Abstract
Organ development requires the coordination of proliferation and differentiation of various cell types. This is particularly challenging in the kidney, where up to 26 different cell types with highly specialized functions are present. Moreover, even though the nephron initially develops from a common progenitor pool, the individual nephron segments are ultimately quite different in respect to cell numbers. This suggests that some cells in the nephron have a higher proliferative index (i.e., cell cycle length) than others. Here, we describe two different immunofluorescence-based approaches to accurately quantify such growth rates in the pronephric kidney of Xenopus laevis. Rapidly dividing cells were identified with the mitosis marker phospho-Histone H3, while slowly cycling cells were labeled using the thymidine analogue EdU. In addition, individual nephron segments were marked using cell type-specific antibodies. To accurately assess the number of positively stained cells, embryos were then serially sectioned and analyzed by immunofluorescence microscopy. Growth rates were established by counting the mitosis or S-phase events in relation to the overall cells present in the nephron segment of interest. This experimental design is very reproducible and can easily be modified to fit other animal models and organ systems.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Stanger BZ (2008) The biology of organ size determination. Diabetes Obes Metab 10(Suppl 4):16–22
Neufeld TP (2003) Body building: regulation of shape and size by PI3K/TOR signaling during development. Mech Dev 120:1283–1296
Nyengaard JR, Bendtsen TF (1992) Glomerular number and size in relation to age, kidney weight, and body surface in normal man. Anat Rec 232:194–201
Meijer AJ, Codogno P (2008) Nutrient Âsensing: TOR’s ragtime. Nat Cell Biol 10:881–883
Grantham JJ, Cook LT, Wetzel LH, Cadnapaphornchai MA, Bae KT (2010) Evidence of extraordinary growth in the progressive enlargement of renal cysts. Clin J Am Soc Nephrol 5:889–896
Al-Awqati Q, Oliver JA (2002) Stem cells in the kidney. Kidney Int 61:387–395
Satriano J (2007) Kidney growth, hypertrophy and the unifying mechanism of diabetic complications. Amino Acids 33:331–339
Sinuani I, Beberashvili I, Averbukh Z et al (2010) Mesangial cells initiate compensatory tubular cell hypertrophy. Am J Nephrol 31:326–331
Wessely O, Tran U (2011) Xenopus pronephros development―past, present, and future. Pediatr Nephrol 26:1545–1551
Simons M, Gloy J, Ganner A et al (2005) Inversin, the gene product mutated in nephronophthisis type II, functions as a molecular switch between Wnt signaling pathways. Nat Genet 37:537–543
Tran U, Zakin L, Schweickert A et al (2010) The RNA-binding protein bicaudal C regulates polycystin 2 in the kidney by antagonizing miR-17 activity. Development 137:1107–1116
White JT, Zhang B, Cerqueira DM, Tran U, Wessely O (2010) Notch signaling, wt1 and foxc2 are key regulators of the podocyte gene regulatory network in Xenopus. Development 137:1863–1873
Moody SA (1987) Fates of the blastomeres of the 16-cell stage Xenopus embryo. Dev Biol 119:560–578
Zhou X, Vize PD (2004) Proximo-distal Âspecialization of epithelial transport processes within the Xenopus pronephric kidney tubules. Dev Biol 271:322–338
Raciti D, Reggiani L, Geffers L et al (2008) Organization of the pronephric kidney revealed by large-scale gene expression mapping. Genome Biol 9:R84
Wingert RA, Selleck R, Yu J et al (2007) The cdx genes and retinoic acid control the positioning and segmentation of the zebrafish pronephros. PLoS Genet 3:1922–1938
Vize PD, Jones EA, Pfister R (1995) Development of the Xenopus pronephric Âsystem. Dev Biol 171:531–540
Hendzel MJ, Wei Y, Mancini MA et al (1997) Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric Âheterochromatin during G2 and spreads in an ordered fashion coincident with mitotic Âchromosome condensation. Chromosoma 106:348–360
Salic A, Mitchison TJ (2008) A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci USA 105:2415–2420
Gratzner HG (1982) Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: a new reagent for detection of DNA replication. Science 218:474–475
Vogetseder A, Palan T, Bacic D, Kaissling B, Le Hir M (2007) Proximal tubular epithelial cells are generated by division of differentiated cells in the healthy kidney. Am J Physiol Cell Physiol 292:C807–C813
Sive HL, Grainger RM, Harland RM (2000) Early development of Xenopus laevis: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Nieuwkoop PD, Faber J (1994) Normal table of Xenopus laevis. Garland Publishing, Inc, New York
Acknowledgments
We would like to thank U. Tran and V. Kumar for critically reviewing the manuscript. D.R. is a recipient of a DFG postdoctoral Âfellowship (RO4124/1-1). This work was supported by NIH/NIDDK (7RO1DK080745-03) to O.W.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Romaker, D., Zhang, B., Wessely, O. (2012). An Immunofluorescence Method to Analyze the Proliferation Status of Individual Nephron Segments in the Xenopus Pronephric Kidney. In: Michos, O. (eds) Kidney Development. Methods in Molecular Biology™, vol 886. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-851-1_11
Download citation
DOI: https://doi.org/10.1007/978-1-61779-851-1_11
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-850-4
Online ISBN: 978-1-61779-851-1
eBook Packages: Springer Protocols