Use of dual section mRNA in situ hybridisation/immunohistochemistry to clarify gene expression patterns during the early stages of nephron development in the embryo and in the mature nephron of the adult mouse kidney

Abstract

The kidney is the most complex organ within the urogenital system. The adult mouse kidney contains in excess of 8,000 mature nephrons, each of which can be subdivided into a renal corpuscle and 14 distinct tubular segments. The histological complexity of this organ can make the clarification of the site of gene expression by in situ hybridisation difficult. We have defined a panel of seven antibodies capable of identifying the six stages of early nephron development, the tubular nephron segments and the components of the renal corpuscle within the embryonic and adult mouse kidney. We have analysed in detail the protein expression of Wt1, Calb1 Aqp1, Aqp2 and Umod using these antibodies. We have then coupled immunohistochemistry with RNA in situ hybridisation in order to precisely identify the expression pattern of different genes, including Wnt4, Umod and Spp1. This technique will be invaluable for examining at high resolution, the structure of both the developing and mature nephron where standard in situ hybridisation and histological techniques are insufficient. The use of this technique will enhance the expression analyses of genes which may be involved in nephron formation and the function of the mature nephron in the mouse.

Claudin-7, -16, and -19 during mouse kidney development

“…24 CLDN16 and 19 were strongly expressed in both the primitive and the mature Loop of Henle at E16, P1 and P28 and showed overlapping expression with Uromodulin that also demarcates the Loop of Henle 24 (Figs. 4 and 5).…”

“…These early and late tubular structures give rise to the Loop of Henle that progresses from an anlage, to a primitive loop, and then to an immature loop prior to its final differentiation. [24][25] Protein expression was examined by performing immunohistochemistry on paraffin-embedded sections and immunofluorescence on cryosections (Figs. 3-6).…”

“…Similar expression was noted at P28, but here CLDN7 was predominantly detected in distal tubular segments based on its overlapping expression with Calbindin-D-28K and with Uromodulin. 24 Calbindin-D-28K protein is expressed by the distal convoluted tubule, the connecting tubule and the collecting duct, while Tamm Horsfall or Uromodulin protein is expressed by the thin and thick ascending limbs of the Loop of Henle.…”

Hepatocyte nuclear factor 1β controls nephron tubular development

“…2B). In addition, the characteristic mutually exclusive distal/proximal expression pattern of specific genes (Georgas et al, 2008) was preserved. In both control and mutant embryos, vesicles were characterized by polarized expression of Jag1 in the distal portion of the vesicle, whereas Wt1 expression was restricted to the proximal compartment ( Fig.…”

Tissue‐ and development‐dependent expression of the small GTPase Arf6 in mice

“…The ureteric buds grow and branch to form tubules with polarized epithelial cells. By E15.5, most of the kidney components appear as immature anlages, which include the S-shaped body, loop of Henle, and collecting ducts (Stuart and Nigam, 1995;Brunskill et al, 2008;Georgas et al, 2008). The expression of Arf6 begins earlier than E13.5.…”