Kirsi Sainio

Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins

Lymphatic vessels are essential for immune surveillance, tissue fluid homeostasis and fat absorption. Defects in lymphatic vessel formation or function cause lymphedema. Here we show that the vascular endothelial growth factor C (VEGF-C) is required for the initial steps in lymphatic development. In Vegfc−/− mice, endothelial cells commit to the lymphatic lineage but do not sprout to form lymph vessels. Sprouting was rescued by VEGF-C and VEGF-D but not by VEGF, indicating VEGF receptor 3 specificity. The lack of lymphatic vessels resulted in prenatal death due to fluid accumulation in tissues, and Vegfc+/− mice developed cutaneous lymphatic hypoplasia and lymphedema. Our results indicate that VEGF-C is the paracrine factor essential for lymphangiogenesis, and show that both Vegfc alleles are required for normal lymphatic development.

ACTIVIN DISRUPTS EPITHELIAL BRANCHING MORPHOGENESIS IN DEVELOPING GLANDULAR ORGANS OF THE MOUSE

We report that activin profoundly alters epithelial branching morphogenesis of embryonic mouse salivary gland, pancreas and kidney rudiments in culture, indicating that it may play a role as a morphogen during mammalian organogenesis. In developing pancreas and salivary gland rudiments, activin causes severe disruption of normal lobulation patterns of the epithelium whereas follistatin, an activin-binding protein, counteracts the effect of activin. In the kidney, activin delays branching of the ureter bud and reduces the number of secondary branches. TGF-beta induces a pattern of aberrant branching in the ureter bud derived epithelium distinct from that seen for activin. Reverse-transcriptase polymerase chain reaction, Northern hybridization and in situ hybridization analyses indicate that these developing tissues express the mRNA transcripts for activin subunits, follistatin or activin receptors. Our results are suggestive of a potential role for the activin-follistatin system as an intrinsic regulator of epithelial branching morphogenesis during mammalian organogenesis.

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.

Integrin-linked kinase is an adaptor with essential functions during mouse development

The development of multicellular organisms requires integrin-mediated interactions between cells and their extracellular environment. Integrin binding to extracellular matrix catalyses assembly of multiprotein complexes, which transduce mechanical and chemical signals that regulate many aspects of cell physiology. Integrin-linked kinase (Ilk) is a multifunctional protein that binds β-integrin cytoplasmic domains and regulates actin dynamics by recruiting actin binding regulatory proteins such as α- and β-parvin. Ilk has also been shown to possess serine/threonine kinase activity and to phosphorylate signalling proteins such as Akt1 and glycogen synthase kinase 3β (Gsk3β) in mammalian cells; however, these functions have been shown by genetic studies not to occur in flies and worms. Here we show that mice carrying point mutations in the proposed autophosphorylation site of the putative kinase domain and in the pleckstrin homology domain are normal. In contrast, mice with point mutations in the conserved lysine residue of the potential ATP-binding site of the kinase domain, which mediates Ilk binding to α-parvin, die owing to renal agenesis. Similar renal defects occur in α-parvin-null mice. Thus, we provide genetic evidence that the kinase activity of Ilk is dispensable for mammalian development; however, an interaction between Ilk and α-parvin is critical for kidney development.

Canonical WNT/β-catenin signaling is required for ureteric branching

WNT/beta-catenin signaling has an established role in nephron formation during kidney development. Yet, the role of beta-catenin during ureteric morphogenesis in vivo is undefined. We generated a murine genetic model of beta-catenin deficiency targeted to the ureteric bud cell lineage. Newborn mutant mice demonstrated bilateral renal aplasia or renal dysplasia. Analysis of the embryologic events leading to this phenotype revealed that abnormal ureteric branching at E12.5 precedes histologic abnormalities at E13.5. Microarray analysis of E12.5 kidney tissue identified decreased Emx2 and Lim1 expression among a small subset of renal patterning genes disrupted at the stage of abnormal branching. These alterations are followed by decreased expression of genes downstream of Emx2, including Lim1, Pax2, and the ureteric tip markers, c-ret and Wnt 11. Together, these data demonstrate that beta-catenin performs essential functions during renal branching morphogenesis via control of a hierarchy of genes that control ureteric branching.

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.

A mutation in hairless dogs implicates FOXI3 in ectodermal development

Mexican and Peruvian hairless dogs and Chinese crested dogs are characterized by missing hair and teeth, a phenotype termed canine ectodermal dysplasia (CED). CED is inherited as a monogenic autosomal semidominant trait. With genomewide association analysis we mapped the CED mutation to a 102–kilo–base pair interval on chromosome 17. The associated interval contains a previously uncharacterized member of the forkhead box transcription factor family (FOXI3), which is specifically expressed in developing hair and teeth. Mutation analysis revealed a frameshift mutation within the FOXI3 coding sequence in hairless dogs. Thus, we have identified FOXI3 as a regulator of ectodermal development.

The tip-top branching ureter.

Organ rudiments with their epithelial bud and adjacent mesenchyme look much the same at their initial stage of differentiation. The subsequent branching of the epithelial anlagen determines the final pattern of the organs, but the mesenchyme provides essential signals for epithelial differentiation. Glial cell line derived neurotrophic factor (GDNF) has recently been shown to regulate ureteric branching morphogenesis and is thereby the first defined signalling molecule in the embryonic metanephric kidney. GDNF is expressed by the mesenchyme, binds to the tip of the ureteric bud and functions in both bud induction and bud orientation. The active receptor complex for GDNF includes the receptor tyrosine kinase Ret and a novel class of glycosylphosphatidylinositol-linked receptors, called GDNF family receptor alpha s.

Expression of neurotrophin receptors during rat tooth development is developmentally regulated, independent of innervation, and suggests functions in the regulation of morphogenesis and innervation.

Low‐affinity neurotrophin receptor (LANR) and trk receptor tyrosine kinases (trks) serve as low‐ and high‐affinity receptors for neurotrophins. Besides promoting the development and maintenance of the mammalian nervous system, it has been suggested that neurotrophins may have broader functions in the development of non‐neuronal tissues. To evaluate the possible roles of neurotrophic factors in tooth development, we performed a detailed examination of the expression patterns of neurotrophin receptors during development of the rat tooth from initiation to completion of crown morphogenesis. mRNA expression was studied by in situ hybridisation and LANR protein was localised by immunohistochemistry. Furthermore, dissected tooth germs were cultured in vitro to examine the role of trigeminal innervation in the expression of neurotrophin receptors. mRNAs for LANR, trkB, and trkC, but not trkA, were detected in developing teeth. LANR and the truncated form of trkB, which lacks the intracellular tyrosine kinase domain, were expressed throughout tooth morphogenesis and their expression patterns were largely non‐overlapping and changed spatio‐temporally. trkC was expressed after birth, and it was restricted to dental papilla mesenchyme. The expression of all receptors correlated with the development of innervation, but, in addition, the expression of LANR and trkB appeared to be associated with cell differentiation and epithelial‐mesenchymal interactions. The patterns of LANR, trkB, and trkC in teeth which underwent morphogenesis in organ culture were similar to those in vivo, which indicates that the expression of these neurotrophin receptors is not regulated by and does not depend on trigeminal innervation. The data suggest that neurotrophin receptors have roles in the development of tooth innervation, but that they also have non‐neuronal, organogenetic functions.