The authors would like to thank Dr. George Gittes for advice and expertise throughout this study. SSL was supported by an American Heart Association fellowship (11POST7330002). Further to this SSL and this study was supported by an NIDDK Mentored Research Scientist Development Award (DK096996) and by the Children&#8217;s Hospital of Pittsburgh.

NOTE: The University of Pittsburgh Institutional Animal Care and Use Committee approved all experiments.
1. Preparation of Ultrasound-microinjection Instruments and Embryos
<ol>
	<li>Set up stage, mount, and probe (Figure 1) as well as surgical instruments (Figure 2). Place phosphate buffered saline (PBS) solution (pH7.4) in a 37 &#176;C warming bath. Fill microinjection needle entirely with mineral oil, using 1 ml syringe attached to a second flexible 25 G needle, through its b...

<ol>
	<li>Abrahamson, D. R., Robert, B., Hyink, D. P., St John, P. L., Daniel, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+and+formation+of+microvasculature+in+the+developing+kidney.">Origins and formation of microvasculature in the developing kidney.</a> Kidney international. Supplement. 67, S7-S11 (1998).</li><li>Sims-Lucas, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Progenitors+Exist+within+the+Kidney+and+Lung+Mesenchyme.">Endothelial Progenitors Exist within the Kidney and Lung Mesenchyme.</a> PloS one. 8 (6), e65993 (2013).</li><li>Dressler, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+early+kidney+specification,+development+and+patterning.">Advances in early kidney specification, development and patterning.</a> Development. 136 (23), 3863-3874 (2009).</li><li>Costantini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+controls+and+cellular+behaviors+in+branching+morphogenesis+of+the+renal+collecting+system.">Genetic controls and cellular behaviors in branching morphogenesis of the renal collecting system.</a> Wiley Interdiscip Rev Dev Biol. 1 (5), 693-713 (2012).</li><li>Costantini, F., Kopan , R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+a+complex+organ:+branching+morphogenesis+and+nephron+segmentation+in+kidney+development.">Patterning a complex organ: branching morphogenesis and nephron segmentation in kidney development.</a> Dev Cell. 18 (5), 698-712 (2010).</li><li>Das, A., 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=Stromal-epithelial+crosstalk+regulates+kidney+progenitor+cell+differentiation.">Stromal-epithelial crosstalk regulates kidney progenitor cell differentiation.</a> Nat Cell Biol. 15 (9), 1035-1044 (2013).</li><li>Rymer, 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=Renal+blood+flow+and+oxygenation+drive+nephron+progenitor+differentiation.">Renal blood flow and oxygenation drive nephron progenitor differentiation.</a> Am J Physiol Renal Physiol. , (2014).</li><li>Shah, S. R., 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=Embryonic+mouse+blood+flow+and+oxygen+correlate+with+early+pancreatic+differentiation.">Embryonic mouse blood flow and oxygen correlate with early pancreatic differentiation.</a> Developmental biology. 349 (2), 342-349 (2011).</li><li>Andres, A. 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=EphB4+receptor+tyrosine+kinase+transgenic+mice+develop+glomerulopathies+reminiscent+of+aglomerular+vascular+shunts.">EphB4 receptor tyrosine kinase transgenic mice develop glomerulopathies reminiscent of aglomerular vascular shunts.</a> Mech Dev. 120 (4), 511-516 (2003).</li><li>Wagner, R., 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=High-resolution+imaging+of+kidney+vascular+corrosion+casts+with+Nano-CT.">High-resolution imaging of kidney vascular corrosion casts with Nano-CT.</a> Microsc Microanal. 17 (2), 215-219 (2011).</li></ol>

Microinjection anesthesia and time frame
With regards to anesthetization of the mother, it is essential to keep airflow constant (2-3 L/min) and at low PSI. The flow of the sedative must be held at approximately 1.75-2 L/min. Simultaneously, timeframes in which the injections take place must be closely monitored and controlled for with each litter. For each litter the injection procedure should be kept under 45 min. The importance of this time limit is paramount to the experiment, as each embr...

During embryonic development two discrete, yet simultaneous, vascular processes take place: angiogenesis, the process whereby a vessel grows from a major pre-existing vessel, and vasculogenesis, which is a de novo formation of vessels from residential endothelial progenitors1,2. Respectively, the former is synonymous with blood flow, while the latter is thought to largely take place in the absence of it.
Simultaneous to blood vessel formation, a cyclical and dynamic process of kidney progenitor cell synthesis, proliferation, and differentiation begins to unfold on embryonic day 9.5 (E9.5). At this point the ureteric bud (UB) invades dorsally into surrounding metanephric mesenchyme (MM), and continues until birth3. Repeated branching of the UB into rapidly condensing metanephric cap mesenchyme begins the formation of the functional units of the kidney, the nephron. With every new generation of UB and nephron, older generations are displaced into inner cortical and medullary regions, where they then undergo further maturation and differentiation within primarily vascular-dense environments. As evidenced by Dressler et al.3, this embryological process is precipitated by inductive signaling, such as crosstalk between UB and MM, and a myriad of extracellular factors 3-6. Two recently investigated extracellular factors within the developing pancreas and kidneys include oxygen tension and blood flow7,8. The latter will be discussed in further detail below with relation to kidney development.
In order to expose the inductive role that blood flow potentially plays in nephron progenitor cell differentiation, as well as in other organogenesis processes, precise and accurate methods of embryonic blood flow mapping is imperative.
Alternative methods of gauging blood flow include the prescription of ultrasound imaging and resin casts9,10. Conclusively, these modes have been shown to be inherently lacking in their capacity to contemporaneously unveil temporal and spatial juxtapositions between blood flow and stem cell differentiation. Resin casts, for example, provide a valid model of vessel patterning within adult tissues, however in immature vessels, such as with embryonic time points, vessels are grossly underdeveloped and leaky. Therefore, resin casts fail to hold within the tiny, oftentimes porous, vessels.
For these apparent obstacles, among others, we chose to incorporate ultrasound-guided in vivo intra-cardiac embryonic tomato lectin (TL) microinjections into our investigations of kidney development. In this procedure we utilize an ultrasound probe to synchronously guide a mounted micropipette needle filled with 2.5 &#956;l of TL solution into the left ventricle of mouse embryos at E11.5, E13.5, E15.5, and E17.5 time points. E17.5 is the latest developmental age as the needles are not strong enough to penetrate the more developed embryo.
The advantages of this microinjection method are abundant. Ultrasound-guided microinjection allows precise positioning of an injection needle within the embryonic left ventricle, passive and controlled expulsion of solution into the beating heart of the animal, minimal damage to heart and surrounding tissues, and the avoidance of sudden cardiac failure and death of the embryo prior to full-body perfusion. With the use of a FITC-labeled TL, any perfused vasculature will maintain the marker along its endothelial apical membrane. In combination with immunohistochemistry, utilizing Pecam (CD31, Platelet endothelial cell adhesion molecule) and various other vascular markers, we are able to clearly distinguish between perfused and un-perfused vessels, as well as characterize any aberrant staining of surrounding tissues.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Name of Material/ Equipment</td>
			<td style="width:292px;">Company</td>
			<td style="width:164px;">Catalog Number</td>
			<td style="width:225px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Sigma Aldrich</td>
			<td>022M4004V</td>
			<td>concentration 1:5000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pecam</td>
			<td>BD Biosciences</td>
			<td>553370</td>
			<td>concentration 1:100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FITC-Tomato Lectin</td>
			<td>Vector Laboratories</td>
			<td>FL-1321</td>
			<td>concentration 2.5&#181;L / embryo</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa Fluor-594 (Donkey Anti-Rat )</td>
			<td>Jackson Immunoresearch</td>
			<td>712-585-150</td>
			<td>concentration 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microinjection Needle</td>
			<td>Origio Mid Atlantic Devices</td>
			<td>C060609</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mineral Oil</td>
			<td style="width:292px;">Fisher Scientific</td>
			<td style="width:164px;">BP26291</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mL syringe</td>
			<td style="width:292px;">Fisher Scientific</td>
			<td style="width:164px;">03-377-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;Clay Blocks</td>
			<td style="width:292px;">Fisher Scientific</td>
			<td style="width:164px;">HR4-326</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical Tape</td>
			<td style="width:292px;">Fisher Scientific</td>
			<td style="width:164px;">18-999-380</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td style="width:292px;">Fisher Scientific</td>
			<td style="width:164px;">NC9763655</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hair Removal Product</td>
			<td style="width:292px;">Fisher Scientific</td>
			<td style="width:164px;">NC0132811</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical Scissors</td>
			<td style="width:292px;">Fine Science tools</td>
			<td style="width:164px;">14084-08</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fine Forceps</td>
			<td style="width:292px;">Fine Science tools</td>
			<td style="width:164px;">11064-07</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;Surgical Marking Pen</td>
			<td style="width:292px;">Fine Science tools</td>
			<td style="width:164px;">18000-30</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;Right angle forceps (for hysterectomy)</td>
			<td style="width:292px;">Fine Science tools</td>
			<td style="width:164px;">11151-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

in utero intra-cardiac tomato-lectin injections on mouse embryos to gauge renal blood flow

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis (which is the branching off of major vessels) and vasculogenesis (de novo vessel formation), perfusion mapping techniques such as resin casts, in vivo ultrasound imaging, and micro-dissection have been limited in demonstrating the intimate relationships between these two processes and developing renal structures within the embryo. Here, we describe the procedure of in utero intra-cardiac ultrasound-guided FITC-labeled tomato lectin microinjections on mouse embryos to gauge the ontogeny of renal perfusion. Tomato lectin (TL) was perfused throughout the embryo and kidneys harvested. Tissues were co-stained for various kidney structures including: nephron progenitors, nephron structures, ureteric epithelium, and vasculature. Starting at E13.5 large caliber vessels were perfused, however peripheral vessels remained unperfused. By E15.5 and E17.5, small peripheral vessels as well as glomeruli started to become perfused. This experimental technique is critical for studying the role of vasculature and blood flow during embryonic development.

Vascular formation precedes flow in developing kidney
A majority of embryonic tissue (including the kidney) contains a dense vasculature (both unperfused and perfused), even at early embryonic time points. To better gauge and analyze blood flow within the developing kidney we utilized a method of in utero embryonic intracardiac microinjections. With the use of a high-resolution ultrasound to identify the embryonic heart at E11.5 through E17.5, and following the extraction and exposure of a sin...

Watch this Scientific Journal Video about In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow at JoVE.com

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney.

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis ...

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9.6K vistas.  Children's Hospital of Pittsburgh of UPMC. This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney.