Maturation of growth hormone stimulation of kidney ornithine decarboxylase in the rat

The effect of ovine growth hormone (GH) on kidney ornithine decarboxylase (ODC) was studied in newborn, preweanling and young adult rats. Basal kidney ODC activity was very low from 4 to 22 days after birth but rose 20-fold by day 25; it remained elevated through day 45. GH failed to stimulate ODC in the first two weeks after birth. GH did however stimulate ODC markedly from 20 through 45 days. Kidney ODC was stimulated in the neonate by vasopressin and by isoproterenol, but not angiotensin II. Liver ODC remained relatively low and stable during development, and was responsive to GH at all ages studied. We conclude that a) the pattern of development of basal kidney ODC appears to be unique to this tissue and may be related to the postnatal maturation of renal morphology and/or function, b) neonatal kidney ODC is unresponsive to certain hormones but is not completely refractory to stimulation. These findings may have implications for the role of hormones in the maturation of the kidney and in the regulation of early renal function.     

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.     

Contrasting expression patterns of three members of the myc family of protooncogenes in the developing and adult mouse kidney

The myc family of protooncogenes encode similar but distinct nuclear proteins. Since N-myc, c-myc, and L-myc have been found to be expressed in the newborn kidney, we studied their expression during murine kidney development. By organ culture studies and in situ hybridization of tissue sections, we found that each of the three members of the myc gene family shows a remarkably distinct expression pattern during kidney development. It is known that mesenchymal stem cells of the embryonic kidney convert into epithelium if properly induced. We demonstrate the N-myc expression increases during the first 24 h of in vitro culture as an early response to induction. Moreover, the upregulation was transient and expression levels were already low during the first stages of overt epithelial cell polarization. In contrast, neither c-myc nor L-myc were upregulated by induction of epithelial differentiation. c-myc was expressed in the uninduced mesenchyme but subsequently became restricted to the newly formed epithelium and was not expressed in the surrounding loose mesenchyme. At onset of terminal differentiation c-myc expression was turned off also from the epithelial tubules. We conclude that N-myc is a marker for induction and early epithelial differentiation states. That the undifferentiated mesenchyme, unlike stromal cells of later developmental stages, express c-myc demonstrates that the undifferentiated mesenchymal stem cells are distinct from the stromal cells. The most astonishing finding, however, was the high level of L-myc mRNA in the ureter, ureter-derived renal pelvis, papilla, and collecting ducts. In the ureter, expression increased, rather than decreased, with advancing maturation and was highest in adult tissue. Our results suggest that each of the three members of the myc gene family are involved in quite disparate differentiation processes, even within one tissue.     

Developmental Changes in the Folate-Dependent Enzymes of De Novo Purine Biosynthesis in Rat Brain

Abstract: The activities of the two folate-dependent enzymes in the de nova purine biosynthetic pathway (e.g., glycinamide ribonucleotide transformylase and aminoimidazolecarboxamide ribonucleotide transformylase), have been evaluated as a function of age in crude extracts from rat brain, liver, kidney, and spleen. The activities of the enzymes in brain are similar to those found in liver and kidney. In all tissues the activity of both enzymes was higher during early development, more than nine times above adult levels. In the CNS the enzymatic activities are apparently related to the periods of increased nucleic acid synthesis, with different activities being found in different regions during development. Our findings lend strong support to the suggestion that folic acid-dependent metabolism plays an important role during early development of the brain.     

Time-dependent expression of cytochrome p450 epoxygenases during human prenatal development

There is growing evidence that some members of cytochrome P450 enzymes contribute to regulation of normal prenatal development. CYP epoxygenases (CYP2C and CYP2J subfamilies) convert arachidonic acid into four regioisomeric epoxyeicosatrienoic acids (EETs), biologically active molecules involved in mitogenesis and cell signaling. Almost nothing is known about localization of their expression in tissues during human prenatal development. The spatio-temporal expression pattern of CYP2C8, CYP2C9, CYP2C19 and CYP2J2 in human embryonic/fetal intestines, liver, and kidney was investigated by immunohistochemical method. CYP epoxygenases are expressed already in early stages of development in these embryonic/fetal tissues (as early as 7th week of IUD in the intestines, 5th week of IUD in the liver, and 6th week of IUD in the kidney). In kidney, CYP epoxygenases are expressed in the metanephrogenic blastema (but not in the uninduced mesenchyme) and in the tubular system. In the intestines, diverse CYP epoxygenases distribution along crypt-villus axis could suggest role in cell differentiation. Moreover, we detected higher CYP2J2 level in these organs than in adult tissue samples.     

Time-dependent expression of cytochrome p450 epoxygenases during human prenatal development

There is growing evidence that some members of cytochrome P450 enzymes contribute to regulation of normal prenatal development. CYP epoxygenases (CYP2C and CYP2J subfamilies) convert arachidonic acid into four regioisomeric epoxyeicosatrienoic acids (EETs), biologically active molecules involved in mitogenesis and cell signaling. Almost nothing is known about localization of their expression in tissues during human prenatal development. The spatio-temporal expression pattern of CYP2C8, CYP2C9, CYP2C19 and CYP2J2 in human embryonic/fetal intestines, liver, and kidney was investigated by immunohistochemical method. CYP epoxygenases are expressed already in early stages of development in these embryonic/fetal tissues (as early as 7th week of IUD in the intestines, 5th week of IUD in the liver, and 6th week of IUD in the kidney). In kidney, CYP epoxygenases are expressed in the metanephrogenic blastema (but not in the uninduced mesenchyme) and in the tubular system. In the intestines, diverse CYP epoxygenases distribution along crypt-villus axis could suggest role in cell differentiation. Moreover, we detected higher CYP2J2 level in these organs than in adult tissue samples.     

Urea production and transport in teleost fishes

Teleosts appear to have retained the genes for the urea cycle enzymes. A few species express the full complement of enzymes and are ureotelic (e.g., Lake Magadi tilapia) or ammoniotelic (e.g., largemouth bass), whereas most species have low or non-detectable enzyme activities in liver tissue and excrete little urea (e.g., adult rainbow trout). It was surprising, therefore, to find the expression of four urea cycle enzymes during early life stages of rainbow trout. The urea cycle may play a role in ammonia detoxification during a critical time of development. Exposure to alkaline water (pH 9.0-9.5) or NH4Cl (0.2 mmol/l) increased urea excretion by several-fold in trout embryos, free embryos and alevin. Urea transport is either by passive simple diffusion or via carried-mediated transport proteins. Molecular studies have revealed that a specialised urea transport protein is present in kidney tissue of elasmobranchs, similar to the facilitated urea transporter found in the mammalian inner medulla of the kidney.     

N-myc proto-oncogene expression during organogenesis in the developing mouse as revealed by in situ hybridization

The N-myc proto-oncogene is expressed during embryogenesis, suggesting that it plays a role in normal development. Since the myc-family oncogenes have been implicated in the control of cell growth, the embryonic expression may reflect rapid proliferation known to occur in development. Alternatively, N-myc expression may be involved in specific differentiation stages. In many embryonic tissues, early and late differentiation events occur in different locations. By in situ hybridization of tissue sections, we now demonstrate a restricted expression of N-myc mRNA to a few tissues and to areas where the first differentiation stages occur. N-myc expression was most strongly expressed in the developing kidney, hair follicles, and in various parts of the central nervous system. In these tissues, expression was restricted to a few cell lineages. In all lineages, expression was confined to early differentiation stages, and, at onset of overt differentiation, the level of expression decreased dramatically. Several rapidly proliferating tissues showed very little, if any, N-myc expression. In the brain, post-mitotic but not yet differentiated cells expressed high levels of N-myc mRNA. Therefore, N-myc expression is not a simple marker for proliferation in the embryo. Rather, N-myc expression seems to be a feature of early differentiation stages of some cell lineages in kidney, brain, and hair follicles, regardless of the proliferative status of the cell. The results raise the possibility that N-myc may participate in the control of these early differentiation events.     

Notch1 and IL-7 receptor interplay maintains proliferation of human thymic progenitors while suppressing non-T cell fates

Notch signaling is critical for T cell development of multipotent hemopoietic progenitors. Yet, how Notch regulates T cell fate specification during early thymopoiesis remains unclear. In this study, we have identified an early subset of CD34high c-kit+ flt3+ IL-7Ralpha+ cells in the human postnatal thymus, which includes primitive progenitors with combined lymphomyeloid potential. To assess the impact of Notch signaling in early T cell development, we expressed constitutively active Notch1 in such thymic lymphomyeloid precursors (TLMPs), or triggered their endogenous Notch pathway in the OP9-Delta-like1 stroma coculture. Our results show that proliferation vs differentiation is a critical decision influenced by Notch at the TLMP stage. We found that Notch signaling plays a prominent role in inhibiting non-T cell differentiation (i.e., macrophages, dendritic cells, and NK cells) of TLMPs, while sustaining the proliferation of undifferentiated thymocytes with T cell potential in response to unique IL-7 signals. However, Notch activation is not sufficient for inducing T-lineage progression of proliferating progenitors. Rather, stroma-derived signals are concurrently required. Moreover, while ectopic IL-7R expression cannot replace Notch for the maintenance and expansion of undifferentiated thymocytes, Notch signals sustain IL-7R expression in proliferating thymocytes and induce IL-7R up-regulation in a T cell line. Thus, IL-7R and Notch pathways cooperate to synchronize cell proliferation and suppression of non-T lineage choices in primitive intrathymic progenitors, which will be allowed to progress along the T cell pathway only upon interaction with an inductive stromal microenvironment. These data provide insight into a mechanism of Notch-regulated amplification of the intrathymic pool of early human T cell progenitors.     

Epithelial-mesenchymal interactions regulate the stage-specific expression of a cell surface proteoglycan, syndecan, in the developing kidney

Morphogenesis of the kidney is regulated by reciprocal tissue interactions between the epithelial ureter bud and the metanephric mesenchyme. The differentiation of the kidney involves profound changes in the extracellular matrix, and therefore matrix receptors may have an important role in this process. We studied the expression of syndecan, a cell surface proteoglycan acting as a receptor for interstitial matrix materials, by using a monoclonal antibody against the core protein of the molecule. Syndecan was not detected in the uninduced metanephric mesenchyme. During the formation of the ureter bud from the Wolffian duct, syndecan appeared in the mesenchymal cells around the invaginating bud. Simultaneously with the first branching of the ureter bud, the whole nephric mesenchyme became syndecan positive, but a 3- to 10-cell-thick layer around the branching ureter bud, representing the presumptive tubular cells, was most intensely stained. During the assembly of the mesenchyme cells into pretubular aggregates, syndecan was detected in these aggregates and, to a lesser degree, in the morphologically undifferentiated mesenchyme. Thereafter syndecan was found only in the differentiating epithelium, from which it was gradually lost during maturation of the nephron. It was last detected in the periphery of the kidney, where tubulogenesis still continued. In transfilter cultures we showed that syndecan appeared in the nephric mesenchyme during the period when the mesenchyme becomes programmed to transform into epithelial structures. By using interspecies recombinations and a species-specific antibody we excluded the possibility that syndecan in the mesenchyme would originate from the inductor. We conclude that syndecan expression is regulated by epithelial-mesenchymal interactions. The findings that syndecan appeared as an early response to induction and that its distribution showed both spatial and temporal correlation with kidney morphogenesis suggest an important role for this molecule in development.     

Calcineurin inhibitors block dorsal-side signaling that affect late-stage development of the heart, kidney, liver, gut and somitic tissue during Xenopus embryogenesis

Calcineurin, a calcium/calmodulin-dependent serine/threonine protein phosphatase, is a key constituent of signaling pathways involved in antigen-dependent T-cell activation and development of the mammalian heart. In addition, calcineurin constitutes a part of the Wnt/calcium-signaling pathway that regulates early stages of dorsoventral axis formation in Xenopus embryos. Although some of the Wnt family members are involved in organ formation at relatively late stages of Xenopus development, the involvement of calcineurin in the development of those organs remains unclear. In the present study, we demonstrate that calcineurin inhibitors (cyclosporine A, FK506, and FK520), but not non-calcineurin inhibitors (rapamycin and GPI1046) that bind the same intracellular receptor as that for FK506, induce edema and gut coiling disruption and exhibit teratogenesis in the kidney, heart, gut, liver, and somitic tissue during Xenopus development. The same effects were observed by injecting the calcineurin inhibitors into the dorsal side, but not ventral side, of blastomeres at the 4-cell stage, although the inhibitors did not affect dorsoventral axis formation. These results suggest that calcineurin is involved in dorsal-side signaling that leads to the formation of the heart, kidney, liver, gut and somitic tissue during Xenopus embryogenesis.     

Characterisation and trophic functions of murine embryonic macrophages based upon the use of a Csf1r-EGFP transgene reporter

All solid organs contain resident monocyte-derived cells that appear early in organogenesis and persist throughout life. These cells are critical for normal development in some organs. Here we report the use of a previously described transgenic line, with EGFP driven by the macrophage-restricted Csf1r (c-fms) promoter, to image macrophage production and infiltration accompanying organogenesis in many tissues. Using microarray analysis of FACS-isolated EGFP-positive cells, we show that fetal kidney, lung and brain macrophages show similar gene expression profiles irrespective of their tissue of origin. EGFP-positive cells appeared in the renal interstitium from 12 days post coitum, prior to nephrogenesis, and maintain a close apposition to renal tubules postnatally. CSF-1 added to embryonic kidney explants increased overall renal growth and ureteric bud branching. Expression profiling of tissue macrophages and of CSF-1-treated explants showed evidence of the alternate, pro-proliferative (M2) activation profile, including expression of macrophage mannose receptor (CD206), macrophage scavenger receptor 2 (Msr2), C1q, CD163, selenoprotein P, CCL24 and TREM2. This response has been associated with the trophic role of tumour-associated macrophages. These findings suggest a trophic role of macrophages in embryonic kidney development, which may continue to play a similar role in postnatal repair.     

Suppression of the allograft response by implants of mature lymphoid tissues in larval Xenopus laevis

One hundred and twenty-two larvae of Xenopus laevis, the South African clawed toad, at developmental stages 48, 50, 52 and 54, were implanted in the tail with two allografts from adult tissues. In each case, one allograft was from kidney, while the other was either from kidney, thymus, spleen, or liver. In any particular host the two implants were always from the same donor and the implants were all visually matched in size. The experimental period was a maximum of nine days, so as to minimize the large numbers of changes normally accompanying larval progress from stage to stage. We are concerned with the timing of allograft response initiation under the implant conditions of each experimental group at a particular point in development. An allograft response was defined as an infiltration and accumulation of small lymphocytes in the “test” kidney allograft. Larvae of all stages developed allograft responses within one week post-implantation when the variable implant was from kidney, but implants from spleen and thymus suppressed both the timing of initiation and the subsequent intensity of the response. Spleen was more effective in this regard than thymus and both were more effective in the earlier larval stages. Liver proved to be toxic to the larvae. The relationship between the maturation of the lymphomyeloid tissues and external morphological staging is also discussed.