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The vertebrate kidney develops through a series of mesenchymal–epithelial interactions between the ureteric bud and the metanephrogenic mesenchyme to form nephrons and the collecting system, which are both embedded in the renal interstitium. The interstitial stromal cells are an essential prerequisite for regular kidney development, but their origin and function is poorly understood. They are found in the kidney periphery and the medulla and are likely derived from the kidney mesenchyme and/or from migrating neural crest cells. During late kidney development, stromal cells are lost through massive apoptosis. We have identified a novel marker of kidney stroma cells, Snep (stromal nidogen extracellular matrix protein), that is additionally expressed in mesenchymal cells of other embryonic tissues and within the nervous system. Of interest, Snep transcripts are also found at sites of embryonic apoptosis. Furthermore, comparative expression analysis of kidney stroma markers suggests that Snep is expressed in a specific subpopulation of stromal cells and may provide environmental cues to support regular development. Developmental Dynamics 230:371-377, 2004 © 2004 Wiley-Liss, Inc.

The interaction of neighboring cells via Notch signalling leads to cell fate determination, differentiation and patterning of highly organized tissues. Mice with targeted disruption of genes from the Notch signal transduction pathway display defects in the developing somites, neurogenic structures, blood vessels, heart and other organs. Recent studies have added requirements for Notch signalling during kidney, pancreas and thymus morphogenesis. Here, we describe the expression of all four receptors (Notch1–4), the five transmembrane ligands (Dll1, 3, 4, Jag1 and Jag2), intracellular effectors (the Hey genes) and extracellular modulators (Lfng, Mfng, Rfng) in the developing mouse metanephros. Our results point to a Lfng-dependent role for Notch signalling in the development of nephron segments, especially the proximal tubules.

Kidney development has often served as a model for epithelial–mesenchymal cell interaction where the branching epithelium of the ureteric bud induces the metanephrogenic mesenchyme to form epithelial nephrons. In a screen for genes differentially expressed during kidney development, we have identified a novel gene that is dynamically expressed in the branching ureter and the developing nephrons. It was designated Emu1 since it shares an N-terminal cysteine-rich domain with Emilin1/2 and Multimerin. This highly conserved EMI domain is also found in another novel protein (Emu2) of similar protein structure: an N-terminal signal peptide followed by the EMI domain, an interrupted collagen stretch, and a conserved C-terminal domain of unknown function. We identified two further secreted EMI domain proteins, prompting us to compare their gene and protein structures, the EMI domain phylogeny, as well as the embryonic expression pattern of known (Emilin1/2, Multimerin) and novel (Emu1/2, Emilin3, Multimerin2) Emu gene family members. Emu1 and Emu2 not only show a similar structural organization, but furthermore a striking complementary expression in organs developing through epithelial–mesenchymal interactions. In these tissues, Emu1 is restricted to epithelial and Emu2 to mesenchymal cells. Preliminary biochemical analysis of Emu1/2 confirmed that they are secreted glycoproteins which are attached to the extracellular matrix and capable of forming homo- and heteromers via disulfide bonding. The widespread, but individually distinct expression patterns of all Emu gene family members suggest multiple functions during mouse embryogenesis. Their multidomain protein structure may indicate that Emu proteins interact with several different extracellular matrix components and serve to connect and integrate the function of multiple partner molecules.

Gridlock (grl) is one of the first mutations characterized from the large zebrafish mutagenesis screens, and it results in an arterial (aortic) maturation defect, which was proposed to resemble aortic coarctation, a clinically important human malformation. While the grl mutation appears to be a hypomorph, grl knockdown experiments have shown even stronger effects on arterial development. We have generated a knockout of the murine Hey2 (gridlock) gene to analyze the mammalian phenotype. Surprisingly, Hey2 loss does not affect aortic development, but it instead leads to a massive postnatal cardiac hypertrophy with high lethality during the first 10 days of life. This cardiomyopathy is ameliorated with time in surviving animals that do not appear to be manifestly impaired during adult life. These differences in phenotypes suggest that changes in expression or function of genes during evolution may lead to quite different pathological phenotypes, if impaired.

We have identified a novel subfamily of mammalian hairy/Enhancer of split (E(spl))-related basic helix-loop-helix (bHLH) genes together with a putative Drosophila homologue. While hairy/E(spl) proteins are characterized by an invariant proline residue in the basic domain and a carboxyterminal groucho-binding WRPW motif, our genes encode a carboxyterminal KPYRPWG sequence and were thus designated as Hey genes (Hairy/E(spl)-related with YRPW motif). Furthermore, they bear a unique C-terminal TE(I/V)GAF motif and the characteristic proline is changed in all Hey family members to glycine. RNA in situ hybridization analysis revealed specific expression of Hey1 during development of the nervous system, the somites, the heart and the craniofacial region. Hey2 is similarly expressed in the somites whereas it shows a complementary expression in the heart, the craniofacial region and the nervous system. The diversity of expression patterns implies unique functions in neurogenesis, somitogenesis and organogenesis.

Development of the metanephric kidney is an experimental model system to analyze interactions between mesenchymal and epithelial cells and mesenchymal-epithelial transition. To study the underlying genetic mechanisms we employed organ culture and differential display PCR to identify genes regulated upon induction of mesenchymal cells. One of the genes found encodes the secreted frizzled related protein 2 (sFRP2) that is upregulated within 2 days of in vitro development. In vivo sFRP2 expression was likewise found in mesenchymal condensates and subsequent epithelial structures. Detailed in situ hybridization analysis revealed sFRP2 expression during development of the eye, brain, neural tube, craniofacial mesenchyme, joints, testis, pancreas and below the epithelia of oesophagus, aorta and ureter where smooth muscles develop. In a comparative analysis transcripts of the related sFRP1 and sFRP4 genes were frequently found in the same tissues as sFRP2 with their expression domains overlapping in some instances, but mutually exclusive in others. While sFRP1 is specifically expressed in the embryonic metanephros, eye, brain, teeth, salivary gland and small intestine, there is only weak expression of sFRP4 except for the developing teeth, eye and salivary gland. The interpretation of the highly specific spatial and temporal expression patterns of sFRP genes will partly depend on a better functional understanding of the interaction between wnt, fz and sFRP family members. Nevertheless, sFRP genes must play quite distinct roles in the morphogenesis of several organ systems.

Gridlock (grl) is one of the first mutations characterized from the large zebrafish mutagenesis screens, and it results in an arterial (aortic) maturation defect, which was proposed to resemble aortic coarctation, a clinically important human malformation. While the grl mutation appears to be a hypomorph, grl knockdown experiments have shown even stronger effects on arterial development. We have generated a knockout of the murine Hey2 (gridlock) gene to analyze the mammalian phenotype. Surprisingly, Hey2 loss does not affect aortic development, but it instead leads to a massive postnatal cardiac hypertrophy with high lethality during the first 10 days of life. This cardiomyopathy is ameliorated with time in surviving animals that do not appear to be manifestly impaired during adult life. These differences in phenotypes suggest that changes in expression or function of genes during evolution may lead to quite different pathological phenotypes, if impaired.

In vertebrates Notch signaling regulates cell fate decisions and boundary formation and it underlies several murine and human diseases. Gene targeting experiments point to key roles of Notch receptors, ligands, modulators and downstream targets in somitogenesis, neurogenesis and vascular development. Here we report the embryonic expression of the hairy-related basic helix-loop-helix gene HeyL in wild-type and Notch pathway mutant mice. We show that HeyL is strongly expressed in the presomitic mesoderm, the somites, the peripheral nervous system and smooth muscle of all arteries. Loss of HeyL expression at the level of nascent somites in Notch1 and Delta-like1 knockout mutants implicates HeyL as a Notch effector during somite formation. Furthermore, HeyL expression in vascular smooth muscle cells and in the thymus strikingly overlaps with that of Notch3, mutations of which underlie the CADASIL vascular disorder.

Many basic helix-loop-helix (bHLH) transcription factors are known as key regulators of embryonic development or differentiation in various species. We have isolated and characterized three new hairy-related bHLH transcription factor genes from mouse and human (hairy and Enhancer-of-split related with YRPW motif; HEY1, HEY2, and HEYL). All three HEY genes have a similar genomic structure with five exons. Together with a highly related Drosophila homologue, they form a new bHLH gene subfamily that is different from both hairy and the known vertebrate Hes and Her genes. While the overall structure with the bHLH domain, Orange domain, and WRPW motif is similar, the last motif is changed to KPYRPWG in Hey1/2 and absent in HeyL. This and other sequence features suggest Hey proteins to have unique functional properties. The genes were mapped by fluorescence in situ hybridization and RH mapping to the following human chromosomes: (HEY1) 8q21, (HEY2) 6q21, and (HEYL) 1p34.3. Based on expression patterns and map location, HEY genes are candidates for several human or mouse disease loci. However, initial screening of DNA from affected individuals for two human disorders and four mouse mutants did not reveal any diagnostic alterations in the coding regions.

Little is known about the mechanisms underlying the generation of various cell types in the hair follicle. To investigate the role of the Notch pathway in this process, transgenic mice were generated in which an active form of Notch1 (NotchΔE) was overexpressed under the control of the mouse hair keratin A1 (MHKA1) promoter. MHKA-NotchΔE is expressed only in one precursor cell type of the hair follicle, the cortex. Transgenic mice could be easily identified by the phenotypes of curly whiskers and wavy, sheen pelage hair. No effects of activated Notch on proliferation were detected in hair follicles of the transgenic mice. We find that activating Notch signaling in the cortex caused abnormal differentiation of the medulla and the cuticle, two neighboring cell types that did not express activated Notch. We demonstrate that these non-autonomous effects are likely caused by cell-cell interactions between keratinocytes within the hair follicle and that Notch may function in such interactions either by directing the differentiation of follicular cells or assisting cells in interpreting a gradient emanating from the dermal papilla.

Kidney development starts with the reciprocal induction of mesenchymal and ureteric bud cells which leads to condensation, epithelialization, and nephron formation in the mesenchyme. To identify changes in gene expression during these processes, we compared differential display polymerase chain reaction (PCR) profiles of uninduced and induced mesenchymal cells. In vitro kidney development in the form of the transfilter organ culture system was used to generate homogeneous cell populations for this type of comparison. Here we describe the isolation of known and novel genetags from this screening. Among the known genes the ufo receptor tyrosine kinase, sFRP2, and the groucho related gene (grg) were verified as being upregulated upon induction. With four of eight novel genes tested, Northern blot analysis proved to be sensitive enough to confirm differential expression. To improve sensitivity and gain additional spatial information, in situ hybridization was performed. Expression analysis of two differential display PCR products, designated C0-5 and M2-4, demonstrated the cell-specific and dynamic expression of these novel genetags in the developing kidney and other tissues. C0-5 transcripts were expressed in the ureteric bud, S-shaped bodies, and in the collecting system. Signals for M2-4, a gene not detectable by Northern blot analysis, were only found in condensing mesenchymal cells and early differentiation stages, but not in the collecting ducts. The large fraction of novel genetags from the present screening that have not yet been analyzed provides a rich resource to clone genetic networks regulating early nephrogenesis.