with 70% ethanol.</li>
	<li>Make a longitudinal incision in the skin at the midline abdomen and open it by pulling the skin apart. Open the peritoneum with forceps and scissors without touching the digestive tract. Pull out the bifid uterus and separate it from the vagina with the scissors.</li>
	<li>Place the uterus in a 90 x 15 mm Petri dish containing 40-50 ml DPBS and cut transversally between each embryo&#8217;s decidua.</li>
	<li>With the pair of fine tip scissors and a fine forceps remove the muscle membrane of the uterus, take out the embryos by cutting between the placenta and the yolk sac, and remove the amnios, which is retaining the embryos.</li>
	<li>Place the embryos in a Petri dish 90 x 15 mm containing HBSS + 1% fetal calf serum (FCS). For embryos older than E15, remove the heads with a pair of scissors before any further dissection.</li>
</ol>
3. Isolation of the Thymus
<ol>
	<li>Under the binocular magnifying lens, place the embryo, lying in supine position (ventral view), on a wet gauze bedding.</li>
	<li>Insert one forceps longitudinally along the cartilage of the sternum, pinch and open the thoracic grid. With curved forceps, pull the thoracic wall aside to visualize the thymic lobes on each side of the trachea.</li>
	<li>Carefully hold each lobe by pinching underneath with a fine forceps and place them in a Petri dish containing 1 ml HBSS + 1% FCS. Isolate the lobes and remove surrounding connective tissue with two 1 ml syringes + 26 G &#8540;&#34; needles, without damaging the capsule.</li>
	<li>Wash the lobes in 3 ml of HBSS + 1% FCS to eliminate blood cells. 
	NOTE: Before E15, thymic lobes are located on each side of the trachea and later fuse in the middle to constitute a bi-lobar organ.</li>
</ol>
4. Cell Suspensions
<ol>
	<li>Tease apart the lobes under the binocular magnifying lens, with two 26 G needles mounted on 1 ml syringes, in HBSS + 1% FCS. Filter the cell suspension through a nylon mesh.</li>
</ol>
5. Staining with Fluorescent Antibodies and Cell Sorting
NOTE: All antibodies are previously titrated to obtain optimal definition of the populations (the titration can vary with the antibody batch).
<ol>
	<li>Incubate the thymocytes (E13 or E18) for 15-30 min at 4 &#176;C with a 100 &#956;l of a cocktail of biotinylated antibodies to stain lineage positive cells (anti-CD19, anti-Gr1, anti-Ter119, anti-NK1.1, anti- CD11c, anti-CD3, anti-CD4, anti-CD8, anti-CD25).</li>
	<li>To wash away excess of antibodies spin down the tubes with 4 ml HBSS + 1% FCS at 280 x g for 7 min, eliminate the supernatant and re-suspending the pellet in fresh HBSS + 1% FCS. Repeat the centrifugation.</li>
	<li>Incubate the E18 biotin-labeled cells with streptavidin microbeads during 15 min at 4 &#176;C and wash the cells twice in HBSS 1% FCS. Re-suspend the cells in 2 ml HBSS + 1% FCS. Most E13 thymocytes are lineage negative and therefore do not need MACS enrichment prior to cell sorting.</li>
	<li>Pass the cells on the LS column, according to the manufacturer&#8217;s instructions. When all the cell suspension entered the column add 2 ml HBSS + 1% FCS. Recover the 4 ml of the column flow through and centrifuge the cells for 7 min at 280 x g.</li>
	<li>Re-suspend the pellet of either depleted E18 or total E13 thymocytes in 50 &#956;l of the TSP antibody mix (anti-CD117 APC, anti-CD135 PE, anti-CD127 PECy7, anti-CD24 FITC, anti-CD44 APCCy7 and streptavidin Pacific Blue) and incubate for 20 min at 4 &#176;C in the dark.</li>
	<li>Wash the cells with 4 ml HBSS + 1% FCS and re-suspend the cells in 1 ml HBSS + 1% FCS with propidium iodide.</li>
	<li>Sort the TSPs Lin-CD44+CD117+CD24lowCD127+CD135+ cells in a FACS (with 4 lasers) onto a 1.5 ml tubes containing 200 &#956;l complete medium + 20% FCS. Gate first the cells according to their size and granularity on the FSC and SSC channels. Eliminate dead cells (staining with propidium iodide), gate out doublets and score the fluorescence in exponential scales. Around 100 or 600 TSPs can be obtained from one E13 or one E18 thymus, respectively.</li>
</ol>
6. Colonization of E14 Thymic Lobes with Progenitors: Hanging Drop Technique
<ol>
	<li>Irradiate (30 Grays = 30 Gy) E14 thymic lobes from CD45.2 embryos, 3 hr before culture. 
	NOTE: E14 or E15 thymic lobes have been successfully used as recipient of T cell progenitors. Using recipient thymic lobes of later gestational days results in premature necrosis due to the bigger size of the organ while thymic lobes at earlier gestational days develop poorly, probably due to incomplete development of the epithelial cells.</li>
	<li>Centrifuge sorted TSPs and re-suspend the cells in culture medium (OPTI-MEM complete medium with 10% FCS, penicillin (50 units/ml), streptomycin (50 &#181;g/ml) and 2&#946;-mercaptoethanol (50 &#181;M)) such that 35 &#956;l contain 500 TSP.</li>
	<li>Place a 35 &#956;l drop of cell suspension every other well of a 60 well Terasaki plate. 
	NOTE: 35 &#956;l drops can fuse if they are placed in contiguous wells.</li>
	<li>Place one irradiated lobe on the surface of each drop. Close the Terasaki plate and turn the plate upside down to let the cells reach the apical pole of the drop by gravity.</li>
	<li>Hit gently the upper side of the inverted plate to force the lobes to slide to the apical edge of the drop. Incubate the inverted Terasaki plate in a humidified incubator for 48 hr at 37 &#176;C + 5% CO2. After colonization, either culture E14 thymi in FTOC7, or graft under the kidney capsule of a recipient adult mouse 8.</li>
</ol>
7. Fetal Thymic Organ Culture
<ol>
	<li>Place 3 ml of complete medium on a 35 mm Petri dish. Place an isopore membrane filter floating on the medium. Place the reconstituted lobes on the edge of the membrane with a forceps (no more than 8 lobes on one membrane).</li>
	<li>Place the Petri dishes containing the thymic lobes inside a 90 mm Petri dish, together with a 35 mm dish, without cover, containing 2 ml of water, to ensure optimal humidity. Incubate 12 days at 37 &#176;C + 5% CO2.</li>
</ol>
8. Grafts Under the Kidney Capsule Under Sterile Conditions
NOTE: The kidney parenchyma is surrounded by connective tissue forming a capsule. The sub-capsular region is particularly rich in blood and lymphatic vessels thereby providing a suitable environment for the development of grafts (e.g. thymic lobes, pancreatic islets or newborn hearts). Grafts are usually done on the left kidney because it is more accessible than the right kidney. CD3-/- male mice were used as recipients thus avoiding graft versus host reaction due to minor histocompatibility antigens linked to the Y chromosome because sex determination before E15 is not easily done.
<ol>
	<li>Sterilize the instruments and wear sterile gloves along the surgery. Anesthetize the mouse by intra peritoneal injection of a solution of Ketamine 10 mg/ml + Xylazine 1 mg/ml diluted in PBS: (50 to 100 &#956;l per 10 g body weight), using a 1 ml syringe. Verify anesthesia by checking lack of interdigital reflexes. Place a drop of hydro-Optimune gel on each eye to prevent dryness during anesthesia. 
	NOTE: The solution should be prepared extemporaneously and warmed at room temperature before injection. The anesthesia lasts at least 20 min. The degree of anesthesia should be controlled all along the surgery. The anesthesia can be prolonged by intra peritoneal injection of half dose every 20 min.</li>
	<li>Place the mouse on its right side, under a horizontal lamina flow hood. Sterilize the surgical field with 70% ethanol and 10% iodide dermic solution. With a forceps part the mouse hair along a 2 cm length just above the joint of the hind leg. Shave the surgical area.</li>
	<li>With a scalpel, make an incision 1.5 cm length of first, the cutaneous tissue, then with scissors cut the muscular tissue. Apply a slight pressure to both sides of the incision, which will force the kidney out of the abdominal cavity.</li>
	<li>Apply saline with a cotton-bud to keep the kidney moist. If you have difficulties in exposing the organ, use a flat forceps to pull out the surrounding adipocyte tissue to which the kidney is attached. Maintain the kidney out of the retroperitoneal cavity by a cotton bud placed underneath the organ.</li>
	<li>With two fine forceps, make a 2-3 mm hole in the capsule (avoid touching the parenchyma to prevent bleeding). During the whole surgical procedure keep the exposed kidney capsule continuously moistened.</li>
	<li>Insert carefully the lobes under the kidney capsule, by maintaining the wall of the capsule opened and slide the graft under the capsule towards the edge pole. Place up to 6 lobes per kidney.</li>
	<li>Replace the kidney in the retro-peritoneal cavity. Suture the muscle aponeuroses, then the skin with individual surgeon knots. Wet the wound with 10% iodide povidone solution. Place the mouse on a heated pad at 37 &#176;C.</li>
	<li>Survey the transplanted mouse until full recovery from anesthesia by control of cardiac, respiratory and whisker movements, mucosal colors until total recovery seen by self contained movements. 
	NOTE: We recommend injection of analgesic solution (Buprenorphin, 0.1 mg/kg) intra muscle just after the surgery and the 2 days following surgery to prevent post-surgery pain.</li>
	<li>Keep the operated animal in isolation until fully recovered. Inspect every other day the stitches of the wound to verify and prevent infection. Never observed wound infection following this procedure. 
	NOTE: Weekly analysis of peripheral blood cells from the CD3-/- grafted mice allow us to detect the thymic derived populations originated from either E13 or E18 progenitors using the CD45 allotypic marker and T cell lineage specific antibodies (Figure 3).</li>
</ol>
9. Analysis of the TSP progeny by Flow Cytometry
<ol>
	<li>Tease the FTOCs individually to obtain single cell suspensions (see protocol section 4).</li>
	<li>Stain cell suspensions from individual lobes with an antibody mixture containing antibodies recognizing CD45.1 PECy7, CD45.2 AmCyan, CD4 APCCy7, CD8 APC, CD3 Pacific Blue, V&#947;5 FITC, V&#948;1 PE and incubate as described in section&#160;5.</li>
	<li>Re-suspend the cells in HBSS + 1% FCS + propidium iodide and analyze in a flow cytometer (see results in Figure 2).</li>
</ol>

<ol>
	<li>Rodewald, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymus+organogenesis.">Thymus organogenesis.</a> Annu Rev Immunol. 26, 355-388 (2008).</li><li>Takahama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Journey+through+the+thymus:+stromal+guides+for+T-cell+development+and+selection.">Journey through the thymus: stromal guides for T-cell development and selection.</a> Nat Rev Immunol. 6, 127-135 (2006).</li><li>Anderson, G., Jenkinson, E. J., Moore, N. C., Owen, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+class+II-positive+epithelium+and+mesenchyme+cells+are+both+required+for+T-cell+development+in+the+thymus.">MHC class II-positive epithelium and mesenchyme cells are both required for T-cell development in the thymus.</a> Nature. 362, 70-73 (1993).</li><li>Douagi, I., Andre, I., Ferraz, J. C., Cumano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+T+cell+precursor+activity+in+the+murine+fetal+thymus:+evidence+for+an+input+of+T+cell+precursors+between+days+12+and+14+of+gestation.">Characterization of T cell precursor activity in the murine fetal thymus: evidence for an input of T cell precursors between days 12 and 14 of gestation.</a> Eur J Immunol. 30, 2201-2210 (2000).</li><li>Calderon, L., Boehm, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic,+context-dependent,+and+hierarchical+functions+of+epithelial+components+in+thymic+microenvironments.">Synergistic, context-dependent, and hierarchical functions of epithelial components in thymic microenvironments.</a> Cell. 149, 159-172 (2012).</li><li>Adolfsson, J., 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=Identification+of+Flt3++lympho-myeloid+stem+cells+lacking+erythro-megakaryocytic+potential+a+revised+road+map+for+adult+blood+lineage+commitment.">Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment.</a> Cell. 121, 295-306 (2005).</li><li>Kondo, M., Weissman, I. L., Akashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+clonogenic+common+lymphoid+progenitors+in+mouse+bone+marrow.">Identification of clonogenic common lymphoid progenitors in mouse bone marrow.</a> Cell. 91, 661-672 (1997).</li><li>Bhandoola, A., von Boehmer, H., Petrie, H. T., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commitment+and+developmental+potential+of+extrathymic+and+intrathymic+T+cell+precursors:+plenty+to+choose+from.">Commitment and developmental potential of extrathymic and intrathymic T cell precursors: plenty to choose from.</a> Immunity. 26, 678-689 (2007).</li><li>Ramond, C., 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=Two+waves+of+distinct+hematopoietic+progenitor+cells+colonize+the+fetal+thymus.">Two waves of distinct hematopoietic progenitor cells colonize the fetal thymus.</a> Nat Immunol. 15, 27-35 (2014).</li><li>Allison, J. P., Havran, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunobiology+of+T+cells+with+invariant+gamma+delta+antigen+receptors.">The immunobiology of T cells with invariant gamma delta antigen receptors.</a> Annu Rev Immunol. 9, 679-705 (1991).</li><li>Anderson, G., Jenkinson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+thymus+organ+culture.">Fetal thymus organ culture.</a> CSH Protoc. , (2007).</li><li>Schmitt, T. M., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+T+cell+development+from+hematopoietic+progenitor+cells+by+delta-like-1+in+vitro.">Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro.</a> Immunity. 17, 749-756 (2002).</li><li>Barbee, S. D., 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=Skint-1+is+a+highly+specific,+unique+selecting+component+for+epidermal+T+cells.">Skint-1 is a highly specific, unique selecting component for epidermal T cells.</a> Proc Natl Acad Sci U S A. 108, 3330-3335 (2011).</li><li>Douagi, I., Colucci, F., Di Santo, J. 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The authors declare that they have no competing financial interests.

Two main assays can be used to analyze T cell differentiation ex vivo. The most recently reported is the co-culture of hematopoietic progenitors with BM stromal cells, OP9, expressing the ligands of Noth1, delta like 1 or 412. This 2-D assay is easy to perform, highly efficient and sensitive, allowing analysis at the single cell level. However, it neither supports T cell development beyond the stage of DP nor the generation of &#947;&#948; DETC13, both of which require direct interactions w...

T lymphocytes, bearing the &#945;&#946; or the &#947;&#948; T cell receptor (TcR), differentiate in a specialized organ, the thymus. The fully developed thymus is organized into two distinct regions: the cortex, where thymic progenitors develop and where thymocytes that productively rearrange the TcR &#946; and &#945; chain genes are rescued from programmed death (a process known as positive selection); and the medulla, where selected thymocytes with too strong reactivity to self-ligands are deleted (negative selection)1,2. The thymus originates from the endodermal layer of the third pharyngeal pouch that is later surrounded by mesenchymal cells3. It is colonized by hematopoietic progenitors starting at embryonic day E12 and, thereafter, continuous recruitment is required for normal T cell development4. Thymic immigrants evolve through successive developmental stages, orchestrated by a tightly regulated program, initiated and maintained by the activation of the Notch signaling pathway on thymocytes upon interaction with its ligand, delta like 4, expressed on thymic epithelial cells (TECs)5.
Thymocyte development starts at the so-called CD4-CD8- double negative (DN) stages. DN thymocytes can be further subdivided according to the expression of CD25 and CD44 into DN1 (CD25-CD44+), DN2 (CD25+CD44+), DN3 (CD25+CD44-) and DN4 (CD25-CD44-). CD24 (HSA) and CD117 (c-Kit) further subdivides the DN1 compartment into 5 subsets where DN1a and b correspond to early thymic progenitors (ETP). Thymocytes rearrange the TcR &#948;, &#946; and &#945; chains at the DN stage and undergo pre-TcR selection (DN3-DN4 stages). They further transit to the CD4+CD8+ double positive (DP) compartment where the TcR &#945; chain rearranges prior to positive and negative selection. At this stage most thymocytes are eliminated and only a small percentage (3-5%) reach the CD4+ or CD8+ mature T cell compartment.
The lymphoid differentiation pathway progresses through the stages of HSCs that generate multipotent progenitors (MPP) and lymphoid-primed multipotent progenitors (LMPP) that lost the erythrocyte and megakaryocyte potential6. LMPP are phenotypically defined by the absence of differentiated blood cell markers (lineage negative, Lin-), the expression of c-Kit (CD117), Sca-1 and Flt3/Flk2 (CD135) and the absence of detectable levels of the interleukin (IL)-7 receptor &#945; chain (IL-7r&#945; or CD127). LMPPs further differentiate into common lymphoid progenitors (CLP)7 that by that stage have lost the capacity to generate myeloid cells. CLP retain lymphocyte (B and T cell), NK cell, DC and innate lymphoid cell (ILC) potential, and differ from LMPP by the expression of CD127 and the absence of high levels of Sca-1.
Although the nature of the thymic settling progenitors (TSP) has been extensively debated8, it became recently clear that TSP change phenotype, differentiation potential and function, throughout development9. We performed in vitro and in vivo assays to characterize the TSP, isolated by FACS cell sorting from either E13 (first wave) or E18 (second wave). Fetal thymic organ cultures (FTOC) with irradiated thymic lobes colonized by equal numbers of E13 and E18 progenitors, bearing different allotypic markers, allowed following their progeny in a similar developmental environment and revealed cell intrinsic properties, different between both types of progenitors. Thymic lobes colonized by either E13 or E18 TSP allowed development without selection due to competition between both progenitors. In vivo transplantation of the colonized thymic lobes further showed that also the mature progeny of E13 and E18 TSP have different biologic properties in vivo. TSPs from the first wave rapidly generate T cells but give rise to low numbers of &#945;&#946; and &#947;&#948; T cells. Among the latter we detected V&#947;5V&#948;1 dendritic epithelial T cells (DETC), that have an invariant TcR, migrate to the epidermis where they exert a function in wound healing and are only produced during embryonic development10. In contrast, TSP from the second wave take longer time to generate high numbers of TcR+ T cells and are unable to generate DETC.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>90 x 15 mm and 35 x 15 mm plastic tissue culture petri dishes.</td>
			<td>TPP</td>
			<td>T93100/T9340</td>
			<td>Sampling</td>
		</tr>
		<tr>
			<td>26GA 3/8 IN 0.45x10mm syringes with needles Beckton-Dickinson Plastipak.</td>
			<td>BD Plastipak</td>
			<td>300015</td>
			<td>Cell suspension</td>
		</tr>
		<tr>
			<td>Nylon mesh bolting cloth sterilized 50/50 mm pieces.</td>
			<td>SEFAR NITEX</td>
			<td>03-100/32</td>
			<td>Filtration of cells</td>
		</tr>
		<tr>
			<td>Ethanol 70%.</td>
			<td>VWR</td>
			<td>83801.36</td>
			<td>Sterility Actions</td>
		</tr>
		<tr>
			<td>Iodide Povidone 10% (Betadine)&#160;</td>
			<td>MEDA Pharma</td>
			<td>314997.8</td>
			<td>Sterility Actions</td>
		</tr>
		<tr>
			<td>Ketamine 100mg/ml (stock solution)</td>
			<td>MERIAL</td>
			<td>&#160;-</td>
			<td>Anesthesic</td>
		</tr>
		<tr>
			<td>Xylazine 100mg/ml in PBS (stock solution)</td>
			<td>Sigma</td>
			<td>X1251-1G</td>
			<td>Anesthesic and muscle relaxant</td>
		</tr>
		<tr>
			<td>Buprenorphin 0.3mg/ml stock solution</td>
			<td>AXIENCE</td>
			<td>&#160;-</td>
			<td>Morphinic analgesic</td>
		</tr>
		<tr>
			<td>Ophtalmic gel (0.2% cyclosporin)</td>
			<td>Schering-Plough Animal Health</td>
			<td>&#160;-</td>
			<td>Eyes protection</td>
		</tr>
		<tr>
			<td>DPBS (+ CaCl2, MgCl2)</td>
			<td>GIBCO Life Technology</td>
			<td>14040-174</td>
			<td>to isolate embryos</td>
		</tr>
		<tr>
			<td>HBSS Hanks&#39; Balanced Salt Solution (+ CaCl2, MgCl2)</td>
			<td>GIBCO Life Technology</td>
			<td>24020-091</td>
			<td>to wash out the blood and dissect the embryos</td>
		</tr>
		<tr>
			<td>OPTI-MEM I GlutaMAX I</td>
			<td>GIBCO Life Technology</td>
			<td>51985-026</td>
			<td>Medium for cultures</td>
		</tr>
		<tr>
			<td>F&#339;tal Calf serum</td>
			<td>EUROBIO</td>
			<td>CVFSVF00-0U</td>
			<td>additive for cultures</td>
		</tr>
		<tr>
			<td>Penicilin and Streptomycin</td>
			<td>GIBCO Life Technology</td>
			<td>15640-055</td>
			<td>Antibiotics for cultures</td>
		</tr>
		<tr>
			<td>2 b mercapto ethanol</td>
			<td>GIBCO Life Technology</td>
			<td>31350010</td>
			<td>additive for cultures</td>
		</tr>
		<tr>
			<td>LEICA MZ6 Dissection microscope</td>
			<td>LEICA</td>
			<td>MZ6 10445111</td>
			<td>Occular W-Pl10x/23</td>
		</tr>
		<tr>
			<td>Cold lamp source</td>
			<td>SCHOTT VWR</td>
			<td>KL1500 compact</td>
			<td>Two goose neck fibers adapted</td>
		</tr>
		<tr>
			<td>Silicone elastomer</td>
			<td>World Precision Instruments</td>
			<td>SYLG184</td>
			<td>Dissecting Pad</td>
		</tr>
		<tr>
			<td>Spoon, round and perforated</td>
			<td>Fine Science Tools</td>
			<td>10370-18</td>
			<td>Dissection tools</td>
		</tr>
		<tr>
			<td>Fine Iris Scissors</td>
			<td>Fine Science Tools</td>
			<td>14090-09</td>
			<td>Dissection tools</td>
		</tr>
		<tr>
			<td>Vannas spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15018-10</td>
			<td>Dissection tools</td>
		</tr>
		<tr>
			<td>Forceps : Dumont #5/45 Inox 11 cm</td>
			<td>F.S.T.</td>
			<td>11253-25</td>
			<td>Dissection tools</td>
		</tr>
		<tr>
			<td>Two pairs of fine straight watchmakers&#8217; forceps Dumont #5 11 cm fine tips.</td>
			<td>Fine Science Tools</td>
			<td>11295-20</td>
			<td>Dissection tools</td>
		</tr>
		<tr>
			<td>Polyamid thread with needle 6-0&#160;&#160; C-3 3/8c</td>
			<td>ETHICON</td>
			<td>F2403</td>
			<td>for sutures</td>
		</tr>
		<tr>
			<td>Needle-holder</td>
			<td>MORIA/F.S.T.</td>
			<td>12060-02</td>
			<td>for sutures</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>VWR</td>
			<td>100229-100</td>
			<td>To maintain mouse temperature during anesthesy</td>
		</tr>
		<tr>
			<td>Membrane Isopore RTTPO2500</td>
			<td>DUTSCHER</td>
			<td>44210</td>
			<td>For FTOC</td>
		</tr>
		<tr>
			<td>Terasaki 60 wells plates</td>
			<td>FISHER</td>
			<td>1x270 10318801</td>
			<td>For hanging drop technique</td>
		</tr>
		<tr>
			<td>Gauze swabs steriles 7.5cmx7.5cm</td>
			<td>Hydrex</td>
			<td>11522</td>
			<td>To apply disinfecting solution</td>
		</tr>
		<tr>
			<td>Fluorescence or biotin labelled antibodies</td>
			<td>BD Biosciences, Biolegend or e-Biocsiences&#160;</td>
			<td>Clone Number see table below</td>
			<td>Staining cells</td>
		</tr>
		<tr>
			<td>MACS Columns/Streptavidin Microbeads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401/130-048-101</td>
			<td>Cell depletion&#160;</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Charles Rivers Laboratories (CD45.1) and Janvier Labs&#160; (CD45.2)</td>
			<td>C57BL/6 CD45.1 OR CD45.2</td>
			<td>Source of cells and thymic lobes&#160;</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>CDTA Orl&#233;ans, France</td>
			<td>CD 3 epsilon Ko CD45.2</td>
			<td>&#160;Grafting experiment</td>
		</tr>
	</tbody>
</table>

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.

In order to choose a method to deplete thymic lobes of endogenous thymocytes allowing the best development of colonizing progenitors, we compared the levels of T cell reconstitution in thymic lobes colonized after irradiation or a 5-day deoxy-guanosine (d-Gua) treatment. The results show that while there is no difference at day 9 of culture, irradiated lobes contained more T cells than those treated with d-Gua, at day 12. Thus, irradiation is more appropriate than d-Gua treatment to obtain T cell development after thymic...

Watch this Scientific Journal Video about Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays at JoVE.com

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.

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 ...

characterization-thymic-settling-progenitors-mouse-embryo-using-vivo

Research

JoVE Journal

Developmental Biology

8.2K Views.  Institut Cochin. 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. 

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

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

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.

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 ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

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 ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

Spatiotemporal Subcellular Manipulation of the Microtubule Cytoskeleton in the Living Preimplantation Mouse Embryo using Photostatins

The microtubule cytoskeleton forms the framework of a cell and is fundamental for intracellular transport, cell division, and signal transduction. Traditional pharmacological disruption of the ubiquitous microtubule network using, for instance, nocodazole can have devastating consequences for any cell. Reversibly photoswitchable microtubule inhibitors have the potential to overcome the limitations by enabling drug effects to be implemented in a spatiotemporally-controlled manner. One such family of drugs is the azobenzene-based photostatins (PSTs). These compounds are inactive in dark conditions, and upon illumination with UV light, they bind to the colchicine-binding site of &#946;-tubulin and block microtubule polymerization and dynamic turnover. Here, the application of PSTs in the 3-dimensional (3D) live preimplantation mouse embryo is set out to disrupt the microtubule network on a subcellular level. This protocol provides instructions for the experimental setup, as well as light activation and deactivation parameters for PSTs using live-cell confocal microscopy. This ensures reproducibility and enables others to apply this procedure to their research questions. Innovative photoswitches like PSTs may evolve as powerful tools to advance the understanding of the dynamic intracellular microtubule network and to non-invasively manipulate the cytoskeleton in real-time. Furthermore, PSTs may prove useful in other 3D structures such as organoids, blastoids, or embryos of other species.

Typical microtubule inhibitors, used widely in basic and applied research, have far-reaching effects on cells. Recently, photostatins emerged as a class of photoswitchable microtubule inhibitors, capable of instantaneous, reversible, spatiotemporally precise manipulation of microtubules. This step-by-step protocol details the application of photostatins in a 3D live preimplantation mouse embryo.