Role of fibroblast growth factor receptor signaling in kidney development

Fibroblast growth factor receptors (Fgfrs) consist of four signaling family members and one nonsignaling "decoy" receptor, Fgfr-like 1 (Fgfrl1), all of which are expressed in the developing kidney. Several studies have shown that exogenous fibroblast growth factors (Fgfs) affect growth and maturation of the metanephric mesenchyme (MM) and ureteric bud (UB) in cultured tissues. Transgenic and conditional knockout approaches in whole animals have shown that Fgfr1 and Fgfr2 (predominantly the IIIc isoform) in kidney mesenchyme are critical for early MM and UB formation. Conditional deletion of the ligand, Fgf8, in nephron precursors or global deletion of Fgfrl1 interrupts nephron formation. Fgfr2 (likely the IIIb isoform signaling downstream of Fgf7 and Fgf10) is critical for ureteric morphogenesis. Moreover, Fgfr2 appears to act independently of Frs2α (the major signaling adapter for Fgfrs) in regulating UB branching. Loss of Fgfr2 in the MM leads to many kidney and urinary tract anomalies, including vesicoureteral reflux. Thus Fgfr signaling is critical for patterning of virtually all renal lineages at early and later stages of development.     

Integration of FGF and TWIST in calvarial bone and suture development

Mutations in the FGFR1-FGFR3 and TWIST genes are known to cause craniosynostosis, the former by constitutive activation and the latter by haploinsufficiency. Although clinically achieving the same end result, the premature fusion of the calvarial bones, it is not known whether these genes lie in the same or independent pathways during calvarial bone development and later in suture closure. We have previously shown that Fgfr2c is expressed at the osteogenic fronts of the developing calvarial bones and that, when FGF is applied via beads to the osteogenic fronts, suture closure is accelerated (Kim, H.-J., Rice, D. P. C., Kettunen, P. J. and Thesleff, I. (1998) Development 125, 1241-1251). In order to investigate further the role of FGF signalling during mouse calvarial bone and suture development, we have performed detailed expression analysis of the splicing variants of Fgfr1-Fgfr3 and Fgfr4, as well as their potential ligand Fgf2. The IIIc splice variants of Fgfr1-Fgfr3 as well as the IIIb variant of Fgfr2 being expressed by differentiating osteoblasts at the osteogenic fronts (E15). In comparison to Fgf9, Fgf2 showed a more restricted expression pattern being primarily expressed in the sutural mesenchyme between the osteogenic fronts. We also carried out a detailed expression analysis of the helix-loop-helix factors (HLH) Twist and Id1 during calvaria and suture development (E10-P6). Twist and Id1 were expressed by early preosteoblasts, in patterns that overlapped those of the FGF ligands, but as these cells differentiated their expression dramatically decreased. Signalling pathways were further studied in vitro, in E15 mouse calvarial explants. Beads soaked in FGF2 induced Twist and inhibited Bsp, a marker of functioning osteoblasts. Meanwhile, BMP2 upregulated Id1. Id1 is a dominant negative HLH thought to inhibit basic HLH such as Twist. In Drosophila, the FGF receptor FR1 is known to be downstream of Twist. We demonstrated that in Twist(+/)(-) mice, FGFR2 protein expression was altered. We propose a model of osteoblast differentiation integrating Twist and FGF in the same pathway, in which FGF acts both at early and late stages. Disruption of this pathway may lead to craniosynostosis.     

Fgf9 signaling regulates inner ear morphogenesis through epithelial-mesenchymal interactions

The mammalian inner ear comprises the cochleovestibular labyrinth, derived from the ectodermal otic placode, and the encasing bony labyrinth of the temporal bone. Epithelial-mesenchymal interactions are thought to control inner ear development, but the modes and the molecules involved are largely unresolved. We show here that, during the precartilage and cartilage stages, Fgf9 is expressed in specific nonsensory domains of the otic epithelium and its receptors, Fgfr1(IIIc) and Fgfr2(IIIc), widely in the surrounding mesenchyme. To address the role of Fgf9 signaling, we analyzed the inner ears of mice homozygous for Fgf9 null alleles. Fgf9 inactivation leads to a hypoplastic vestibular component of the otic capsule and to the absence of the epithelial semicircular ducts. Reduced proliferation of the prechondrogenic mesenchyme was found to underlie capsular hypoplasticity. Semicircular duct development is blocked at the initial stages, since fusion plates do not form. Our results show that the mesenchyme directs fusion plate formation and they give direct evidence for the existence of reciprocal epithelial-mesenchymal interactions in the developing inner ear. In addition to the vestibule, in the cochlea, Fgf9 mutation caused defects in the interactions between the Reissner's membrane and the mesenchymal cells, leading to a malformed scala vestibuli. Together, these data show that Fgf9 signaling is required for inner ear morphogenesis.     

Cleavage in vivo and in culture in the Dasyurid marsupial Antechinus stuartii (macleay)

Cleavage in the brown marsupial mouse, Antechinus stuartii, from the zygote to the unilaminar blastocyst, was observed in vivo and in culture and in sections of embryos. The first three divisions were meridional and passed from the yolk pole to the opposite pole. Deutoplasmolysis, resulting in a distinct yolk mass, occurred during the first two divisions. Prior to the third and fourth divisions, the blastomeres elongated and flattened against the zona pellucida. The fourth division was latitudinal and resulted in two histologically distinct rings of eight blastomeres which were at first rounded and then became flattened against the zona. Further divisions and flattening of the blastomeres resulted in a complete unilaminar blastocyst by the time the blastocyst numbered 22 to 30 cells. Some expansion, causing compression of the zona and mucoid layers, occurred before completion of the blastocyst, but most expansion occurred once the blastocyst was complete. No histological differences could be detected between the blastomeres at this stage. Embryos were successfully cultured from the zygote to the rounded four-cell stage and from the flattened four-cell stage to the completed unilaminar blastocyst of around 32 cells. Total estimated cleavage times were slower in culture than in vivo, but the relative lengths of time for each division were approximately the same.     

Fgfr1 Inactivation in the Mouse Telencephalon Results in Impaired Maturation of Interneurons Expressing Parvalbumin

Fibroblast growth factors (Fgfs) and their receptors (Fgfr) are expressed in the developing and adult CNS. Previous studies demonstrated a decrease in cortical interneurons and locomotor hyperactivity in mice with a conditional Fgfr1 deletion generated in radial glial cells during midneurogenesis (Fgfr1^f/f;hGfapCre+). Here, we report earlier and more extensive inactivation of Fgfr1 in neuroepithelial cells of the CNS (Fgfr1^f/f;NesCre+). Similar to findings in Fgfr1^f/f;hGfapCre+ mice, parvalbumin positive (PV+) cortical interneurons are also decreased in the neocortex of Fgfr1f/f;NesCre+ mice when compared to control littermates (Fgfr1f/f). Fgfr1f/f;NesCre+ embryos do not differ from controls in the initial specification of GABAergic cells in the ganglionic eminence (GE) as assessed by in situ hybridization for Dlx2, Mash1 and Nkx2. Equal numbers of GABAergic neuron precursors genetically labeled with green fluorescent protein (GFP) were observed at P0 in Fgfr1^f/f;hGfapCre+;Gad1-GFP mutant mice. However, fewer GFP+ and GFP+/PV+ interneurons were observed in these mutants at adulthood, indicating that a decrease in cortical interneuron markers is occurring postnatally. Fgfr1 is expressed in cortical astrocytes in the postnatal brain. To test whether the astrocytes of mice lacking Fgfr1 are less capable of supporting interneurons, we co-cultured wild type Gad1-GFP+ interneuron precursors isolated from the medial GE (MGE) with astrocytes from Fgfr1f/f control or Fgfr1^f/f;hGfapCre+ mice. Interneurons grown on Fgfr1 deficient astrocytes had small soma size and fewer neurites per cell, but no differences in cell survival. Decreased soma size of Gad67 immunopositive interneurons was also observed in the cortex of adult Fgfr1^f/f;NesCre+ mice. Our data indicate that astrocytes from Fgfr1 mutants are impaired in supporting the maturation of cortical GABAergic neurons in the postnatal period. This model may elucidate potential mechanisms of impaired PV interneuron maturation relevant to neuropsychiatric disorders that develop in childhood and adolescence.     

The differential response to Fgf signalling in cells internalized at different times influences lineage segregation in preimplantation mouse embryos

Lineage specification in the preimplantation mouse embryo is a regulative process. Thus, it has been difficult to ascertain whether segregation of the inner-cell-mass (ICM) into precursors of the pluripotent epiblast (EPI) and the differentiating primitive endoderm (PE) is random or influenced by developmental history. Here, our results lead to a unifying model for cell fate specification in which the time of internalization and the relative contribution of ICM cells generated by two waves of asymmetric divisions influence cell fate. We show that cells generated in the second wave express higher levels of Fgfr2 than those generated in the first, leading to ICM cells with varying Fgfr2 expression. To test whether such heterogeneity is enough to bias cell fate, we upregulate Fgfr2 and show it directs cells towards PE. Our results suggest that the strength of this bias is influenced by the number of cells generated in the first wave and, mostly likely, by the level of Fgf signalling in the ICM. Differences in the developmental potential of eight-cell- and 16-cell-stage outside blastomeres placed in the inside of chimaeric embryos further support this conclusion. These results unite previous findings demonstrating the importance of developmental history and Fgf signalling in determining cell fate.     

Specification process of animal plate in the sea urchin embryo

The most animal part of the ciliated band of sea urchin larvae, the animal plate, is a specialized region in which elongated cells form long and non-beating cilia. To learn how this region is specified, animal halves were isolated from the early cleavage to pregastrulation stages. As is well known, the animal half that is isolated at the eight-cell stage develops into a 'dauerblastula', which forms long and non-beating cilia all around the surface. The region with long cilia, however, became restricted toward the animal pole when separation was delayed. If separated before primary mesenchyme ingression, even a small animal-pole-side fragment formed a normal-sized animal plate. Thus, the prospective animal plate region is gradually restricted by some signal from the vegetal hemisphere, and the specification process terminates before the mesenchyme blastula stage. It was also known that a normal-sized animal plate was formed in micromere-less embryos, indicating that the signal does not depend on micromeres or their descendants. Further, the animal-pole-side fragments were isolated from embryos in which the third cleavage plane was shifted toward the vegetal pole. They formed a normal-sized animal plate, containing more than 75% of the egg volume from the animal pole. This indicates that the egg cytoplasm delivered to veg₁ -lineage blastomeres plays an important role in the animal plate specification. Interestingly, the an1-less embryo formed long and non-beating cilia at its top region, but thickening did not occur. The cytoplasm near the animal pole might contain some factors necessary for the animal plate to become thick.     

An FGF autocrine loop initiated in second heart field mesoderm regulates morphogenesis at the arterial pole of the heart

In order to understand how secreted signals regulate complex morphogenetic events, it is crucial to identify their cellular targets. By conditional inactivation of Fgfr1 and Fgfr2 and overexpression of the FGF antagonist sprouty 2 in different cell types, we have dissected the role of FGF signaling during heart outflow tract development in mouse. Contrary to expectation, cardiac neural crest and endothelial cells are not primary paracrine targets. FGF signaling within second heart field mesoderm is required for remodeling of the outflow tract: when disrupted, outflow myocardium fails to produce extracellular matrix and TGFbeta and BMP signals essential for endothelial cell transformation and invasion of cardiac neural crest. We conclude that an autocrine regulatory loop, initiated by the reception of FGF signals by the mesoderm, regulates correct morphogenesis at the arterial pole of the heart. These findings provide new insight into how FGF signaling regulates context-dependent cellular responses during development.     

Mechanism of First Cleavage Specification in the Mouse Egg: Is Our Body Plan Set at Day 0?

In most animals the body axis is specified in the egg. Because of their highlyregulative capacity after experimental manipulations,1-4 mammalianpreimplantation embryos have long been thought to be an exception to this rule,lacking polarity until the blastocyst stage. However, it has recently been suggested5-7 that the embryonic-abembryonic (Em-Ab) axis of the mouse blastocyst arisesperpendicular to the first cleavage plane. Considering the second polar body (2pb)as a stationary marker for the “animal pole (A-pole)” during preimplantationdevelopment,5,6 the authors concluded that the polarity of the mouse embryo isalready specified in the egg, as is the case for most non-mammalian animals.5-7However, the results of our recent time-lapse recordings have shown8 that in 50 %of the embryos the first cleavage occurs at a considerable distance from the“animal-vegetal (A-V) axis” and that the 2pb moves towards the first cleavageplane, in contrast to the previous claims. Thus, there is no predetermined axis in themouse egg. We also presented a novel model for specification of the first cleavageplane: this is defined as the plane separating the two apposing pronuclei that havemoved to the center of the egg. In this review we will elucidate the discrepancybetween the previous model and our model, and discuss the possible causes.     

Cell fate in the polar trophectoderm of mouse blastocysts as studied by microinjection of cell lineage tracers

Horseradish peroxidase (HRP), together with Fast Green or rhodamine-conjugated dextran (RDX), was used as an intracellular lineage tracer to determine cell fate in the polar trophectoderm of 3.5-day-old mouse embryos. In HRP-injected midstage (approximately 39-cell) and expanded (approximately 65-cell) blastocysts incubated for 24 hr, the central polar trophectoderm cell was displaced from the embryonic pole an average of 20 micron (5% of blastocyst circumference) and 29 micron (6% of blastocyst circumference), respectively. Expanded blastocysts injected with HRP + Fast Green and incubated for 24 hr or with HRP + RDX and incubated for 48 hr showed a displacement of 24 micron (4% of blastocyst circumference) and 88 micron (14% of blastocyst circumference), respectively. Up to 10 HRP-positive trophectoderm cells were observed among embryos incubated for 48 hr, indicating that in those cases, the labeled progenitor cells had divided at least three times. Our observations show that the central polar trophectoderm cell divides in the plane of the trophectoderm in expanded blastocysts and, along with its descendants, is displaced toward the mural trophectoderm. The systematic tandem displacement of labeled cells and their descendants toward the abembryonic pole suggests the presence of a proliferative area at the embryonic pole of the blastocyst. Large shifts in inner cell mass (ICM) position in relation to the trophectoderm do not occur during blastocyst expansion. Furthermore, random movements within the polar trophectoderm population do not account for the replacement of labeled cells by unlabeled polar trophectoderm cells. Rather, we propose the hypothesis that the ICM contributes these replacement cells to the polar trophectoderm during blastocyst expansion.     

Genetic mosaic analysis reveals FGF receptor 2 function in terminal end buds during mammary gland branching morphogenesis

FGF signaling is associated with breast cancer and is required for mammary placode formation in the mouse. In this study, we employed a genetic mosaic analysis based on Cre-mediated recombination to investigate FGF receptor 2 (Fgfr2) function in the postnatal mammary gland. Mosaic inactivation of Fgfr2 by the MMTV-Cre transgene enabled us to compare the behavior of Fgfr2 null and Fgfr2 heterozygous cells in the same gland. Fgfr2 null cells were at a competitive disadvantage to their Fgfr2 heterozygous neighbors in the highly proliferative terminal end buds (TEBs) at the invasion front, owing to a negative effect of loss of Fgfr2 function on cell proliferation. However, Fgfr2 null cells were tolerated in mature ducts. In these genetic mosaic mammary glands, the epithelial network is apparently built by TEBs that over time are composed of a progressively larger proportion of Fgfr2-positive cells. However, subsequently, most cells lose Fgfr2 function, presumably due to additional rounds of Cre-mediated recombination. Using an independent strategy to create mosaic mammary glands, which employed an adenovirus-Cre that acts only once, we confirmed that Fgfr2 null cells were out-competed by neighboring Fgfr2 heterozygous cells. Together, our data demonstrate that Fgfr2 functions in the proliferating and invading TEBs, but it is not required in the mature ducts of the pubertal mammary gland.     

Fgfr1 and Fgfr2 have distinct differentiation- and proliferation-related roles in the developing mouse skull vault

Fibroblast growth factor receptors (FGFRs) play major roles in skeletogenesis, and activating mutations of the human FGFR1, FGFR2 and FGFR3 genes cause premature fusion of the skull bones (craniosynostosis). We have investigated the patterns of expression of Fgfr1, Fgfr2 and Fgfr3 in the fetal mouse head, with specific reference to their relationship to cell proliferation and differentiation in the frontal and parietal bones and in the coronal suture. Fgfr2 is expressed only in proliferating osteoprogenitor cells; the onset of differentiation is preceded by down-regulation of Fgfr2 and up-regulation of Fgfr1. Following up-regulation of the differentiation marker osteopontin, Fgfr1, osteonectin and alkaline phosphatase are down-regulated, suggesting that they are involved in the osteogenic differentiation process but not in maintaining the differentiated state. Fgfr3 is expressed in the cranial cartilage, including a plate of cartilage underlying the coronal suture, as well as in osteogenic cells, suggesting a dual role in skull development. Subcutaneous insertion of FGF2-soaked beads onto the coronal suture on E15 resulted in up-regulation of osteopontin and Fgfr1 in the sutural mesenchyme, down-regulation of Fgfr2, and inhibition of cell proliferation. This pattern was observed at 6 and 24 hours after bead insertion, corresponding to the timing and duration of FGF2 diffusion from the beads. We suggest (a) that a gradient of FGF ligand, from high levels in the differentiated region to low levels in the environment of the osteogenic stem cells, modulates differential expression of Fgfr1 and Fgfr2, and (b) that signalling through FGFR2 regulates stem cell proliferation whereas signalling through FGFR1 regulates osteogenic differentiation.     

Early embryonic lethality of mice lacking POLD2

As a highly conserved DNA polymerase (Pol), Pol δ plays crucial roles in chromosomal DNA synthesis and various DNA repair pathways. However, the function of POLD2, the second small subunit of DNA Pol δ (p50 subunit), has not been characterized in vivo during mammalian development. Here, we report for the first time, the essential role of subunit POLD2 during early murine embryogenesis. Although Pold2 mutant mouse embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at gastrulation stages. Outgrowth assays reveal that mutant blastocysts cannot hatch from the zona pellucida, indicating impaired blastocyst function. Notably, these phenotypes can be recapitulated by small interfering RNA (siRNA)-mediated knockdown, which also exhibit slowed cellular proliferation together with skewed primitive endoderm and epiblast allocation during the second cell lineage specification. In summary, our study demonstrates that POLD2 is essential for the earliest steps of mammalian development, and the retarded proliferation and embryogenesis may also alter the following cell lineage specifications in the mouse blastocyst embryos.     

Independent roles of Fgfr2 and Frs2alpha in ureteric epithelium

Mice with conditional deletion of fibroblast growth factor receptor 2 (Fgfr2) in the ureteric bud using a Hoxb7cre line (Fgfr2(UB-/-)) develop severe ureteric branching defects; however, ureteric deletion of fibroblast growth factor receptor substrate 2α (Frs2α), a key docking protein that transmits fibroblast growth factor receptor intracellular signaling (Frs2α(UB-/-)) leads to mild ureteric defects. Mice with point mutations in the Frs2α binding site of Fgfr2 (Fgfr2(LR/LR)) have normal kidneys. The aim of this study was to determine the relationship between Fgfr2 and Frs2α in the ureteric lineage. Mice with ureteric deletion of both Fgfr2 and Frs2α (Fgfr2/Frs2α(UB-/)) were compared with Frs2α(UB-/-) and Fgfr2(UB-/-) mice. To avoid potential rescue of Fgfr1 forming heterodimers with Fgfr2(LR) alleles to recruit Frs2α, compound mutant mice were generated with ureteric deletion of Fgfr1 and with Fgfr2(LR/LR) point mutations (Fgfr1(UB-/-)Fgfr2(LR/LR)). At E13.5, three-dimensional reconstructions and histological assessment showed that, whereas Fgfr2(UB-/-) kidneys had more severe ureteric branching defects than Frs2α(UB-/-), Fgfr2(UB-/-) kidneys were indistinguishable from Fgfr2/Frs2α(UB-/-). At later stages, however, Fgfr2/Frs2α(UB-/-) kidneys were more severely affected than either Fgfr2(UB-/-) or Frs2α(UB-/-) kidneys. Taken together, although Fgfr2 and Frs2α have crucial roles in the ureteric lineage, they appear to act separately and additively.     

The IIIb isoform of fibroblast growth factor receptor 2 is required for proper growth and branching of pancreatic ductal epithelium but not for differentiation of exocrine or endocrine cells

Fibroblast growth factors (Fgfs) and their receptors have been implicated in embryonic pancreas development. Recently it was shown that Fgf10, a major ligand for the IIIb isoform of fibroblast growth factor receptor 2 (Fgfr2b), has an important regulatory role in early pancreas development. The aim of our study was to define the role of Fgfr2b in pancreas development by analyzing the phenotype of Fgfr2b (-/-) mice. Pancreases of Fgfr2b (-/-) embryos were noticeably smaller than the wild type littermates during embryogenesis, and pancreatic ductal branching as well as duct cell proliferation was significantly reduced. However, both exocrine and endocrine pancreatic differentiation occurred relatively normally. Exogenous addition of Fgfr2b ligands (Fgf7 and Fgf10) stimulated duct cell proliferation and inhibited endocrine cell differentiation in the ex vivo embryonic organ cultures of wild type pancreas. Our results thus suggest that Fgfr2b-mediated signaling plays a major role in pancreatic ductal proliferation and branching morphogenesis, but has little effect on endocrine and exocrine differentiation.