Interaction of the receptor FGFRL1 with the negative regulator Spred1

FGFRL1 is a member of the fibroblast growth factor receptor family. It plays an essential role during branching morphogenesis of the metanephric kidneys, as mice with a targeted deletion of the Fgfrl1 gene show severe kidney dysplasia. Here we used the yeast two-hybrid system to demonstrate that FGFRL1 binds with its C-terminal, histidine-rich domain to Spred1 and to other proteins of the Sprouty/Spred family. Members of this family are known to act as negative regulators of the Ras/Raf/Erk signaling pathway. Truncation experiments further showed that FGFRL1 interacts with the SPR domain of Spred1, a domain that is shared by all members of the Sprouty/Spred family. The interaction could be verified by coprecipitation of the interaction partners from solution and by codistribution at the cell membrane of COS1 and HEK293 cells. Interestingly, Spred1 increased the retention time of FGFRL1 at the plasma membrane where the receptor might interact with ligands. FGFRL1 and members of the Sprouty/Spred family belong to the FGF synexpression group, which also includes FGF3, FGF8, Sef and Isthmin. It is conceivable that FGFRL1, Sef and some Sprouty/Spred proteins work in concert to control growth factor signaling during branching morphogenesis of the kidneys and other organs.

Developmental alveolarization of the mouse lung

Postnatal lung development is not well characterized in mice, especially the time point when alveolarization is completed. Using the total length and the length density of the free septal edge as measured for the formation of new septa, we followed alveolarization throughout postnatal lung development (days 2-125). Furthermore, the alveolar surface area was estimated. The formation of new septa was observed until day 36. Approximately 10% of the septa present in adult mice were formed prenatally by branching morphogenesis, approximately 50% were generated postnatally before and approximately 40% after maturation of the alveolar microvasculature. Approximately 5% of the alveolar surface area present during adulthood was present before alveolarization started, approximately 55% was formed during alveolarization (days 4-36) and approximately 40% afterward due to growth processes. We conclude that alveolarization continues until young adulthood and that the maturation of the alveolar microvasculature does not preclude further alveolarization.

The murine Fgfrl1 receptor is essential for the development of the metanephric kidney

Fgfrl1 is a novel member of the fibroblast growth factor receptor family. Its extracellular domain resembles the four conventional Fgfrs, while its intracellular domain lacks the tyrosine kinase domain necessary for Fgf mediated signal transduction. During embryonic development Fgfrl1 is expressed in the musculoskeletal system, in the lung, the pancreas and the metanephric kidney. Targeted disruption of the Fgfrl1 gene leads to the perinatal death of the mice due to a hypoplastic diaphragm, which is unable to inflate the lungs. Here we show that Fgfrl1-/- embryos also fail to develop the metanephric kidney. While the rest of the urogenital system, including bladder, ureter and sexual organs, develops normally, a dramatic reduction of ureteric branching morphogenesis and a lack of mesenchymal-to-epithelial transition in the nephrogenic mesenchyme result in severe renal dysgenesis. The failure of nephron induction might be explained by the absence of the tubulogenic markers Wnt4, Fgf8, Pax8 and Lim1 at E12.5 of the mutant animals. We also observed a loss of Pax2 positive nephron precursor cells and an increase of apoptosis in the cortical zone of the remnant kidney. Fgfrl1 is therefore essential for mesenchymal differentiation in the early steps of nephrogenesis.

Lung structure at preterm and term birth

When lung development is not interrupted by premature birth and unaffected by genetic or environmental disturbances, all components develop with complex control to form a functional organ with a predictable timeline during fetal development. In this chapter we describe the relationship between morphological development and function in both physiological and pathological conditions in human lung development. Tree-like growth of the lung begins during the first few weeks postconception, with the embryonic stage characterized by branching morphogenesis in both the airways and blood vessels, separately in the left and right lung buds, which appear near day 26 postcoitus (p.c.). Branching continues through the embryonic stage, with proliferation of mesenchymal and epithelial cells and apoptosis near branch points and in the areas of new formation. The pseudoglandular stage (weeks 5–17 p.c.) is characterized by accelerated cellular proliferation and airway and vascular branching, with epithelial differentiation in proximal and distal airways. Further epithelial differentiation, angiogenesis of the parenchymal capillary network, and the first formation of the air–blood barrier characterize the canalicular stage (16–26 weeks p.c.), just before the completion of branching morphogenesis (saccular stage, weeks 24–38 p.c.) and the start of alveolarization (week 36 through adolescence).

Overexpression of EphB4 in the mammary epithelium shifts the differentiation pathway of progenitor cells and promotes branching activity and vascularization

Postnatally, the mammary gland undergoes continuous morphogenesis and thereby is especially prone to malignant transformation. Thus, the maintenance of the epithelium depends on a tight control of stem cell recruitment. We have previously shown that epithelial overexpression of the EphB4 receptor results in defective mammary epithelial development and conferred a metastasizing tumor phenotype on experimental mouse mammary tumors accompanied by a preponderance of progenitor cells. To analyze the effect of EphB4 overexpression on mammary epithelial cell fate, we have used Fluorescence Activated Cell Sorting (FACS) analyses to quantify epithelial sub-populations and repopulation assays of cleared fat pads to investigate their regenerative potential. These experiments revealed that deregulated EphB4 expression leads to an augmentation of bi-potent progenitor cells and to a shift of the differentiation pathway towards the luminal lineage. The analyses of the ductal outgrowths indicated that EphB4 overexpression leads to enforced branching activity, impedes ductal differentiation and stimulates angiogenesis. To elucidate the mechanisms forwarding EphB4 signals, we have compared the expression profile of defined cell populations between EphB4 transgene and wild type mammary glands concentrating on the wnt signaling pathway and on genes implicated in cell migration. With respect to wnt signaling, the progenitor cell population was the most affected, whereas the stem cell-enriched population showed the most pronounced deregulation of migration-associated genes. Thus, the luminal epithelial EphB4 signaling contributes, most likely via wnt signaling, to the regulation of migration and cell fate of early progenitors and is involved in the determination of branching points along the ductal tree.

A novel allele of FILAMENTOUS FLOWER reveals new insights on the link between inflorescence and floral meristem organization and flower morphogenesis

Background The Arabidopsis FILAMENTOUS FLOWER (FIL) gene encodes a YABBY (YAB) family putative transcription factor that has been implicated in specifying abaxial cell identities and thus regulating organ polarity of lateral organs. In contrast to double mutants of fil and other YAB genes, fil single mutants display mainly floral and inflorescence morphological defects that do not reflect merely a loss of abaxial identity. Recently, FIL and other YABs have been shown to regulate meristem organization in a non-cell-autonomous manner. In a screen for new mutations affecting floral organ morphology and development, we have identified a novel allele of FIL, fil-9 and characterized its floral and meristem phenotypes. Results The fil-9 mutation results in highly variable disruptions in floral organ numbers and size, partial homeotic transformations, and in defective inflorescence organization. Examination of meristems indicates that both fil-9 inflorescence and floral meristems are enlarged as a result of an increase in cell number, and deformed. Furthermore, primordia emergence from these meristems is disrupted such that several primordia arise simultaneously instead of sequentially. Many of the organs produced by the inflorescence meristems are filamentous, yet they are not considered by the plant as flowers. The severity of both floral organs and meristem phenotypes is increased acropetally and in higher growth temperature. Conclusions Detailed analysis following the development of fil-9 inflorescence and flowers throughout flower development enabled the drawing of a causal link between multiple traits of fil-9 phenotypes. The study reinforces the suggested role of FIL in meristem organization. The loss of spatial and temporal organization of fil-9 inflorescence and floral meristems presumably leads to disrupted cell allocation to developing floral organs and to a blurring of organ whorl boundaries. This disruption is reflected in morphological and organ identity aberrations of fil-9 floral organs and in the production of filamentous organs that are not perceived as flowers. Here, we show the role of FIL in reproductive meristem development and emphasize the potential of using fil mutants to study mersitem organization and the related effects on flower morphogenesis.

Site-Specific Expression of Gelatinolytic Activity during Morphogenesis of the Secondary Palate in the Mouse Embryo

Morphogenesis of the secondary palate in mammalian embryos involves two major events: first, reorientation of the two vertically oriented palatal shelves into a horizontal position above the tongue, and second, fusion of the two shelves at the midline. Genetic evidence in humans and mice indicates the involvement of matrix metalloproteinases (MMPs). As MMP expression patterns might differ from sites of activity, we used a recently developed highly sensitive in situ zymography technique to map gelatinolytic MMP activity in the developing mouse palate. At embryonic day 14.5 (E14.5), we detected strong gelatinolytic activity around the lateral epithelial folds of the nasopharyngeal cavity, which is generated as a consequence of palatal shelf elevation. Activity was concentrated in the basement membrane of the epithelial fold but extended into the adjacent mesenchyme, and increased in intensity with lateral outgrowth of the cavity at E15.5. Gelatinolytic activity at this site was not the consequence of epithelial fold formation, as it was also observed in Bmp7-deficient embryos where shelf elevation is delayed. In this case, gelatinolytic activity appeared in vertical shelves at the exact position where the epithelial fold will form during elevation. Mmp2 and Mmp14 (MT1-MMP), but not Mmp9 and Mmp13, mRNAs were expressed in the mesenchyme around the epithelial folds of the elevated palatal shelves; this was confirmed by immunostaining for MMP-2 and MT1-MMP. Weak gelatinolytic activity was also found at the midline of E14.5 palatal shelves, which increased during fusion at E15.5. Whereas MMPs have been implicated in palatal fusion before, this is the first report showing that gelatinases might contribute to tissue remodeling during early stages of palatal shelf elevation and formation of the nasopharynx.

Putative functions of extracellular matrix glycoproteins in secondary palate morphogenesis

Cleft palate is a common birth defect in humans. Elevation and fusion of paired palatal shelves are coordinated by growth and transcription factors, and mutations in these can cause malformations. Among the effector genes for growth factor signaling are extracellular matrix (ECM) glycoproteins. These provide substrates for cell adhesion (e.g., fibronectin, tenascins), but also regulate growth factor availability (e.g., fibrillins). Cleft palate in Bmp7 null mouse embryos is caused by a delay in palatal shelf elevation. In contrast, palatal shelves of Tgf-β3 knockout mice elevate normally, but a cleft develops due to their failure to fuse. However, nothing is known about a possible functional interaction between specific ECM proteins and Tgf-β/Bmp family members in palatogenesis. To start addressing this question, we studied the mRNA and protein distribution of relevant ECM components during secondary palate development, and compared it to growth factor expression in wildtypewild type and mutant mice. We found that fibrillin-2 (but not fibrillin-1) mRNA appeared in the mesenchyme of elevated palatal shelves adjacent to the midline epithelial cells, which were positive for Tgf-β3 mRNA. Moreover, midline epithelial cells started expressing fibronectin upon contact of the two palatal shelves. These findings support the hypothesis that fibrillin-2 and fibronectin are involved in regulating the activity of Tgf-β3 at the fusing midline. In addition, we observed that tenascin-W (but not tenascin-C) was misexpressed in palatal shelves of Bmp7-deficient mouse embryos. In contrast to tenascin-C, tenascin-W secretion was strongly induced by Bmp7 in embryonic cranial fibroblasts in vitro. These results are consistent with a putative function for tenascin-W as a target of Bmp7 signaling during palate elevation. Our results indicate that distinct ECM proteins are important for morphogenesis of the secondary palate, both as downstream effectors and as regulators of Tgf-β/Bmp activity.

The cytoplasmic domain of the ligand ephrinB2 is required for vascular morphogenesis but not cranial neural crest migration

The transmembrane ligand ephrinB2 and its cognate Eph receptor tyrosine kinases are important regulators of vascular morphogenesis. EphrinB2 may have an active signaling role, resulting in bi-directional signal transduction downstream of both ephrinB2 and Eph receptors. To separate the ligand and receptor-like functions of ephrinB2 in mice, we replaced the endogenous gene by cDNAs encoding either carboxyterminally truncated (ephrinB2(DeltaC)) or, as a control, full-length ligand (ephrinB2(WT)). While homozygous ephrinB2(WT/WT) animals were viable and fertile, loss of the ephrinB2 cytoplasmic domain resulted in midgestation lethality similar to ephrinB2 null mutants (ephrinB2(KO)). The truncated ligand was sufficient to restore guidance of migrating cranial neural crest cells, but ephrinB2(DeltaC/DeltaC) embryos showed defects in vasculogenesis and angiogenesis very similar to those observed in ephrinB2(KO/KO) animals. Our results indicate distinct requirements of functions mediated by the ephrinB carboxyterminus for developmental processes in the vertebrate embryo.

Roles of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/venous domains, vascular morphogenesis, and sprouting angiogenesis

Eph receptor tyrosine kinases and their cell-surface-bound ligands, the ephrins, regulate axon guidance and bundling in the developing brain, control cell migration and adhesion, and help patterning the embryo. Here we report that two ephrinB ligands and three EphB receptors are expressed in and regulate the formation of the vascular network. Mice lacking ephrinB2 and a proportion of double mutants deficient in EphB2 and EphB3 receptor signaling die in utero before embryonic day 11.5 (E11.5) because of defects in the remodeling of the embryonic vascular system. Our phenotypic analysis suggests complex interactions and multiple functions of Eph receptors and ephrins in the embryonic vasculature. Interaction between ephrinB2 on arteries and its EphB receptors on veins suggests a role in defining boundaries between arterial and venous domains. Expression of ephrinB1 by arterial and venous endothelial cells and EphB3 by veins and some arteries indicates that endothelial cell-to-cell interactions between ephrins and Eph receptors are not restricted to the border between arteries and veins. Furthermore, expression of ephrinB2 and EphB2 in mesenchyme adjacent to vessels and vascular defects in ephB2/ephB3 double mutants indicate a requirement for ephrin-Eph signaling between endothelial cells and surrounding mesenchymal cells. Finally, ephrinB ligands induce capillary sprouting in vitro with a similar efficiency as angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF), demonstrating a stimulatory role of ephrins in the remodeling of the developing vascular system.