Genetic Dissection of Cadherin Function during Nephrogenesis

ABSTRACT The distinct expression of R-cadherin in the induced aggregating metanephric mesenchyme suggests that it may regulate the mesenchymal-epithelial transition during kidney development. To address whether R-cadherin is required for kidney ontogeny, R-cadherin-deficient mice were generated. These mice appeared to be healthy and were fertile, demonstrating that R-cadherin is not essential for embryogenesis. The only kidney phenotype of adult mutant animals was the appearance of dilated proximal tubules, which was associated with an accumulation of large intracellular vacuoles. Morphological analysis of nephrogenesis in R-cadherin −/− mice in vivo and in vitro revealed defects in the development of both ureteric bud-derived cells and metanephric mesenchyme-derived cells. First, the morphology and organization of the proximal parts of the ureteric bud epithelium were altered. Interestingly, these morphological changes correlated with an increased rate of apoptosis and were further supported by perturbed branching and patterning of the ureteric bud epithelium during in vitro differentiation. Second, during in vitro studies of mesenchymal-epithelial conversion, significantly fewer epithelial structures developed from R-cadherin −/− kidneys than from wild-type kidneys. These data suggest that R-cadherin is functionally involved in the differentiation of both mesenchymal and epithelial components during metanephric kidney development. Finally, to investigate whether the redundant expression of other classic cadherins expressed in the kidney could explain the rather mild kidney defects in R-cadherin-deficient mice, we intercrossed R-cadherin −/− mice with cadherin-6−/− , P-cadherin −/−, and N-cadherin +/− mice. Surprisingly, however, in none of the compound knockout strains was kidney development affected to a greater extent than within the individual cadherin knockout strains.

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Dominant effects of RET receptor misexpression and ligand-independent RET signaling on ureteric bud development

Development ◽

10.1242/dev.126.7.1375 ◽

1999 ◽

Vol 126 (7) ◽

pp. 1375-1386 ◽

Cited By ~ 7

Author(s):

S. Srinivas ◽

Z. Wu ◽

C.M. Chen ◽

V. D'Agati ◽

F. Costantini

Keyword(s):

Kidney Development ◽

Collecting Duct ◽

Target Cells ◽

Metanephric Mesenchyme ◽

Ureteric Bud ◽

Independent Form ◽

Normal Expression ◽

Bud Growth

During kidney development, factors from the metanephric mesenchyme induce the growth and repeated branching of the ureteric bud, which gives rise to the collecting duct system and also induces nephrogenesis. One signaling pathway known to be required for this process includes the receptor tyrosine kinase RET and co-receptor GFR(α)-1, which are expressed in the ureteric bud, and the secreted ligand GDNF produced in the mesenchyme. To examine the role of RET signaling in ureteric bud morphogenesis, we produced transgenic mice in which the pattern of RET expression was altered, or in which a ligand-independent form of RET kinase was expressed. The Hoxb7 promoter was used to express RET throughout the ureteric bud branches, in contrast to its normal expression only at the bud tips. This caused a variable inhibition of ureteric bud growth and branching reminiscent of, but less severe than, the RET knockout phenotype. Manipulation of the level of GDNF, in vitro or in vivo, suggested that this defect was due to insufficient rather than excessive RET signaling. We propose that RET receptors expressed ectopically on ureteric bud trunk cells sequester GDNF, reducing its availability to the normal target cells at the bud tips. When crossed to RET knockout mice, the Hoxb7/RET transgene, which encoded the RET9 isoform, supported normal kidney development in some RET−/− animals, indicating that the other major isoform, RET51, is not required in this organ. Expression of a Hoxb7/RET-PTC2 transgene, encoding a ligand-independent form of RET kinase, caused the development of abnormal nodules, outside the kidney or at its periphery, containing branched epithelial tubules apparently formed by deregulated growth of the ureteric bud. This suggests that RET signaling is not only necessary but is sufficient to induce ureteric bud growth, and that the orderly, centripetal growth of the bud tips is controlled by the spatially and temporally regulated expression of GDNF and RET.

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In vitro and in vivo parameters for identification of landrace pigs with low reproductive performance

Semina Ciências Agrárias ◽

10.5433/1679-0359.2022v43n2p573 ◽

2022 ◽

Vol 43 (2) ◽

pp. 573-584

Author(s):

Fábio da Costa Málaga ◽

◽

Helloa Alaide Siqueira ◽

Lucio Pereira Rauber ◽

Mariana Groke Marques ◽

...

Keyword(s):

Birth Rate ◽

Morphological Changes ◽

Poor Performance ◽

Low Fertility ◽

Retrospective Data ◽

In Vitro Motility ◽

Improvement Programs ◽

The Individual

In pig farming, measurements of production parameters play a fundamental role in the success of the activity. Minimal differences in fertility between breeders can lead to less reproductive efficiency and, less productivity. However, assessing the fertility of each male and the early identification of subfertile males is a difficult task to be performed. Thus, the aim of this study was to evaluate the use of in vitro and in vivo parameters in the identification of subfertile males of the Landrace breed, aiming to collaborate with genetic improvement programs, routine optimization in the Genetic Diffusion Units (GDUs) and the results of performance. In experiment 1, an approach to identify males with subfertility was evaluated based on retrospective data. For this, the results (averages of birth rates, number of total births and average percentages of female and male piglets per litter) were evaluated for a total of 996 matings and 847 parturitions. The inseminations came from ejaculates of 32 males, who had at least 19 females inseminated with homospermic doses in the concentration of 2.5 x 109 total sperm from the same male. As for the birth rate (BR), an average of 85.47% ± 6.05 was observed with a group of median males, seven males that stood out and one individual (M32) with a performance of 58.06% ± 9.0. For the total number of piglets born (PB) the average was 13.41 ± 0.56, with three males with better performance and one (M32) with very poor performance (8.62 ± 0.59). In experiment 2, it was verified whether evaluations of inseminating doses (ID) of semen in vitro (motility and sperm morphology) after 96 hours of storage had correlations with fertility in vivo, which can be used to identify subfertile males. The evaluations were performed on 30 ejaculates regarding the means of BR and PB, considering only those who had at least 7 females inseminated. There were no correlations between the motility assessments and semen morphological changes and the reproductive parameters evaluated. The results obtained in vivo, referring to BR and PB, demonstrated that it was possible to identify differences between males, the individual (M32) had the worst results for the percentages of BR and PB. It is concluded that there are males of high and low fertility and that only the in vitro analyzes carried out in this study are not enough to categorize them, however, the evaluation of retrospective data was efficient for this purpose.

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LIF, the ES-cell inhibition factor, reversibly blocks nephrogenesis in cultured mouse kidney rudiments

Development ◽

10.1242/dev.113.1.193 ◽

1991 ◽

Vol 113 (1) ◽

pp. 193-198 ◽

Cited By ~ 3

Author(s):

J.B. Bard ◽

A.S. Ross

Keyword(s):

Embryonic Stem ◽

Mouse Kidney ◽

Leukaemia Inhibitory Factor ◽

Es Cell ◽

Metanephric Mesenchyme ◽

Ureteric Bud ◽

Cell Inhibition ◽

Striking Effect

Mouse kidney induction proceeds in vitro much as it does in vivo: the ureteric bud bifurcates to give collecting ducts while the mesenchyme condenses into aggregates which epithelialise and then elongate into tubules with glomerular and other nephron structures. We report here that the factor known as LIF (leukaemia inhibitory factor), which regulates the differentiation and growth of embryonic-stem (ES) and other cells in culture, has little effect in vitro on growth or on ureteric-bud morphogenesis other than to stimulate the bifurcation process. It does however exert a striking effect on the mesenchyme. At about four times the concentration required to inhibit ES-cell differentiation, LIF strongly but reversibly blocks the effects of metanephric mesenchyme induction: although mesenchyme condenses around growing duct tips, the number of mature nephrons that form over 6 days is reduced by 75% or more. The few nephrons that do develop in the presence of LIF probably come from mesenchyme already induced at the time of culture and are indistinguishable from those that form in controls as assayed by morphology, by X-gal staining of endogenous galactosidase and by antibodies to brush-border and CD15 antigens. There is a further unexpected feature of rudiments cultured in LIF which is absent in controls: they contain an unexpectedly high number of stable epithelialised aggregates that express laminin around their periphery and which do not develop further. These results argue that the process of nephrogenesis involves at least two distinct stages which can be blocked by LIF: the effect of the initial induction and the future development of epithelialised aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

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The metanephric blastema differentiates into collecting system and nephron epithelia in vitro

Development ◽

10.1242/dev.121.10.3207 ◽

1995 ◽

Vol 121 (10) ◽

pp. 3207-3214 ◽

Cited By ~ 5

Author(s):

J. Qiao ◽

D. Cohen ◽

D. Herzlinger

Keyword(s):

Cell Fate ◽

Mesenchymal Cells ◽

Lacz Gene ◽

Metanephric Mesenchyme ◽

Ureteric Bud ◽

Ureteric Buds ◽

Collecting System ◽

Metanephric Blastema

The kidney forms from two tissue populations derived from intermediate mesoderm, the ureteric bud and metanephric mesenchyme. It is currently accepted that metanephric mesenchyme is committed to differentiating into nephrons while the ureteric bud is restricted to forming the renal collecting system. To test this hypothesis, we transferred lacZ into pure metanephric mesenchyme isolated from gestation day 13 rat embryos. The fate of tagged mesenchymal cells and their progeny was characterized after co-culture with isolated ureteric buds. When induced to differentiate by the native inducer of kidney morphogenesis, lineage-tagged mesenchymal cells exhibit the potential to differentiate into collecting system epithelia, in addition to nephrons. The fate of cells deriving from isolated ureteric buds was also examined and results of these lacZ gene transfer experiments indicate that the majority of ureteric bud cells differentiate into the renal collecting system. These cell fate studies combined with in situ morphological observations raise the possibility that collecting system morphogenesis in vivo occurs by growth of the ureteric bud and recruitment of mesenchymal cells from the metanephric blastema. Thus, metanephric mesenchyme may be a pluripotent renal stem population.

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OrganIn VitroCulture: What Have We Learned about Early Kidney Development?

Stem Cells International ◽

10.1155/2015/959807 ◽

2015 ◽

Vol 2015 ◽

pp. 1-16 ◽

Cited By ~ 22

Author(s):

Aleksandra Rak-Raszewska ◽

Peter V. Hauser ◽

Seppo Vainio

Keyword(s):

Regenerative Medicine ◽

Kidney Development ◽

Ex Vivo ◽

Renal Development ◽

Metanephric Mesenchyme ◽

Ureteric Bud ◽

Chemical Inducers ◽

Open Platform ◽

Artificial Environment

When Clifford Grobstein set out to study the inductive interaction between tissues in the developing embryo, he developed a method that remained important for the study of renal development until now. From the late 1950s on,in vitrocultivation of the metanephric kidney became a standard method. It provided an artificial environment that served as an open platform to study organogenesis. This review provides an introduction to the technique of organ culture, describes how the Grobstein assay and its variants have been used to study aspects of mesenchymal induction, and describes the search for natural and chemical inducers of the metanephric mesenchyme. The review also focuses on renal development, starting with ectopic budding of the ureteric bud, ureteric bud branching, and the generation of the nephron and presents the search for stem cells and renal progenitor cells that contribute to specific structures and tissues during renal development. It also presents the current use of Grobstein assay and its modifications in regenerative medicine and tissue engineering today. Together, this review highlights the importance ofex vivokidney studies as a way to acquire new knowledge, which in the future can and will be implemented for developmental biology and regenerative medicine applications.

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Hoxa11andHoxd11regulate branching morphogenesis of the ureteric bud in the developing kidney

Development ◽

10.1242/dev.128.11.2153 ◽

2001 ◽

Vol 128 (11) ◽

pp. 2153-2161 ◽

Cited By ~ 3

Author(s):

Larry T. Patterson ◽

Martina Pembaur ◽

S. Steven Potter

Keyword(s):

Gene Expression ◽

Kidney Development ◽

Growth Failure ◽

Immunohistochemical Analysis ◽

Branching Morphogenesis ◽

Metanephric Mesenchyme ◽

Ureteric Bud ◽

Metanephric Kidney ◽

Normal Gene ◽

Ureteric Buds

Hoxa11 and Hoxd11 are functionally redundant during kidney development. Mice with homozygous null mutation of either gene have normal kidneys, but double mutants have rudimentary, or in extreme cases, absent kidneys. We have examined the mechanism for renal growth failure in this mouse model and find defects in ureteric bud branching morphogenesis. The ureteric buds are either unbranched or have an atypical pattern characterized by lack of terminal branches in the midventral renal cortex. The mutant embryos show that Hoxa11 and Hoxd11 control development of a dorsoventral renal axis. By immunohistochemical analysis, Hoxa11 expression is restricted to the early metanephric mesenchyme, which induces ureteric bud formation and branching. It is not found in the ureteric bud. This suggests that the branching defect had been caused by failure of mesenchyme to epithelium signaling. In situ hybridizations with Wnt7b, a marker of the metanephric kidney, show that the branching defect was not simply the result of homeotic transformation of metanephros to mesonephros. Absent Bf2 and Gdnf expression in the midventral mesenchyme, findings that could by themselves account for branching defects, shows that Hoxa11 and Hoxd11 are necessary for normal gene expression in the ventral mesenchyme. Attenuation of normal gene expression along with the absence of a detectable proliferative or apoptotic change in the mutants show that one function of Hoxa11 and Hoxd11 in the developing renal mesenchyme is to regulate differentiation necessary for mesenchymal-epithelial reciprocal inductive interactions.

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Signalling Alterations in Bones of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Gene Deficient Mice

International Journal of Molecular Sciences ◽

10.3390/ijms19092538 ◽

2018 ◽

Vol 19 (9) ◽

pp. 2538 ◽

Cited By ~ 8

Author(s):

Gergő Józsa ◽

Vince Szegeczki ◽

Andrea Pálfi ◽

Tamás Kiss ◽

Zsuzsanna Helyes ◽

...

Keyword(s):

Bone Formation ◽

Adenylate Cyclase ◽

Morphological Changes ◽

Bone Matrix ◽

Transverse Diameter ◽

Type I ◽

Pituitary Adenylate Cyclase ◽

Deficient Mice

: Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with diverse developmental roles, including differentiation of skeletal elements. It is a positive regulatory factor of chondrogenesis and osteogenic differentiation in vitro, but little is known about its in vivo role in bone formation. In our experiments, diaphyses of long bones from hind limbs of PACAP gene-deficient mice showed changes in thickness and increased staining intensity. Our main goal was to perform a detailed morphological and molecular biological analysis of femurs from PACAP knockout (KO) and wild type (WT) mice. Transverse diameter and anterior cortical bone thickness of KO femurs showed significant alterations with disturbed Ca2+ accumulation and collagen type I expression. Higher expression and activity of alkaline phosphatase were also observed, accompanied by increased fragility PACAP KO femurs. Increased expression of the elements of bone morphogenic protein (BMP) and hedgehog signalling was also observed, and are possibly responsible for the compensation mechanism accounting for the slight morphological changes. In summary, our results show that lack of PACAP influences molecular and biomechanical properties of bone matrix, activating various signalling cascade changes in a compensatory fashion. The increased fragility of PACAP KO femur further supports the role of endogenous PACAP in in vivo bone formation.

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Matrix metalloproteinases and their inhibitors regulate in vitro ureteric bud branching morphogenesis

AJP Renal Physiology ◽

10.1152/ajprenal.2000.279.5.f891 ◽

2000 ◽

Vol 279 (5) ◽

pp. F891-F900 ◽

Cited By ~ 35

Author(s):

Martin Pohl ◽

Hiroyuki Sakurai ◽

Kevin T. Bush ◽

Sanjay K. Nigam

Keyword(s):

Conditioned Medium ◽

Kidney Development ◽

Gelatin Zymography ◽

Branching Morphogenesis ◽

Complex Model ◽

Northern Analysis ◽

Metanephric Mesenchyme ◽

Ureteric Bud ◽

Direct Role

Mammalian kidney development is initiated by the mutual interaction between embryonic metanephric mesenchyme (MM) and the ureteric bud (UB), leading to tightly controlled UB branching morphogenesis. In a three-dimensional cell culture model, which employs MM cell-derived conditioned medium (BSN-CM) to induce UB cell branching morphogenesis in extracellular matrix (ECM) gels (Sakurai H, Barros EJ, Tsukamoto T, Barasch J, and Nigam SK. Proc Natl Acad Sci USA 94: 6279–6284, 1997), branching morphogenesis was inhibited by both chemical agents (ilomastat and 1,10-orthophenanthroline) and a physiological protein factor [tissue inhibitor of metalloproteinases (TIMP)-2], known to act as matrix metalloproteinase (MMP) inhibitors. In addition, UB branching was inhibited in isolated UB culture (Qiao J, Sakurai H, and Nigam SK. Proc Natl Acad Sci USA96: 7330–7335, 1999) by TIMP-2 and ilomastat, suggesting a direct role for MMPs in UB branching. Gelatin zymography and enzymatic measurement of MMP activity revealed that MMPs could originate from at least three different sources: the conditioned medium, the ECM, and the UB cells themselves. In the UB cells, transcription of several MMPs [gelatinase A (MMP2) and B (MMP9), stromelysin (MMP3), MT1-MMP] and TIMPs was altered by BSN-CM and changed as more complex branching structures formed. The ECM appeared to serve as both a reservoir for MMPs and modulated their expression because different ECM compositions altered the total MMP activity as well as specific subsets of MMPs expressed by the UB cells (as determined by zymography and Northern analysis). In the context of UB branching morphogenesis during kidney development, our data suggest a complex model in which soluble factors produced by the MM, in the context of specific ECM components, modulate the expression of specific subsets of MMPs and TIMPs in the UB, which alter as structures develop and the matrix environment changes. This suggests distinct roles for different subsets of MMPs and their inhibitors during different phases of branching morphogenesis.

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The Venom of the Ectoparasitoid Wasp Pachycrepoideus vindemiae (Hymenoptera: Pteromalidae) Induces Apoptosis of Drosophila melanogaster Hemocytes

Insects ◽

10.3390/insects11060363 ◽

2020 ◽

Vol 11 (6) ◽

pp. 363

Author(s):

Bin Wan ◽

Lei Yang ◽

Jiao Zhang ◽

Liming Qiu ◽

Qi Fang ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Morphological Changes ◽

Venom Component ◽

Limited Effect ◽

Induced Apoptosis ◽

The Individual ◽

Almost All ◽

Crystal Cells

The pupal ectoparasitoid Pachycrepoideus vindemiae injects venom into its fly hosts prior to oviposition. We have shown that this venom causes immune suppression in Drosophila melanogaster pupa but the mechanism involved remained unclear. Here, we show using transgenic D. melanogaster with fluorescent hemocytes that the in vivo number of plasmatocytes and lamellocytes decreases after envenomation while it has a limited effect on crystal cells. After in vitro incubation with venom, the cytoskeleton of plasmatocytes underwent rearrangement with actin aggregation around the internal vacuoles, which increased with incubation time and venom concentration. The venom also decreased the lamellocytes adhesion capacity and induced nucleus fragmentation. Electron microscopy observation revealed that the shape of the nucleus and mitochondria became irregular after in vivo incubation with venom and confirmed the increased vacuolization with the formation of autophagosomes-like structures. Almost all venom-treated hemocytes became positive for TUNEL assays, indicating massive induced apoptosis. In support, the caspase inhibitor Z-VAD-FMK attenuated the venom-induced morphological changes suggesting an involvement of caspases. Our data indicate that P. vindemiae venom inhibits D. melanogaster host immunity by inducing strong apoptosis in hemocytes. These assays will help identify the individual venom component(s) responsible and the precise mechanism(s)/pathway(s) involved.

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Dynamic regulation of sphingosine-1-phosphate homeostasis during development of mouse metanephric kidney

AJP Renal Physiology ◽

10.1152/ajprenal.90232.2008 ◽

2009 ◽

Vol 296 (3) ◽

pp. F634-F641 ◽

Cited By ~ 8

Author(s):

R. Jason Kirby ◽

Ying Jin ◽

Jian Fu ◽

Jimena Cubillos ◽

Debi Swertfeger ◽

...

Keyword(s):

Kinase Inhibitors ◽

Kidney Development ◽

Sphingosine Kinase ◽

Branching Morphogenesis ◽

Lipid Mediator ◽

Metanephric Mesenchyme ◽

Ureteric Bud ◽

Dynamic Regulation ◽

Sphingosine 1 Phosphate ◽

Metanephric Kidney

Branching morphogenesis of the metanephric kidney is critically dependent on the delicate orchestration of diverse cellular processes including proliferation, apoptosis, migration, and differentiation. Sphingosine-1-phosphate (S1P) is a potent lipid mediator influencing many of these cellular events. We report increased expression and activity of both sphingosine kinases and S1P phosphatases during development of the mouse metanephric kidney from induction at embryonic day 11.5 to maturity. Sphingosine kinase activity exceeded S1P phosphatase activity in embryonic kidneys, resulting in a net accumulation of S1P, while kinase and phosphatase activities were similar in adult tissue, resulting in reduced S1P content. Sphingosine kinase expression was greater in the metanephric mesenchyme than in the ureteric bud, while the S1P phosphatase SPP2 was expressed at greater levels in the ureteric bud. Treatment of cultured embryonic kidneys with sphingosine kinase inhibitors resulted in a dose-dependent reduction of ureteric bud tip numbers and increased apoptosis. Exogenous S1P rescued kidneys from apoptosis induced by kinase inhibitors. Ureteric bud tip number was unaffected by exogenous S1P in kidneys treated with N, N-dimethylsphingosine, although tip number increased in those treated with d,l- threo-dihydrosphingosine. S1P1 and S1P2 were the predominant S1P receptors expressed in the embryonic kidney. S1P1 expression increased during renal development while expression of S1P2 decreased, and both receptors were expressed predominantly in the metanephric mesenchyme. These results demonstrate dynamic regulation of S1P homeostasis during renal morphogenesis and suggest that differential expression of S1P metabolic enzymes and receptors provides a novel mechanism contributing to the regulation of kidney development.

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