Estimation of total glomerular number using an integrated disector method in embryonic and postnatal kidneys
© Arsenault et al.; licensee BioMed Central Ltd. 2014
Received: 23 January 2014
Accepted: 14 April 2014
Published: 17 June 2014
Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) are a polymorphic group of clinical disorders comprising the major cause of renal failure in children. Included within CAKUT is a wide spectrum of developmental malformations ranging from renal agenesis, renal hypoplasia and renal dysplasia (maldifferentiation of renal tissue), each characterized by varying deficits in nephron number. First presented in the Brenner Hypothesis, low congenital nephron endowment is becoming recognized as an antecedent cause of adult-onset hypertension, a leading cause of coronary heart disease, stroke, and renal failure in North America. Genetic mouse models of impaired nephrogenesis and nephron endowment provide a critical framework for understanding the origins of human kidney disease. Current methods to quantitate nephron number include (i) acid maceration (ii) estimation of nephron number from a small number of tissue sections (iii) imaging modalities such as MRI and (iv) the gold standard physical disector/fractionator method. Despite its accuracy, the physical disector/fractionator method is rarely employed because it is labour-intensive, time-consuming and costly to perform. Consequently, less rigourous methods of nephron estimation are routinely employed by many laboratories. Here we present an updated, digitized version of the physical disector/fractionator method using free open source Fiji software, which we have termed the integrated disector method. This updated version of the gold standard modality accurately, rapidly and cost-effectively quantitates nephron number in embryonic and post-natal mouse kidneys, and can be easily adapted for stereological measurements in other organ systems.
KeywordsNephron number Disector Nephron endowment Kidney development Kidney disease Stereology
Les anomalies congénitales du rein et des voies urinaires (Congenital Anomalies of the Kidney and Urinary Tract, CAKUT) désignent un groupe polymorphe d’entités cliniques qui constitue la cause la plus fréquente d’insuffisance rénale chez l’enfant. Le CAKUT comprend aussi un grand nombre de malformations développementales, dont le syndrome de Potter, l’hypoplasie rénale, ainsi que la dysplasie rénale (maldifférentiation des tissus rénaux), toutes caractérisées par un déficit de néphrons. On reconnaît de plus en plus une masse néphronique congénitale réduite, d’abord présentée dans l’hypothèse de Brenner, comme une cause de l’hypertension chez l’adulte, de coronaropathie, d’AVC, et d’insuffisance rénale en Amérique du Nord. Les modèles génétiques de souris comportant une détérioration de la fonction rénale et de la masse néphronique fournissent un cadre pour permettre la compréhension de l’origine des néphropathies chez l’humain. Les méthodes actuelles de quantification des néphrons comprennent (i) la macération acide (ii) l’estimation du nombre de néphrons à partir d’une petite quantité de tissus sectionnés (iii) les modes d’imagerie tels que l’IRM et (iv) la technique de référence du disecteur et fractionnement. Malgré sa précision, cette dernière méthode n’est employée que rarement, puisqu’elle requiert main-d’œuvre, temps et argent. Par conséquent, plusieurs laboratoires emploient systématiquement des méthodes moins rigoureuses d’estimation du nombre de néphrons. Nous présentons ici une version mise à jour et numérisée de la technique du disecteur et fractionnement, que nous appelons la technique intégrée du disecteur, en utilisant Fiji, un logiciel ouvert et gratuit. Cette version mise à jour de la modalité de référence permet de quantifier les néphrons de manière précise, rapide et rentable dans les reins de souris à l’état embryonnaire ou postnatal, et peut aisément être adaptée aux mesures stéréologiques d’autres organes.
KeywordsNephron number Disector Nephron endowment Kidney development Kidney disease Stereology
Defects in nephrogenesis and ureteric branching result in CAKUT. With an incidence of 1:400 [1, 2], CAKUT constitute one of the most frequent birth defects in humans, and the major cause of childhood renal failure [3, 4]. These pleiotrophic malformations comprise a multitude of renal phenotypes including renal agenesis, hypoplasia and renal dysplasia, associated with obstructive or refluxive anomalies of the ureter . CAKUT are predicted to have different genetic origins , yet the molecular pathogenesis of CAKUT is not well understood. Nephron deficiency is a hallmark feature of CAKUT. In fact, nephron number varies greatly even within the normal distribution, ranging from 7,000 to 11,000 in mice , and from 200,000 to 1.8 million in humans . Low nephron endowment within this normal range - though asymptomatic early in life, is associated with adult-onset hypertension [8–10], a disease affecting 73 million North Americans and a leading cause of coronary heart disease, stroke, and renal failure in North America [11, 12]. Despite the fundamental importance of nephron endowment to human health and disease, only a minority of molecular mechanisms underlying nephron morphogenesis have been identified. To this end, mouse models of impaired nephrogenesis and nephron endowment provide an important framework for understanding the molecular and developmental origins of human kidney disease.
Four main methods are currently employed to quantitate glomerular number (Nglom) in the kidney . The most accessible methods for estimating glomerular number by virtue of their ease of performance, relative low cost and rapidity, are (i) acid maceration and (ii) quantitation of glomeruli in a small number of histological sections. In the acid maceration method, the kidney is decapsulated, cut into small pieces and incubated in a weakly acidic solution. Tissue fragments are gently disrupted by manual aspiration which dissociates glomeruli in the suspension, and Nglom is counted within a known volume in a chamber similar to a hemocytometer [14, 15]. Acid maceration is a rapid, simple and inexpensive means of obtaining Nglom; however, this method is subject to some of the same sources of counting errors and consequent low precision associated with hemocytometer use, including non-uniform suspensions and non-adherence to convention for counting glomeruli in contact with boundary lines or each other. A tacit assumption in the acid maceration method is that glomerular structural integrity is equivalent between sample groups. While not previously reported in the literature, it is feasible that disease states or genetic mutations that deleteriously affect the structural integrity of the glomerulus would render glomeruli more susceptible to acid digestion. Under such conditions, spurious differences in glomerular number could be obtained due to degradation of glomeruli in suspension, rather than true differences in glomerular number between groups.
A second commonly employed method involves evaluation of stained histological sections of the kidney. A small number of kidney tissue sections are chosen and glomerular number is counted and reported per area of tissue, or cortex. Although straightforward to perform, it is a biased sampling method that does not provide an accurate depiction of glomerular number for the entire kidney. Using this method, a glomerulus that is longer perpendicular to the plane of sectioning has a higher probability of being sampled in any given section than would the same glomerulus orientated parallel to the plane of sectioning. The number of glomeruli counted using this method not only depends on glomerular number and density, but also on their size and shape, which are not equally distributed throughout the kidney [16, 17].
The use of micro-magnetic resonance imaging (micro-MRI), also known as high-field MRI, is a third modality that can provide quantitative measurements in vivo and therefore holds great potential for future human applications. MRI provides both rapid and accurate evaluation of total glomerular number in healthy kidneys [18, 19]. However, access to the micro-MRI system is costly, rendering it less accessible to many laboratories. In addition, the accuracy of MRI is significantly reduced in kidneys with focal and segmental glomerulosclerosis , due to the low retention of the MRI contrasting agent cationic ferritin in the basement membranes of diseased glomeruli. Thus, disease states or genetic mutations that affect the integrity of the glomerular basement membrane may alter the retention of the contrasting agents in affected glomeruli, and can spuriously skew results.
The gold standard method for estimating Nglom is the unbiased physical disector/fractionator method. This stereological method involves exhaustively sectioning the kidney, and using a physical disector (a stereological probe) to count glomeruli in pairs of tissue sections. The application of stereology in biological systems and in the context of the kidney has been well-described [21, 22]. Briefly, this approach uses a pair of projection microscopes, a pencil and paper to manually circle and count glomeruli . The physical disector method is extremely accurate because it is unbiased, but is very labour-intensive to perform, requiring a full day for an experienced technician to complete quantitation of a single rat kidney . Commercially-available software packages offer powerful stereological alternatives to using projection microscopes for the physical disector/fractionator method , but are cost-prohibitive to most laboratories.
Members of the Sry-Related HMG Box (Sox) gene family have been shown to play master regulatory roles in a multitude of developmental processes including dorso-ventral patterning , stemness [25–27], male differentiation [28, 29], neurogenesis [30–32], cardiac outflow tract, B-lymphocyte  and spinal cord development , and skeletogenesis . We have recently identified Sox4 as a critical regulator of normal renal development in vivo  . Using an established Six2-Cre transgenic mouse line [37, 38] we ablated Sox4 function in nephron progenitors and their cellular descendants (Sox4 nephron- mice). Using the acid maceration method, we demonstrated that Sox4 nephron- kidneys exhibit 48% reduction in nephron number compared to wild-type animals. In light of the low precision associated with this method and the inherent limitations associated with each of the existing methods of glomerular quantitation, we sought to develop an accurate, rapid and cost-effective alternative means of quantitating nephron number that could be of use to the nephrology community.
ImageJ is an open source image processing program through the NIH which has been freely available for more than 15 years . Fiji is a free biological imaging package within ImageJ that comes pre-loaded with the TrakEM2 plugin for morphological data mining and three-dimensional modeling, and includes a stereological disector tool [40, 41]. By integrating the stereologic principles of the physical disector/fractionator method with automated light microscopy and ImageJ/Fiji software, we have developed an accurate, rapid, cost-effective method for quantitation of total nephron number in embryonic day (E)17.5 murine kidneys (including S-shaped bodies and maturing glomeruli), and estimation of nephron number in postnatal day (P)7 murine kidneys. Thus, we present the integrated disector method as a highly accessible adaptation of the physical disector fractionator method that can be easily adopted for in house application with minimal set up costs.
The Sox4 null (Sox4 − ) allele , conditional Sox4 (Sox4 C ) allele  and Six2-Cre BAC transgenic line  have been previously characterized. All animal experiments were carried out in accordance with the policies of the Animal Care Committee at the University of Prince Edward Island and according to the Canadian Council on Animal Care guidelines.
Kidneys were isolated from E17.5 and P7 Six2Cre; Sox4 C/- (Sox4 nephron- ) and wild-type mice. Kidneys were fixed in 10% neutral buffered formalin, processed to paraffin blocks and exhaustively sectioned in the sagittal plane at 5 μm. Ribbons of serial E17.5 sections were mounted on glass slides, such that every section of the kidney was captured on a set of slides. Sections were stained with biotinylated peanut agglutinin (PNA) as described previously  with the following modifications: reduced incubation time of the biotinylated PNA to 10 min. and without hematoxylin counterstaining. Paired sections from P7 kidneys were selected using a systematic uniform random sampling approach as described . Sections were stained with periodic acid-Schiff (PAS).
Fiji processing of kidney section images
A similar approach was used to estimate glomerular number in postnatal kidneys. A pilot study was conducted to determine mean maximal glomerular size and the total number of sections through a P7 kidney, and the optimal section sampling fraction was determined to estimate total glomerular number (please see  for detailed protocol). Paired sections were imported, converted to 8-bit colour and manually aligned. The disector tool was then used to identify glomeruli that were either present on both paired sections or on only one of the two. Those present on only one of the two sections were counted as disector particles and used to estimate the total number of glomeruli (please refer to Additional file 2 for detailed instructions).
Two sample t-tests were performed using Minitab 16. Data are presented as mean ± standard deviation.
Results and discussion
Assessment of glomerular number in Sox4nephron- kidneys
Using acid maceration, we previously reported a substantially greater reduction (48%) in glomerular number in Sox4- deficient kidneys at P7. This difference is likely attributable to the high variability (low precision) inherent to the acid maceration technique. Variability in the acid maceration technique of glomerular quantitation is introduced by (i) pipetting errors (ii) non-uniform suspension of glomeruli and most significantly (iii) high standard deviation and its derivative coefficient of variability (CV) arising from the extrapolation of Nglom from a small volume (500 μL) to Nglom in the total volume (30 mL) . Indeed, quantitation of Nglom using acid maceration is associated with a high CV in both wild-type kidneys (12.7%) and in Sox4 mutant kidneys (15.6%), which may largely explain the difference in Nglom obtained using acid maceration vs. integrated disector methodologies. When restricted by cost and resources, acid maceration continues to provide useful insight into kidney development and physiology. This approach lends itself particularly well in instances where large between-group differences exist, as in the case of Sox4- mutant vs. wild-type glomerular number, and has recently been used for validation of new MRI approaches . However, it is generally accepted and understood that stereology is a more robust approach for quantification of glomeruli. Indeed, the high variability inherent to the acid maceration technique was a significant motivation for us to implement cost-effective in house stereological methods for glomerular quantitation.
The integrated disector method is cost-effective and accessible
Several open source biomedical image analysis software packages have recently become available including Endrov , Icy , BioImagXD  and Fiji . Each package has a specialized focus and toolset to facilitate processing of complex biological spatio-temporal image data obtained mainly by microscopy. Certainly, multiple strategies can be developed to perform glomerular quantitation with high accuracy, speed and economy. However, to the best of our knowledge, we are the first to report a means of combining the high accuracy of the physical disector/fractionator gold standard method with the power and economy of open source image analysis software.
Although many groups are studying the effects of genes and various environmental factors on the development and function of the kidney, there is currently no broadly accessible method for accurately determining if there is an effect on nephron endowment. In the present study, we have optimized PNA and PAS staining of histological sections to identify glomeruli in embryonic and postnatal kidneys. We have updated the physical disector/fractionator method by combining semi-automated microscopy image capture with Fiji/TrakEM2 to streamline data collection and image analysis. With minimal setup costs, these tools permit accurate, efficient and rapid stereological measurement of glomerular number in many laboratories, and can be applied from early embryonic development through to the adult mouse. Implementing the integrated disector requires laboratories to follow the same steps associated with the physical disector/fractionator method from tissue embedding to exhaustive sectioning and staining of tissue . The power of our approach is in its ease of set up and accessibility for counting Nglom using a freely available disector tool which allows for unbiased estimation of Nglom. Total time for image acquisition and analysis for an experienced user and full automation would average 2-3 hours per kidney. Smaller laboratories that may not have access to motorized microscopes can easily outsource the image capture component to collaborators while performing the Fiji/TrakEM2 quantitation in-house. Alternatively, researchers can manually capture overlapping partial images of serial sections at the appropriate magnification, and sections can be stitched using the Grid/Collection stitching plugin built-in to Fiji. Stitched sections can then be loaded into the TrakEM2 environment and analysed as described. Other refinements and adaptations can be performed to best suit the needs of individual research teams, including the acquisition of volumetric data, and can be broadly applied towards stereological measurements in other organ systems.
In summary, we have adapted the physical disector/fractionator method of stereological glomerular quantitation using semi-automated microscopy together with open source Fiji software and the TrakEM2 plugin. We present the integrated disector method in its entirety through step-wise video and screen capture, in the hopes of offering the general nephrology community an accurate, rapid, cost-effective and therefore highly-accessible means of glomerular quantification in embryonic and postnatal kidneys.
The authors gratefully acknowledge funding from the Kidney Research Scientist Core Education and National Training (KRESCENT) Program and the Kidney Foundation of Canada to S.H. We thank Daniel Hartwig, Sharon MacKenzie, David MacKenzie, An Soon Pak and Chea Gun Pak for their wonderful support sine qua non. SDG.
Kidney Foundation of Canada, CIHR, Kidney Research Education and National Training Program (KRESCENT); Grant numbers 615368, 615314.
- Nakanishi K, Yoshikawa N: Genetic disorders of human congenital anomalies of the kidney and urinary tract (CAKUT). Pediatr Int 2003,45(5):610–6. 10.1046/j.1442-200X.2003.01779.xView ArticleGoogle Scholar
- Limwongse C, Clarren SK, Cassidy SB: Syndromes and malformations of the urinary tract. In Pediatric nephrology. 4th edition. Edited by: Barratt TM, Avner ED, We H. Baltimore: Lippincott Williams and Wilkins; 1998:415–426.Google Scholar
- Pope JC 4th, Brock JW 3rd, Adams MC, Stephens FD, Ichikawa I: How they begin and how they end: Classic and new theories for the development and deterioration of congenital anomalies of the kidney and urinary tract, CAKUT. J Am Soc Nephrol 1999,10(9):2018–28.Google Scholar
- Seikaly MG, Ho PL, Emmett L, Fine RN, Tejani A: Chronic renal insufficiency in children: The 2001 annual report of the NAPRTCS. Pediatr Nephrol 2003 Aug,18(8):796–804. 10.1007/s00467-003-1158-5View ArticleGoogle Scholar
- Weber S: Novel genetic aspects of congenital anomalies of kidney and urinary tract. Curr Opin Pediatr 2012 Apr,24(2):212–8. 10.1097/MOP.0b013e32834fdbd4View ArticleGoogle Scholar
- Cebrian C, Borodo K, Charles N, Herzlinger DA: Morphometric index of the developing murine kidney. Dev Dyn 2004 Nov,231(3):601–8. 10.1002/dvdy.20143View ArticleGoogle Scholar
- Hoppe CC, Evans RG, Bertram JF, Moritz KM: Effects of dietary protein restriction on nephron number in the mouse. Am J Physiol Regul Integr Comp Physiol 2007 May,292(5):R1768–74. 10.1152/ajpregu.00442.2006View ArticleGoogle Scholar
- Keller G, Zimmer G, Mall G, Ritz E, Amann K: Nephron number in patients with primary hypertension. N Engl J Med 2003 Jan 9,348(2):101–8. 10.1056/NEJMoa020549View ArticleGoogle Scholar
- Reyes L, Manalich R: Long-term consequences of low birth weight. Kidney Int Suppl 2005, (97):S107–11.
- Walker KA, Cai X, Caruana G, Thomas MC, Bertram JF, Kett MM: High nephron endowment protects against salt-induced hypertension. Am J Physiol Renal Physiol 2012 Jul,303(2):F253–8. 10.1152/ajprenal.00028.2012View ArticleGoogle Scholar
- Wilkins K, Campbell NR, Joffres MR, McAlister FA, Nichol M, Quach S, Johansen HL, Tremblay MS: Blood pressure in canadian adults. Health Rep 2010 Mar,21(1):37–46.Google Scholar
- Tedla FM, Brar A, Browne R, Brown C: Hypertension in chronic kidney disease: Navigating the evidence. Int J Hypertens 2011, 2011: 132405.View ArticleGoogle Scholar
- Bertram JF: Estimating glomerular number: Why we do it and how. Clin Exp Pharmacol Physiol 2013 Nov,40(11):785–8. 10.1111/1440-1681.12133View ArticleGoogle Scholar
- MacKay K, Striker LJ, Pinkert CA, Brinster RL, Striker GE: Glomerulosclerosis and renal cysts in mice transgenic for the early region of SV40. Kidney Int 1987 Dec,32(6):827–37. 10.1038/ki.1987.283View ArticleGoogle Scholar
- Godley LA, Kopp JB, Eckhaus M, Paglino JJ, Owens J, Varmus HE: Wild-type p53 transgenic mice exhibit altered differentiation of the ureteric bud and possess small kidneys. Genes Dev 1996 Apr 1,10(7):836–50. 10.1101/gad.10.7.836View ArticleGoogle Scholar
- Samuel T, Hoy WE, Douglas-Denton R, Hughson MD, Bertram JF: Determinants of glomerular volume in different cortical zones of the human kidney. J Am Soc Nephrol 2005 Oct,16(10):3102–9. 10.1681/ASN.2005010123View ArticleGoogle Scholar
- Kanzaki G, Tsuboi N, Utsunomiya Y, Ikegami M, Shimizu A, Hosoya T: Distribution of glomerular density in different cortical zones of the human kidney. Pathol Int 2013 Mar,63(3):169–75. 10.1111/pin.12044View ArticleGoogle Scholar
- Heilmann M, Neudecker S, Wolf I, Gubhaju L, Sticht C, Schock-Kusch D, Kriz W, Bertram JF, Schad LR, Gretz N: Quantification of glomerular number and size distribution in normal rat kidneys using magnetic resonance imaging. Nephrol Dial Transplant 2012 Jan,27(1):100–7. 10.1093/ndt/gfr273View ArticleGoogle Scholar
- Beeman SC, Zhang M, Gubhaju L, Wu T, Bertram JF, Frakes DH, Cherry BR, Bennett KM: Measuring glomerular number and size in perfused kidneys using MRI. Am J Physiol Renal Physiol 2011 Jun,300(6):F1454–7. 10.1152/ajprenal.00044.2011View ArticleGoogle Scholar
- Bennett KM, Zhou H, Sumner JP, Dodd SJ, Bouraoud N, Doi K, Star RA, Koretsky AP: MRI of the basement membrane using charged nanoparticles as contrast agents. Magn Reson Med 2008,60(3):564–74. 10.1002/mrm.21684View ArticleGoogle Scholar
- Nyengaard JR: Stereologic methods and their application in kidney research. J Am Soc Nephrol 1999 May,10(5):1100–23.Google Scholar
- Howard CV, Reed MG: Unbiased stereology. New York: Bios Scientific Publishers; 1998.Google Scholar
- Cullen-McEwen LA, Douglas-Denton RN, Bertram JF: Estimating total nephron number in the adult kidney using the physical disector/fractionator combination. Methods Mol Biol 2012, 886: 333–50. 10.1007/978-1-61779-851-1_30View ArticleGoogle Scholar
- Chen YY, Harris MP, Levesque MP, Nusslein-Volhard C, Sonawane M: Heterogeneity across the dorso-ventral axis in zebrafish EVL is regulated by a novel module consisting of sox, snail1a and max genes. Mech Dev 2012 Mar,129(1–4):13–23.View ArticleGoogle Scholar
- Avilion A, Nicolis S, Pevny L, Perez L, Vivian N, Lovell-Badge R: Multipotent cell lineages in early mouse development depend on SOX2 function. Genes & Develop 2003, 17: 126–140. 10.1101/gad.224503View ArticleGoogle Scholar
- Masui S, Nakatake Y, Toyooka Y, Shimosato D, Yagi R, Takahashi K, Okochi H, Okuda A, Matoba R, Sharov AA, Ko MS, Niwa H: Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nat Cell Biol 2007 Jun,9(6):625–35. 10.1038/ncb1589View ArticleGoogle Scholar
- Wang Z, Oron E, Nelson B, Razis S, Ivanova N: Distinct lineage specification roles for NANOG, OCT4, and SOX2 in human embryonic stem cells. Cell Stem Cell 2012 Apr 6,10(4):440–54. 10.1016/j.stem.2012.02.016View ArticleGoogle Scholar
- Koopman P, Gubbay J, Vivian N, Goodfellow P, Lovell-Badge R: Male development of chromosomally female mice transgenic for sry. Nature 1991, 351: 117–21. 10.1038/351117a0View ArticleGoogle Scholar
- Tea W: Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell 1994, 79: 1111–1120. 10.1016/0092-8674(94)90041-8View ArticleGoogle Scholar
- Bhattaram P, Penzo-Mendez A, Sock E, Colmenares C, Kaneko KJ, Vassilev A, Depamphilis ML, Wegner M, Lefebvre V: Organogenesis relies on SoxC transcription factors for the survival of neural and mesenchymal progenitors. Nat Commun 2010 Apr 12, 1: 9.View ArticleGoogle Scholar
- Bergsland M, Ramskold D, Zaouter C, Klum S, Sandberg R, Muhr J: Sequentially acting sox transcription factors in neural lineage development. Genes Dev 2011 Dec 1,25(23):2453–64. 10.1101/gad.176008.111View ArticleGoogle Scholar
- Mu L, Berti L, Masserdotti G, Covic M, Michaelidis TM, Doberauer K, Merz K, Rehfeld F, Haslinger A, Wegner M, Sock E, Lefebvre V, Couillard-Despres S, Aigner L, Berninger B, Lie DC: SoxC transcription factors are required for neuronal differentiation in adult hippocampal neurogenesis. J Neurosci 2012 Feb 29,32(9):3067–80. 10.1523/JNEUROSCI.4679-11.2012View ArticleGoogle Scholar
- Schilham M, Oosterwegel M, Moerer P, Ya J, Deboer P, van de Wetering M, Verbeek S, Lamers W, Kruisbeek A, Cumano A, Clevers H: Defects in cardiac outflow tract formation and pro-B-lymphocyte expansion in mice lacking sox-4. Nature 1996, 380: 711–714. 10.1038/380711a0View ArticleGoogle Scholar
- Shim S, Kwan KY, Li M, Lefebvre V, Sestan N: Cis-regulatory control of corticospinal system development and evolution. Nature 2012 May 30,486(7401):74–9.Google Scholar
- Akiyama H, Lefebvre V: Unraveling the transcriptional regulatory machinery in chondrogenesis. J Bone Miner Metab 2011 Jul,29(4):390–5. 10.1007/s00774-011-0273-9View ArticleGoogle Scholar
- Huang J, Arsenault M, Kann M, Lopez-Mendez C, Saleh M, Wadowska D, Taglienti M, Ho J, Miao Y, Sims D, Spears J, Lopez A, Wright G, Hartwig S: The transcription factor sry-related HMG box-4 (SOX4) is required for normal renal development in vivo. Dev Dyn 2013 Jun,242(6):790–9. 10.1002/dvdy.23971View ArticleGoogle Scholar
- Kobayashi A, Valerius MT, Mugford JW, Carroll TJ, Self M, Oliver G, McMahon AP: Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development. Cell Stem Cell 2008 Aug 7,3(2):169–81. 10.1016/j.stem.2008.05.020View ArticleGoogle Scholar
- Kobayashi A, Kwan KM, Carroll TJ, McMahon AP, Mendelsohn CL, Behringer RR: Distinct and sequential tissue-specific activities of the LIM-class homeobox gene Lim1 for tubular morphogenesis during kidney development. Development 2005 Jun,132(12):2809–23. 10.1242/dev.01858View ArticleGoogle Scholar
- Schneider CA, Rasband WS, Eliceiri KW: NIH image to ImageJ: 25 years of image analysis. Nat Methods 2012 Jul,9(7):671–5. 10.1038/nmeth.2089View ArticleGoogle Scholar
- Cardona A, Saalfeld S, Schindelin J, Arganda-Carreras I, Preibisch S, Longair M, Tomancak P, Hartenstein V, Douglas RJ: TrakEM2 software for neural circuit reconstruction. PLoS One 2012,7(6):e38011. 10.1371/journal.pone.0038011View ArticleGoogle Scholar
- Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A: Fiji: An open-source platform for biological-image analysis. Nat Methods 2012 Jun 28,9(7):676–82. 10.1038/nmeth.2019View ArticleGoogle Scholar
- Penzo-Mendez A, Dy P, Pallavi B, Lefebvre V: Generation of mice harboring a Sox4 conditional null allele. Genesis 2007 Dec,45(12):776–80. 10.1002/dvg.20358View ArticleGoogle Scholar
- Cullen-McEwen LA, Armitage JA, Nyengaard JR, Bertram JF: Estimating nephron number in the developing kidney using the physical disector/fractionator combination. Methods Mol Biol 2012, 886: 109–19. 10.1007/978-1-61779-851-1_10View ArticleGoogle Scholar
- Shapiro H: Practical flow cytometry. Inc.: John WIley & Sons; 2005.Google Scholar
- Henriksson J, Hench J, Tong YG, Johansson A, Johansson D, Burglin TR: Endrov: An integrated platform for image analysis. Nat Methods 2013 Jun,10(6):454–6. 10.1038/nmeth.2478View ArticleGoogle Scholar
- de Chaumont F, Dallongeville S, Chenouard N, Herve N, Pop S, Provoost T, Meas-Yedid V, Pankajakshan P, Lecomte T, Le Montagner Y, Lagache T, Dufour A, Olivo-Marin JC: Icy: An open bioimage informatics platform for extended reproducible research. Nat Methods 2012 Jun 28,9(7):690–6. 10.1038/nmeth.2075View ArticleGoogle Scholar
- Kankaanpaa P, Paavolainen L, Tiitta S, Karjalainen M, Paivarinne J, Nieminen J, Marjomaki V, Heino J, White DJ: BioImageXD: An open, general-purpose and high-throughput image-processing platform. Nat Methods 2012 Jun 28,9(7):683–9. 10.1038/nmeth.2047View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.