Hannu Sariola

Novel functions and signalling pathways for GDNF

Glial-cell-line-derived neurotrophic factor (GDNF) was originally identified as a survival factor for midbrain dopaminergic neurons. GDNF and related ligands, neurturin (NRTN), artemin (ARTN) and persephin (PSPN), maintain several neuronal populations in the central nervous systems, including midbrain dopamine neurons and motoneurons. In addition, GDNF, NRTN and ARTN support the survival and regulate the differentiation of many peripheral neurons, including sympathetic, parasympathetic, sensory and enteric neurons. GDNF has further critical roles outside the nervous system in the regulation of kidney morphogenesis and spermatogenesis. GDNF family ligands bind to specific GDNF family receptor α (GFRα) proteins, all of which form receptor complexes and signal through the RET receptor tyrosine kinase. The biology of GDNF signalling is much more complex than originally assumed. The neurotrophic effect of GDNF, except in motoneurons, requires the presence of transforming growth factor β, which activates the transport of GFRα1 to the cell membrane. GDNF can also signal RET independently through GFR1α. Upon ligand binding, GDNF in complex with GFRα1 may interact with heparan sulphate glycosaminoglycans to activate the Met receptor tyrosine kinase through cytoplasmic Src-family kinases. GDNF family ligands also signal through the neural cell adhesion molecule NCAM. In cells lacking RET, GDNF binds with high affinity to the NCAM and GFRα1 complex, which activates Fyn and FAK.

Early organogenesis of the kidney

The mammalian permanent kidney consists of three cell lineages of different origin: the epithelial cells of the ureter bud, the mesenchymal cells of the nephric blastema and the endothelial cells of the capillaries. Organogenesis is governed by a cascade of morphogenetic interactions between these cell populations, a reciprocal epithelial-mesenchymal interaction between the branching ureter and the metanephric mesenchyme, homotypic interactions between cells of the tubular anlagen, stimulation of angiogenesis by the differentiating blastema and a mesenchymal-endothelial interaction guiding the migration of the capillary endothelial cells. While the biology of these interactive events is well known, as described in this overview, the molecular mechanisms are less well mapped out.

Differentiation and vascularization of the metanephric kidney grafted on the chorioallantoic membrane.

The origin and development of mouse kidney vasculature were examined in chorioallantoic grafts of early kidney rudiments and of experimentally induced explants of separated metanephric mesenchymes. Whole kidney rudiments developed into advanced stages, expressed the segment-specific antigenic markers of tubules and the polyanionic coat of the glomeruli. In contrast to development in vitro, these grafts regularly showed glomeruli with an endothelial component and a basement membrane expressing type IV collagen and laminin. The glomerular endothelial cells in these grafts were shown to carry the nuclear structure of the host. This confirms the outside origin of these cells and the true hybrid nature of the glomeruli. When in vitro induced mesenchymes were grafted on chorioallantoic membranes, abundant vascular invasion was regularly found but properly vascularized glomeruli were exceptional. Uninduced, similarly grafted mesenchymal explants remained avascular as did the undifferentiated portions of partially induced mesenchymal blastemas. It is concluded that the stimulation of the host endothelial cells to invade into the differentiating mesenchyme requires the morphogenetic tissue interaction between the ureter bud and the mesenchyme. The induced metanephric cells presumably start to produce chemoattractants for endothelial cells at an early stage of differentiation. Kidney development thus seems to require an orderly, synchronized development of the three cell lineages: the branching ureter, the induced, tubule-forming mesenchyme, and the invading endothelial cells of outside origin.

Other neurotrophic factors: glial cell line-derived neurotrophic factor (GDNF).

Glial cell line‐derived neurotrophic factor (GDNF) was first discovered as a potent survival factor for midbrain dopaminergic neurons and was then shown to rescue these neurons in animal models of Parkinsons disease. GDNF is a more potent survival factor for dopaminergic neurons and the noradrenergic neurons of the locus coeruleus than other neurotrophic factors, and an almost 100 times more efficient survival factor for spinal motor neurons than the neurotrophins. The members of the GDNF family, GDNF, neurturin (NTN), persephin (PSP), and artemin (ART), have seven conserved cysteine residues with similar spacing, making them distant members of the transforming growth factor‐β (TGF‐β) superfamily. Like the members of the neurotrophin family, the GDNF‐like growth factors belong structurally to the cysteine knot proteins. Like neurotrophins, GDNF family proteins are responsible for the development and maintenance of various sets of sensory and sympathetic neurons but, in addition, GDNF and NTN are also responsible for the development and survival of the enteric neurons, and NTN for parasympathetic neurons. All neurotrophins bind to the p75 low‐affinity receptor, but their ligand specificity is determined by trk receptor tyrosine kinases. GDNF, NTN, PSP, and ART mediate their signals via a common receptor tyrosine kinase, Ret, but their ligand specificity is determined by a novel class of glycosyl‐phosphatidylinositol (GPI)‐anchored proteins called the GDNF family receptor α (GFRα). GDNF binds preferentially to GFRα1, NTN GFRα2, ART GRFα3, and PSP GFRα4 as a co‐receptor to activate Ret. GFRα4 has until now been described only from chicken. Although the GDNF family members signal mainly via Ret receptor tyrosine kinase, there is recent evidence that they can also mediate their signals via GFRα receptors independently of Ret. The GDNF family of growth factors, unlike neurotrophins, has a well‐defined function outside the nervous system. Recent transgenic and organ culture experiments have clearly demonstrated that GDNF is a mesenchyme‐derived signaling molecule for the promotion of ureteric branching in kidney development. NTN, ART, and PSP are also expressed in the developing kidney, and NTN and PSP induce ureteric branching in vitro, but their true in vivo role in kidney morphogenesis is still unclear. Microsc. Res. Tech. 45:292–302, 1999.

Identification of a neurite outgrowth-promoting domain of laminin using synthetic peptides

We have identified a synthetic peptide derived from the B2‐chain of mouse laminin, Arg‐Asn‐Ile‐Ala‐Glu‐Ile‐Ile‐Lys‐Asp‐Ile (p20), which simulates the neurite outgrowth‐promoting activity of the native molecule. In organotypic cultures, neurons from newborn mouse brain or embryonic peripheral nervous system responded by extensive neurite outgrowth for native laminin or the peptide p20 in the culture medium. If rat cerebellar neurons were grown on laminin, 1–5 μM (1–5 μg/ml) of peptide p20 in the culture medium competed with laminin and inhibited neuronal attachment and neurite outgrowth, whereas higher concentrations (⪢ 50 μM; ⪢ 50 μg/ml) had a specific neurotoxic effect. When peptide p20 was used as the culture substratum, neurite outgrowth in cerebellar cultures was up to 60% of that seen on native laminin. Our results indicate that a neurite outgrowth‐promoting domain of laminin is located in the α‐helical region of the B2‐chain, and is active for both central and peripheral neurons.

Localization of Glial Cell Line‐derived Neurotrophic Factor (GDNF) mRNA in Embryonic Rat by In Situ Hybridization

The localization of glial cell line‐derived neurotrophic factor (GDNF) mRNA was studied by in situhybridization in rat from embryonic (E) day E10 to E15. At E10, GDNF mRNA is found in the urogenital field and the cranial part of the gut. At E11, the most abundant expression of GDNF mRNA is seen in the epithelial cells of the second, third and fourth pharyngeal pouches, the third and fourth pharyngeal arches and pharynx. Also mesenchymal cells of the gut and mesonephric tubules contain GDNF mRNA. At E13, expression is observed in the mesenchymal cell layers of the oesophagus, intestine and stomach, the mesenchymal cells around the condensing cartilages and metanephric kidney mesenchyme. Also, the epithelia of Rathkes pouch and pharynx are intensely labelled. High expression of GDNF mRNA continues at El5 in kidney, gastrointestinal tract and cartilage. At that stage, GDNF mRNA is seen also in whisker pad and skeletal muscles. The distribution of GDNF mRNA in embryonic rat suggests important roles for GDNF in the early differentiation of the kidney tubules, the innervation of the gastrointestinal tract and the differentiation process of the cartilage and muscle. Our results indicate novel functions for GDNF outside the nervous system.

BMP‐4 affects the differentiation of metanephric mesenchyme and reveals an early anterior‐posterior axis of the embryonic kidney

Bone morphogenetic protein‐4 (BMP‐4), a member of the transforming growth factor‐β (TGF‐β) family, regulates several developmental processes during animal development. We have now studied the effects of BMP‐4 in the metanephric kidney differentiation by using organ culture technique. Human recombinant BMP‐4 diminishes the number of ureteric branches and changes the branching pattern. Our data suggest that BMP‐4 affects the ureteric branching indirectly via interfering with the differentiation of the nephrogenic mesenchyme. The clear positional preference of the defects to posterior mesenchyme might reflect an early anterior‐posterior patterning of the metanephric mesenchyme. The smooth muscle α‐actin expressing cell population around the ureteric stalk, highly expressing Bmp‐4 mRNA, is also expanded in kidneys treated with BMP‐4. Thus, BMP‐4 may be a physiological regulator of the development of the periureteric smooth muscle layer and ureteric elongation. Dev Dyn;217:146–158.

Dual origin of glomerular basement membrane.

The histogenesis of renal basement membranes was studied in grafts of avascular, 11-day-old mouse embryonic kidney rudiments grown on chick chorioallantoic membrane (CAM). Vessels of the chick CAM invade the mouse tissue during an incubation period of 7-10 days and eventually hybrid glomeruli composed of mouse epithelium and chick endothelium form. Formation of basement membranes during this development was followed by immunofluorescence and immunoperoxidase stainings using polyclonal and monoclonal antibodies against mouse and chick collagen type IV and against mouse laminin. These antibodies were species-specific as shown in immunochemical and immunohistologic analyses. The glomerular basement membrane contained both mouse and chick collagen type IV, demonstrating its dual cellular origin. All other basement membranes were either exclusively of chick origin (mesangium, vessels) or of mouse origin (tubuli, Bowmans capsule).

Primary structure and expression of a novel human laminin α4 chain

The complete primary structure of a novel human laminin α4 chain was derived from cDNA clones. The translation product contains a 24‐residue signal peptide preceding the mature α4 chain of 1792 residues. The domain structure is similar to that of the recently described α3 chain [Ryan, Tizard, Van Devanter and Carter (1994) J. Biol. Chem. 269, 22779–22787]. Northern analysis of RNA from human fetal and adult tissues revealed developmental regulation of expression. In adult, strong expression was observed in heart as well as lung, ovary, small and large intestines, placenta and liver, whereas weak or no expression was detected in skeletal muscle, kidney, pancreas, testis, prostate or brain. In contrast, fetal lung and kidney revealed high expression. In situ hybridization analysis of human fetal and newborn tissues showed expression of the laminin in α4 chain in certain mesenchymal cells in tissues such as smooth muscle and dermis.

Antibodies to cell surface ganglioside GD3 perturb inductive epithelial-mesenchymal interactions

Most epithelial sheets emerge during embryogenesis by a branching and growth of the epithelium. The surrounding mesenchyme is crucial for this process. We report that branching morphogenesis and the formation of a new epithelium from the mesenchyme in the embryonic kidney can be blocked by a monoclonal antibody reacting with a surface glycolipid, disialoganglioside GD3. In contrast, a more than 10-fold excess of antibodies to adhesive glycoproteins (N-CAM, L-CAM, fibronectin) fails to inhibit morphogenesis. Although the anti-GD3 antibody affected epithelial development, the disialoganglioside GD3 was expressed not in the epithelium, but in the mesenchyme surrounding the developing epithelia. The data raise the intriguing possibility that the anti-GD3 antibody inhibits epithelial development by interfering with epithelial-mesenchymal interactions.