A role for FGF-8 in the initiation and maintenance of vertebrate limb bud outgrowth.

BACKGROUND: The outgrowth of the vertebrate limb bud is the result of a reciprocal interaction between the mesenchyme and a specialized region of the ectoderm, the apical ectodermal ridge (AER), which overlies it. Signals emanating from the AER act to maintain the underlying mesenchyme, called the progress zone, in a highly proliferative and undifferentiated state. Removal of the AER results in the cessation of limb bud growth, thus causing limb truncation. The best candidates for this AER-derived signal are members of the fibroblast growth factor (FGF) family, in particular FGF-4, which can maintain limb bud outgrowth following removal of the AER. However, FGF-4 is only expressed after considerable outgrowth has occurred and a well-developed limb bud has formed, and then only in the posterior part of the AER. Likewise, the other FGFs studied to date are not candidates for this activity. RESULTS: We report evidence that a recently identified member of this family, FGF-8, is expressed in the ectoderm of the prospective limb territory prior to morphological outgrowth of the limb bud in both mouse and chick. Thereafter, expression is maintained throughout the AER during limb development. We have produced and purified the FGF-8 protein, and shown that it will substitute for the AER in maintaining limb bud outgrowth in mouse embryos from which the AER has been surgically removed. FGF-8 does not, however, maintain expression of the sonic hedgehog gene. CONCLUSIONS: These results indicate that FGF-8 is an AER-derived mitogen that stimulates limb bud outgrowth. Moreover, our data suggest that FGF-8 may also be an ectodermally derived mitogen that stimulates the onset of limb bud outgrowth (budding) in the absence of a morphological AER, and indicate the possible involvement of FGF-8 in the establishment of the limb field.

Elevation of D4 dopamine receptor mRNA in postmortem schizophrenic brain.

The D4 dopamine (DA) receptor has been proposed to be a target for the development of a novel antipsychotic drug based on its pharmacological and distribution profile. There is much interest in whether D4 DA receptor levels are altered in schizophrenia, but the lack of an available receptor subtype-specific radioligand made this difficult to quantitate. In this study, we examined whether D4 mRNA levels are altered in different brain regions of schizophrenics compared to controls. Ribonuclease protection assays were carried out on total RNA samples isolated postmortem from frontal cortex and caudate brain regions of schizophrenics and matched controls. 32P-labelled RNA probes to the D4 DA receptor and to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), were hybridised with the RNA samples, digested with ribonucleases to remove unhybridised probe, and separated on 6% sequencing gels. Densitometer analysis on the subsequent autoradiogams was used to calculate the relative optical density of D4 mRNA compared to G3PDH mRNA. Statistical analysis of the data revealed a 3-fold higher level (P<0.011) of D4 mRNA in the frontal cortex of schizophrenics compared to controls. No increase was seen in caudate. D4 receptors could play a role in mediating dopaminergic activity in frontal cortex, an activity which may be malfunctioning in schizophrenia.

Identification and characterization of a truncated variant of the 5-hydroxytryptamine(2A) receptor produced by alternative splicing.

We have identified an alternatively spliced 5-hydroxytryptamine 2A receptor (5-HT(2A)-R) transcript by PCR of human brain cDNA using degenerate oligonucleotide primers to transmembrane (TM) domains 3 and 7 of the 5-HT(2)-R subfamily. The variant contains a 118-bp insertion at the exon II/III boundary of the 5-HT(2A)-R, which produces a frame shift in the coding sequence and a premature stop codon. PCR analysis showed that the truncated receptor (5-HT(2A-tr)) and native 5-HT(2A)-R were co-expressed in most brain tissues, with the highest levels being found in hippocampus, corpus collosum, amygdala and caudate nucleus. Western blot analysis of HEK-293 cells transfected transiently with a 5-HT(2A-tr) construct showed that a 30-kDa protein was expressed on cell membranes. Co-transfection studies showed no effect of the 5-HT(2A-tr) variant on 3H-ketanserin binding to the native 5-HT(2A)-R or on functional coupling of the 5-HT(2A)-R to 5-HT-stimulated Ca(2+) mobilization. The functional significance of the 5-HT(2A-tr) variant and other truncated receptors remains to be established.

Fibroblast growth factor signalling and cyclin D1 function are necessary for normal mammary gland development during pregnancy. A transgenic mouse approach.

A number of growth factors, growth factor receptors and cell cycle regulatory proteins have been implicated in the genesis of mammary carcinomas both in animal models as well as in human breast tumour samples. Studies on the development of the mammary gland has revealed that several of the proto-oncogenes, or their closely related gene-family members, have a function in the normal growth and differentiation of the gland. In this review the role of fibroblast growth factor signalling and the critical requirement for the cell cycle regulator, cyclin D1 is discussed with respect to their normal function in mammary gland development and abnormal role in mammary carcinogenesis.

A role for fibroblast growth factor signaling in the lobuloalveolar development of the mammary gland.

The inappropriate expression of growth factors, or activating mutations of their receptors, have been implicated as causative factors in mouse and human mammary cancer. For example, it has been known for some time that three members of the fibroblast growth factor (FGF) family behave like oncogenes in virally induced mammary cancer of mice. In normal circumstances, signaling via FGF receptors is known to mediate growth, differentiation, and patterning, during embryogenesis and fetal development. A powerful approach to dissecting the roles for these signaling pathways is to determine the developmental consequences of abrogating their function in transgenic mice. In this review, we describe the use of dominant negative FGF receptors to evaluate the contribution of specific FGF signals in normal mammary gland development. These studies have revealed that normal lobuloalveolar development requires FGF signaling to the mammary epithelium, a function that is presumably usurped by MMTV in mouse mammary tumorigenesis.

Receptor binding and mitogenic properties of mouse fibroblast growth factor 3. Modulation of response by heparin.

fgf3 has been implicated in the embryonic and fetal development of the mouse and as an oncogene in murine breast cancer. We describe a procedure to purify the product of the mouse fgf3 gene and show it to be a potent mitogen for some epithelial cell lines. Using a receptor binding competition assay, Fgf3 was shown to bind with high affinity to the IIIb isoforms of Fgf receptor (FgfR) 1 and FgfR2 (ID50 = approximately 0.8 nM) and with a lower affinity to the IIIc variant of FgfR2 (ID50 = approximately 9 nM). No competition for the binding of 125I-Fgf1 was observed for FgfR1 (IIIc), FgfR3 (IIIb and IIIc), or FgfR4. Mitogenicity assays using BaF3 cells containing individual Fgf receptors showed a pattern of response in agreement with the receptor binding results. A comparison of two mammary epithelial cell lines showed a marked difference of potency and dependence upon heparin in their response to mouse Fgf3, suggesting a complex interaction between the ligand and its low and high affinity receptors.

Fibroblast growth factor receptor signalling has a role in lobuloalveolar development of the mammary gland.

We have used the mouse mammary tumor virus promoter to express two dominant negative (DN) fibroblast growth factor receptor (FGFR) isoforms in the mammary epithelium of transgenic mice. While expression of DN-FGFR1(IIIc) showed no discernible phenotype, a similar kinase negative form of FGFR2(IIIb) caused a marked impairment of lobuloalveolar development. The growth retardation was apparent by mid-pregnancy and persisted in the post-partum glands. Despite the substantial underdevelopment of the mammary gland there was a measurable lactational response, but it was insufficient to properly sustain the new-born pups. These findings demonstrate that fibroblast growth factor signalling is necessary for pregnancy dependent lobuloalveolar development of the mammary gland.