Disruption of polycystin-1 function interferes with branching morphogenesis of the ureteric bud in developing mouse kidneys

The polycystic kidney disease (PKD1) gene-encoded protein, polycystin-1, is developmentally regulated, with highest expression levels seen in normal developing kidneys, where it is distributed in a punctate pattern at the basal surface of ureteric bud epithelia. Overexpression in ureteric epithelial cell membranes of an inhibitory pMyr-GFP-PKD1 fusion protein via a retroviral (VVC) delivery system and microinjection into the ureteric bud lumen of embryonic day 11 mouse metanephric kidneys resulted in disrupted branching morphogenesis. Using confocal quantitative analysis, significant reductions were measured in the numbers of ureteric bud branch points and tips, as well as in the total ureteric bud length, volume and area, while significant increases were seen as dilations of the terminal branches, where significant increases in outer diameter and volumes were measured. Microinjection of an activating 5TM-GFP-PKD1 fusion protein had an opposite effect and showed significant increases in ureteric bud length and area. These are the first studies to experimentally manipulate polycystin-1 expression by transduction in the embryonic mouse kidney and suggest that polycystin-1 plays a critical role in the regulation of epithelial morphogenesis during renal development.     

Real-time analysis of ureteric bud branching morphogenesis in vitro

While it is clear that the normal branching morphogenesis of the ureteric bud (UB) is critical for development of the metanephric kidney, the specific patterns of branching and growth have heretofore only been inferred from static images. Here, we present a systematic time-lapse analysis of UB branching morphogenesis during the early development of the mouse kidney in organ culture. Metanephric primordia from Hoxb7/GFP transgenic embryos were cultured for 3-4 days, and GFP images of the UB taken every 30 min were assembled into movies. Analysis of these movies (available as )revealed that the UB is a highly plastic structure, which can branch in a variety of complex patterns, including terminal bifid, terminal trifid, and lateral branching. To examine kinetic parameters of branching and elongation, skeletal representations of the UB were used to measure the number of segments and branch points and the length of each segment as a function of time and of branch generation. These measurements provide a baseline for future studies on mutant kidneys with defects in renal development. To illustrate how these quantitative methods can be applied to the analysis of abnormal kidney development, we examined the effects of the MEK1 inhibitor PD98059 on renal organ cultures and confirmed a previous report that the drug has a specific inhibitory effect on UB branching as opposed to elongation.     

Low Ambient O2 Enhances Ureteric Bud Branching In Vitro

Hypoxia exists widely in developing embryos where it may regulate blood vessel formation. VEGF and FGF2 produced in developing renal primordia (metanephroi) stimulate microvessel formation from embryonic thoracic aorta cultured under hypoxic conditions (HC) relative to room air (RA). The aim of the present study was to provide insight into the participation of hypoxia in a process that occurs concomitant with metanephros vascularization in vivo, ureteric bud (UB) branching. To this end, the arborization of the UB and growth of metanephroi were measured in metanephroi grown in serum-free organ culture for 2 days under RA or HC. When metanephroi were cultured under HC the arborization of UB was stimulated relative to RA. In the presence of anti-VEGF neutralizing antibody (amVEGF), or anti-FGF2 neutralizing antibody (ahFGF2) UB branching was inhibited under both RA and HC. When both amVEGF and ahFGF2 were added, the inhibition was enhanced. Addition of exogenous VEGF or FGF2 to cultures stimulated UB branching under RA and HC and addition of both stimulated it further. These findings provide evidence for roles of hypoxia and metanephric VEGF and FGF2, as regulators not only for vascularization but also for UB bud branching during renal organogenesis.     

Low Ambient O2 Enhances Ureteric Bud Branching In Vitro

Hypoxia exists widely in developing embryos where it may regulate blood vessel formation. VEGF and FGF2 produced in developing renal primordia (metanephroi) stimulate microvessel formation from embryonic thoracic aorta cultured under hypoxic conditions (HC) relative to room air (RA). The aim of the present study was to provide insight into the participation of hypoxia in a process that occurs concomitant with metanephros vascularization in vivo, ureteric bud (UB) branching. To this end, the arborization of the UB and growth of metanephroi were measured in metanephroi grown in serum-free organ culture for two days under RA or HC. When metanephroi were cultured under HC the arborization of UB was stimulated relative to RA. In the presence of anti-VEGF neutralizing antibody (αmVEGF), or anti-FGF2 neutralizing antibody (αhFGF2) UB branching was inhibited under both RA and HC. When both αmVEGF and αhFGF2 were added, the inhibition was enhanced. Addition of exogenous VEGF or FGF2 to cultures stimulated UB branching under RA and HC and addition of both stimulated it further. These findings provide evidence for roles of hypoxia and metanephric VEGF and FGF2, as regulators not only for vascularization but also for UB bud branching during renal organogenesis.Key Words: metanephroi, embryogenesis, fibroblast growth factor, vascular endothelial growth factor     

Novel expression and regulation of the renin-angiotensin system in metanephric organ culture

To evaluate the presence and regulation of the renin-angiotensin system (RAS) in metanephric organ culture, embryonic day 14 (E14) rat metanephroi were cultured for 6 days. mRNAs for renin and both ANG II receptors (AT(1) and AT(2)) are expressed at E14, and all three genes continue to be expressed in culture. Renin mRNA is localized to developing tubules and ureteral branches in the cultured explants. At E14, renin immunostaining is found in isolated cells scattered within the mesenchyme. As differentiation progresses, renin localizes to the ureteric epithelium, developing tubules and glomeruli. E14 metanephroi contain ANG II, and peptide production persists in culture. Renin activity is present at E14 (6.13 +/- 0.61 pg ANG I. kidney(-1). h(-1)) and in cultured explants (28.84 +/- 1. 13 pg ANG I. kidney(-1). h(-1)). Renin activity in explants is increased by ANG II treatment (70.1 +/- 6.36 vs. 40.97 +/- 1.94 pg ANG I. kidney(-1). h(-1) in control). This increase is prevented by AT(1) blockade, whereas AT(2) antagonism has no effect. These studies document an operational local RAS and a previously undescribed positive-feedback mechanism for renin generation in avascular, cultured developing metanephroi. This novel expression pattern and regulatory mechanism highlight the unique ability of developing renal cells to express an active RAS.     

Sema4C-Plexin B2 signalling modulates ureteric branching in developing kidney

Semaphorins, originally identified as axon guidance molecules, have also been implicated in angiogenesis, function of the immune system and cancerous growth. Here we show that deletion of Plexin B2 (Plxnb2), a semaphorin receptor that is expressed both in the pretubular aggregates and the ureteric epithelium in the developing kidney, results in renal hypoplasia and occasional double ureters. The rate of cell proliferation in the ureteric epithelium and consequently the number of ureteric tips are reduced in the kidneys lacking Plexin B2 (Plxnb2-/-). Semaphorin 4C, a ligand for Plexin B2, stimulates branching of the ureteric epithelium in wild type and Plxnb2+/- kidney explants, but not in Plxnb2-/- explants. As shown by co-immunoprecipitation Plexin B2 interacts with the Ret receptor tyrosine kinase, the receptor of Glial-cell-line-derived neurotrophic factor (Gdnf), in embryonic kidneys. Isolated Plxnb2-/- ureteric buds fail to respond to Gdnf by branching, but this response is rescued by Fibroblast growth factor 7 and Follistatin as well as by the metanephric mesenchyme. The differentiation of the nephrogenic mesenchyme, its morphology and the rate of apoptosis in the Plxnb2-/- kidneys are normal. Plexin B2 is co-expressed with Plexin B1 (Plxnb1) in the kidney. The double homozygous Plxnb1-Plxnb2-deficient mice show high embryonic lethality prior to onset of nephrogenesis. The only double homozygous embryo surviving to E12 showed hypoplastic kidneys with ureteric branches and differentiating mesenchyme. Taken together, our results show that Sema4C-Plexin B2 signalling regulates ureteric branching, possibly through modulation of Gdnf signalling by interaction with Ret, and suggest non-redundant roles for Plexin B1 and Plexin B2 in kidney development.     

Independent roles of Fgfr2 and Frs2alpha in ureteric epithelium

Mice with conditional deletion of fibroblast growth factor receptor 2 (Fgfr2) in the ureteric bud using a Hoxb7cre line (Fgfr2(UB-/-)) develop severe ureteric branching defects; however, ureteric deletion of fibroblast growth factor receptor substrate 2α (Frs2α), a key docking protein that transmits fibroblast growth factor receptor intracellular signaling (Frs2α(UB-/-)) leads to mild ureteric defects. Mice with point mutations in the Frs2α binding site of Fgfr2 (Fgfr2(LR/LR)) have normal kidneys. The aim of this study was to determine the relationship between Fgfr2 and Frs2α in the ureteric lineage. Mice with ureteric deletion of both Fgfr2 and Frs2α (Fgfr2/Frs2α(UB-/)) were compared with Frs2α(UB-/-) and Fgfr2(UB-/-) mice. To avoid potential rescue of Fgfr1 forming heterodimers with Fgfr2(LR) alleles to recruit Frs2α, compound mutant mice were generated with ureteric deletion of Fgfr1 and with Fgfr2(LR/LR) point mutations (Fgfr1(UB-/-)Fgfr2(LR/LR)). At E13.5, three-dimensional reconstructions and histological assessment showed that, whereas Fgfr2(UB-/-) kidneys had more severe ureteric branching defects than Frs2α(UB-/-), Fgfr2(UB-/-) kidneys were indistinguishable from Fgfr2/Frs2α(UB-/-). At later stages, however, Fgfr2/Frs2α(UB-/-) kidneys were more severely affected than either Fgfr2(UB-/-) or Frs2α(UB-/-) kidneys. Taken together, although Fgfr2 and Frs2α have crucial roles in the ureteric lineage, they appear to act separately and additively.     

Patterning parameters associated with the branching of the ureteric bud regulated by epithelial-mesenchymal interactions

The mechanisms by which the branching of epithelial tissue occurs and is regulated to generate different organ structures are not well understood. In this work, image analyses of the organ rudiments demonstrate specific epithelial branching patterns for the early lung and kidney; the lung type typically generating several side branches, whereas kidney branching was mainly dichotomous. Parameters such as the number of epithelial tips, the angle of the first branch, the position index of the first branch (PIFB) in a module, and the percentage of epithelial module type (PMT) were analysed. The branching patterns in the cultured lung and kidney, and in homotypic tissue recombinants recapitulated their early in vivo branching patterns. The parameters were applied to heterotypic tissue recombinants between lung mesenchyme and ureteric bud, and tip number, PIFB and PMT values qualified the change in ureter morphogenesis and the reprogramming of the ureteric bud with lung mesenchyme. All the values for the heterotypic recombinant between ureteric bud and lung mesenchyme were significantly different from those for kidney samples but similar to those of the lung samples. Hence, lung mesenchyme can instruct the ureteric bud to undergo aspects of early lung-type epithelial morphogenesis. Different areas of the lung mesenchyme, except the tracheal region, were sufficient to promote ureteric bud growth and branching. In conclusion, our findings provide morphogenetic parameters for monitoring epithelial development in early embryonic lung and kidney and demonstrate the use of heterotypic tissue recombinants as a model for studying tissue-specific epithelial branching during organogenesis.     

Effect of NSAIDs and Diuretics on Nephrogenesis in an Ex Vivo Embryogenic Kidney Model

The kidney is one of the key organs in clearing foreign compounds. The effects of drugs on the developing kidney are relatively unknown. We studied the direct effect of furosemide, hydrochlorothiazide, ibuprofen, and indomethacin on kidney development in an ex vivo embryonic kidney model. At embryonic day 13, metanephroi were dissected from mice and cultured in control media or media supplemented with various clinically relevant concentrations of drugs. The ureteric tree was visualized by whole‐mount staining and branching was evaluated by counting. Additionally, gene expression levels of Wt1, Sox9, Bmp7, Fgf8, and Gdnf were investigated. No distinct differences were noted on either ureteric tip development or gene expression analysis for each drug after 24 hr of exposure. Even though short‐term exposure to clinically relevant concentrations seems not to disturb renal development, future research is needed to study prolonged or repeated exposures.     

A transgenic mouse that reveals cell shape and arrangement during ureteric bud branching

Understanding the cellular events that underlie epithelial morphogenesis is a key problem in developmental biology. Here, we describe a new transgenic mouse line that makes it possible to visualize individual cells specifically in the Wolffian duct and ureteric bud, the epithelial structures that give rise to the collecting system of the kidney. myr‐Venus, a membrane‐associated form of the fluorescent protein Venus, was expressed in the ureteric bud lineage under the control of the Hoxb7 promoter. In Hoxb7/myr‐Venus mice, the outlines of all Wolffian duct and ureteric bud epithelial cells are strongly labeled at all stages of urogenital development, allowing the shapes and arrangements of individual cells to be readily observed by confocal microscopy of freshly excised or cultured kidneys. This strain should be extremely useful for studies of cell behavior during ureteric bud branching morphogenesis in wild type and mutant mouse lines. genesis 47:61–66, 2009. © 2008 Wiley‐Liss, Inc.     

TGF-beta superfamily members modulate growth, branching, shaping, and patterning of the ureteric bud

Protein-rich fractions inhibitory for isolated ureteric bud (UB) growth were separated from a conditioned medium secreted by cells derived from the metanephric mesenchyme (MM). Elution profiles and immunoblotting indicated the presence of members of the transforming growth factor-beta (TGF-beta) superfamily. Treatment of cultured whole embryonic kidney with BMP2, BMP4, activin, or TGF-beta1 leads to statistically significant differences in the overall size of the kidney, the number of UB branches, the length and angle of the branches, as well as in the thickness of the UB stalks. Thus, the pattern of the ureteric tree is altered. LIF, however, appeared to have only minimal effect on growth and development of the whole embryonic kidney in organ culture. The factors all directly inhibited, in a concentration-dependent fashion, the growth and branching of the isolated UB, albeit to different extents. Antagonists of some of these factors reduced their inhibitory effect. Detailed examination of TGF-beta1-treated UBs revealed only a slight increase in the amount of apoptosis in tips by TUNEL staining, but diminished proliferation throughout by Ki67 staining. These data suggest an important direct modulatory role for BMP2, BMP4, LIF, TGF-beta1, and activin (as well as their antagonists) on growth and branching of the UB, possibly in shaping the growing UB by playing a role in determining the number of branches, as well as where and how the branches occur. In support of this notion, UBs cultured in the presence of fibroblast growth factor 7 (FGF7), which induces the formation of globular structures with little distinction between the stalk and ampullae [Mech. Dev. 109 (2001) 123], and TGF-beta superfamily members lead to the formation of UBs with clear stalks and ampullae. This indicates that positive (i.e., growth and branch promoting) and negative (i.e., growth and branch inhibiting) modulators of UB morphogenesis can cooperate in the formation of slender arborized UB structures similar to those observed in the intact developing kidney or in whole embryonic kidney organ culture. Finally, purification data also indicate the presence of an as yet unidentified soluble non-heparin-binding activity modulating UB growth and branching. The data suggest how contributions of positive and negative growth factors can together (perhaps as local bipolar morphogenetic gradients existing within the mesenchyme) modulate the vectoral arborization pattern of the UB and shape branches as they develop, thereby regulating both nephron number and tubule/duct caliber. We suggest that TGF-beta-like molecules and other non-heparin-binding inhibitory factors can, in the appropriate matrix context, facilitate "braking" of the branching program as the UB shifts from a rapid branching stage (governed by a feed-forward mechanism) to a stage where branching slows down (negative feedback) and eventually stops.     

The tyrosine phosphatase Shp2 acts downstream of GDNF/Ret in branching morphogenesis of the developing mouse kidney

The tyrosine phosphatase Shp2 acts downstream of various growth factors, hormones or cytokine receptors. Mutations of the Shp2 gene are associated with several human diseases. Here we have ablated Shp2 in the developing kidneys of mice, using the ureteric bud epithelium-specific Hoxb7/Cre. Mutant mice produced a phenotype that is similar to mutations of the genes of the GDNF/Ret receptor system, that is: strongly reduced ureteric bud branching and downregulation of the Ret target genes Etv4 and Etv5. Shp2 mutant embryonic kidneys also displayed reduced cell proliferation at the branch tips and branching defects, which could not be overcome by GDNF in organ culture. We also examined compound mutants of Shp2 and Sprouty1, which is an inhibitor of receptor tyrosine kinase signaling in the kidney. Sprouty1 single mutants produce supernumerary ureteric buds, which branch excessively. Sprouty1 mutants rescued branching deficits in Ret^−/− and GDNF^−/− kidneys. Sprouty1; Shp2 double mutants showed no rescue of kidney branching. Our data thus indicate an intricate interplay of Shp2 and Sprouty1 in signaling downstream of receptor tyrosine kinases during kidney development. Apparently, Shp2 mediates not only GDNF/Ret but also signaling by other receptor tyrosine kinases in branching morphogenesis of the embryonic kidney.     

Retinal Vessel Width Measurement at Branchings Using an Improved Electric Field Theory-Based Graph Approach

The retinal vessel width relationship at vessel branch points in fundus images is an important biomarker of retinal and systemic disease. We propose a fully automatic method to measure the vessel widths at branch points in fundus images. The method is a graph-based method, in which a graph construction method based on electric field theory is applied which specifically deals with complex branching patterns. The vessel centerline image is used as the initial segmentation of the graph. Branching points are detected on the vessel centerline image using a set of detection kernels. Crossing points are distinguished from branch points and excluded. The electric field based graph method is applied to construct the graph. This method is inspired by the non-intersecting force lines in an electric field. At last, the method is further improved to give a consistent vessel width measurement for the whole vessel tree. The algorithm was validated on 100 artery branchings and 100 vein branchings selected from 50 fundus images by comparing with vessel width measurements from two human experts.     

Vascularization of the brains of the Atlantic and Pacific hagfishes, Myxine glutinosa and Eptatretus stouti: a scanning electron microscope study of vascular corrosion casts

The microvascularization of the brains of the hagfishes, Myxine glutinosa L. and Eptatretus stouti, were studied by scanning electron microscopy (SEM) of microvascular corrosion casts. Sections of these casts were used to determine the vascular territories of defined brain areas. Histological serial sections (10 microm) of the brains served for correlation of findings. Analysis of the microvascular casts of both species revealed that the blood supply to and from these brains arose ventrally and dorsally, respectively. Neither species possesses an arterial circle (Circulus Willisi) and both have similar microvascular patterns. The only difference between Myxine and Eptatretus was that the posterior cerebral artery in Myxine divides into mesencephalic and rhombencephalic branches, and in Eptatretus a third branch, termed telencephalic branch, arises from the posterior cerebral artery. 3D-morphometry revealed that luminal diameters of: 1) intracerebral arteries and arterioles range from 35.11 +/- 5.66 microm (mean +/- SEM) in the hypothalamus to 92.69 +/- 14.48 microm in the thalamus; 2) capillaries range from 17.8 +/- 0.44 microm in the olfactory bulb to 21.70 +/- 0.87 microm in the basal ganglia; and 3) intracerebral venules and veins range from 49.38 +/- 4.17 microm in the hypothalamus to 75.58 +/- 6.59 microm in the rhombencephalon. Interbranching distances of arteries and arterioles range from 179.19 +/- 11.32 microm in the optic tectum to 235.19 +/- 94.64 microm in the hypothalamus. Capillaries range from 91.07 +/- 6.22 microm in the hypothalamus to 116.15 +/- 9.45 microm in the thalamus, and venules and veins range from 137.30 +/- 18.11 microm in the hypothalamus to 189.83 +/- 17.47 microm in the optic tectum. Intervascular distances range from 70.58 +/- 3.58 microm in the olfactory bulb to 89.52 +/- 5.74 microm in the optic tectum. Branching angles of arteries and arterioles range from 38.39 +/- 10.9 degrees in the olfactory bulb to 100.73 +/- 9.4 degrees in the optic tectum, and the branching angles of capillaries range from 74.40 +/- 5.42 degrees in the optic tectum to 90.24 +/- 4.66 degrees in the olfactory bulb. Finally, the branching angles of the venules and veins range from 67.84 +/- 6.83 degrees in the tegmentum of the mesencephalon to 92.30 +/- 6.35 degrees in the optic tectum.     

Vertex analysis of Purkinje cell dendritic trees in the cerebellum of the rat

All networks are made up of vertices (points interconnected by segments), which include terminals interconnected by terminal segments, nodes interconnected by link segments and the root point connected to the tree by the root segment. All nodes may be classified into unique types according to the number of terminal and link segments they drain. For example, there are three distinct dichotomous nodes, a 'primary' node draining two terminal segments, a 'secondary' node draining one terminal segment and a link segment, and a 'tertiary' node draining two link segments. The numbers of primary and tertiary nodes approximate to equality in large networks and thus the ratio of primary to secondary nodes defines topology. All higher order nodes ( trichotomous and beyond) may be resolved into dichotomous forms and incorporated into the analysis. Different forms of growth may thus be analysed by comparing the frequency distributions of nodes with those generated by computer simulated growth models. Moreover, all vertices can be ordered so that metrical parameters are easily incorporated and the hierarchical arrangements of vertices of different order discerned. The dendritic trees of 48 Purkinje cells, taken from folia along the primary fissure, were analysed using vertex analysis. The mean number of segments in Purkinje cell trees was 881 +/- 23 (s.e.) and mean total dendritic length 7959 +/- 233 (s.e.) micrometers. Segment lengths were longest over proximal segments but over most of the tree segment lengths were constant at 10 +/- 0.2 (s.e.) micrometers. Vertex, segment and terminal frequency distributions of equivalent orders were all normal with a slight positive skew. Peak frequencies were recorded at the 12th equivalent order. The mean primary/secondary nodal vertex ratio was 0.93 and the proportion of trichotomous branch points in the tree was 5%. Comparison of the frequency distribution of all vertices with computer generated models showed that growth of the Purkinje cell was most closely simulated by a random terminal growth model, incorporating 5% trichotomy , in which the branching of high order terminals was more likely than low order terminals. It was concluded that growth of the Purkinje cell tree could proceed by random terminal branching with growth occurring preferentially over a front composed of terminals that are ascending through a corridor in the molecular layer whose margins are defined by neighbouring trees.     

PS-100 and NF 70-200 double immunolabeling for human digital skin meissner corpuscle 3D imaging.

For detailed study of complex structures such as corpuscular mechanoreceptors, confocal microscopy can be used with multiple immunolabeling that identifies specifically different subcomponents. In addition, anatomic interpretation is enhanced by three-dimensional reconstruction. Confocal laser micrographs, reconstructed from serial images 1 microm thick of human skin Meissner corpuscles simultaneously immunostained for neurofilaments (NF 70-200) and protein S-100 (PS-100), clearly reveal the complex 3D relationship between Schwann-related lamellar cells immunoreactive for PS-100 and the nerve fibers marked by NF 70-200. The nerve fiber, after branching into the corpuscle, divides into several ramifications, presenting discoidal expansions and flattened fringed sections. The mean nerve diameter was 4 microm +/- 1 (2-5 microm) and the mean size of the discoidal expansions was 15 microm +/- 1 (7-30 microm). Corpuscle size varied from 30-140 +/- 1 microm in length and from 20-60 +/- 1 microm in diameter. This study confirms the presence of neural discoidal areas in Meissner's corpuscles, which are probably involved to some extent with the transduction process. Despite the accuracy of immunolabeling and imaging, an extracorpuscular neural network was never observed in the vicinity of corpuscles, thus giving doubt as to their existence. (J Histochem Cytochem 48:295-302, 2000)     

Renal collecting system growth and function depend upon embryonic γ1 laminin expression

In order to understand the functions of laminins in the renal collecting system, the Lamc1 gene was inactivated in the developing mouse ureteric bud (UB). Embryos bearing null alleles exhibited laminin deficiency prior to mesenchymal tubular induction and either failed to develop a UB with involution of the mesenchyme, or developed small kidneys with decreased proliferation and branching, delayed renal vesicle formation and postnatal emergence of a water transport deficit. Embryonic day 12.5 kidneys revealed an almost complete absence of basement membrane proteins and reduced levels of α6 integrin and FGF2. mRNA levels for fibroblast growth factor 2 (FGF2) and mediators of the GDNF/RET and WNT11 signaling pathway were also decreased. Furthermore, collecting duct cells derived from laminin-deficient kidneys and grown in collagen gels were found to proliferate and branch slowly. The laminin-deficient cells exhibited decreased activation of growth factor- and integrin-dependent pathways, whereas heparin lyase-treated and β1 integrin-null cells exhibited more selective decreases. Collectively, these data support a requirement of γ1 laminins for assembly of the collecting duct system basement membrane, in which immobilized ligands act as solid-phase agonists to promote branching morphogenesis, growth and water transport functions.     

Transplantation of rat metanephroi into mice

To determine whether transplanted metanephroi grow and differentiate after implantation into the omentum in hosts of a different species, we implanted metanephroi from embryonic day 15 (E15) rat embryos into uninephrectomized mice (hosts). Some host mice received human CTLA4Ig (hCTLA4Ig), anti-CD45RB, and anti-CD154 (tolerance-inducing agents). E15 metanephroi contained only metanephric blastema, segments of ureteric bud, and primitive nephrons with no glomeruli. Rat metanephroi did not grow or differentiate in mice that received no tolerance-inducing agents. However, by 2 wk posttransplantation in mice that received hCTLA4Ig, anti-CD45RB, and anti-CD154, metanephroi from E15 rats had enlarged, become vascularized, and formed mature tubules and glomeruli. Rat metanephroi contained cells that stained specifically for mouse CD31, a marker for sprouting endothelial cells. Some rat glomerular capillary loops stained positively for mouse CD31. Here, we show that chimeric kidneys develop from metanephroi transplanted rat-->mouse and that glomeruli are vascularized, at least in part, by host vessels.     

Evaluating a non-destructive method for calibrating tree biomass equations derived from tree branching architecture

Key message

Functional branch analysis (FBA) is a promising non-destructive method that can produce accurate tree biomass equations when applied to trees which exhibit fractal branching architecture.

Abstract

Functional branch analysis (FBA) is a promising non-destructive alternative to the standard destructive method of tree biomass equation development. In FBA, a theoretical model of tree branching architecture is calibrated with measurements of tree stems and branches to estimate the coefficients of the biomass equation. In this study, species-specific and mixed-species tree biomass equations were derived from destructive sampling of trees in Western Kenya and compared to tree biomass equations derived non-destructively from FBA. The results indicated that the non-destructive FBA method can produce biomass equations that are similar to, but less accurate than, those derived from standard methods. FBA biomass prediction bias was attributed to the fact that real trees diverged from fractal branching architecture due to highly variable length–diameter relationships of stems and branches and inaccurate scaling relationships for the lengths of tree crowns and trunks assumed under the FBA model.