Akt/PKB regulates laminin and collagen IV isotypes of the basement membrane.

Basement membranes are important for epithelial differentiation, cell survival, and normal and metastatic cell migration. Much is known about their breakdown and remodeling, yet their positive regulation is poorly understood. Our previous analysis of a fibroblast growth factor (FGF) receptor mutation raised the possibility that protein kinase B (Akt/PKB) activated by FGF is connected to the expression of certain laminin and type IV collagen isotypes. Here we test this hypothesis and demonstrate that constitutively active Akt/PKB, an important downstream element of phosphoinositide 3'-kinase signaling, induces the synthesis of laminin-1 and collagen IV isotypes and causes their translocation to the basement membrane. By using promoter-reporter constructs, we show that constitutively active phosphoinositide 3'-kinase-p110 or Akt/PKB activates, whereas dominant negative Akt/PKB inhibits, transcription of laminin beta1 and collagen IV alpha1 in differentiating C2 myoblast- and insulin-induced Chinese hamster ovary-T cell cultures. These results suggest that Akt/PKB activated by receptor tyrosine kinases is involved in the positive regulation of basement membrane formation. The possible role of Akt/PKB-induced laminin and collagen IV synthesis in cell survival and differentiation will be discussed.

Fibroblast growth factor signaling and basement membrane assembly are connected during epithelial morphogenesis of the embryoid body.

Fibroblast growth factors and receptors are intimately connected to the extracellular matrix by their affinity to heparan sulfate proteoglycans. They mediate multiple processes during embryonic development and adult life. In this study, embryonic stem cell-derived embryoid bodies were used to model fibroblast growth factor signaling during early epithelial morphogenesis. To avoid redundancy caused by multiple receptors, we employed a dominant negative mutation of Fgfr2. Mutant-derived embryoid bodies failed to form endoderm, ectoderm, and basement membrane and did not cavitate. However, in mixed cultures they displayed complete differentiation induced by extracellular products of the normal cell. Evidence will be presented here that at least one of these products is the basement membrane or factors connected to it. It will be shown that in the mutant, collagen IV and laminin-1 synthesis is coordinately suppressed. We will demonstrate that the basement membrane is required for embryoid body differentiation by rescuing columnar ectoderm differentiation and cavitation in the mutant by externally added basement membrane proteins. This treatment induced transcription of Eomesodermin, an early developmental gene, suggesting that purified basement membrane proteins can activate inherent developmental programs. Our results provide a new paradigm for the role of fibroblast growth factor signaling in basement membrane formation and epithelial differentiation.

Fibroblast growth factor (FGF) signaling through PI 3-kinase and Akt/PKB is required for embryoid body differentiation.

The role of FGF signaling in early epithelial differentiation was investigated in ES (embryonic stem) cell derived embryoid bodies. A dominant negative fibroblast growth factor receptor (FGFR) mutation was created by stably introducing into ES cells an Fgfr2 cDNA, truncated in its enzymatic domains. These cells failed to differentiate into cystic embryoid bodies. No epithelial differentiation and cavitation morphogenesis could be observed, in the mutant, although its rate of cell proliferation remained unchanged. This phenotype was associated with a significant decrease in the activation of Akt/PKB and PLCgamma-1, as compared to the wild type, while the activation of MAPK/Erk was less affected. Requirement for PI 3-kinase signaling in embryoid body differentiation was demonstrated by specific inhibitors. Akt/PKB activation was abrogated by wortmannin in short-term experiments. In long-term cultures Ly294002 inhibited the differentiation of ES cells into embryoid bodies. Our data demonstrate that for early epithelial differentiation FGF signaling is required through the PI 3-kinase-Akt/ PKB pathway.

Fgfr2 is required for limb outgrowth and lung-branching morphogenesis.

The aim of this study was to clarify the role of Fgfr2 during later stages of embryonic development. Of two previously reported gene-targeting experiments, the more extensive Fgfr2 deletion was lethal shortly after implantation, because of trophoblast defects, whereas the less extensive one survived until midgestation with placental insufficiency and defective limb outgrowth [Xu, X., Weinstein, M., Li, C., Naski, M., Cohen, R. I., Ornitz, D. M., Leder, P. & Deng, C. (1998) Development (Cambridge, U.K.) 125, 753-765]. Fgfr2 in the early embryo is expressed in the trophectoderm, and this extra-embryonic localization persists into mid- and late gestation, when Fgfr2 also is expressed in multiple developing organs. To gain insight into the later functions of Fgfr2, fusion chimeras were constructed from homozygous mutant embryonic stem cells and wild-type tetraploid embryos. This allowed survival until term and revealed that Fgfr2 is required for both limb outgrowth and branching lung morphogenesis. The use of fusion chimeras demonstrated that early lethality was indeed because of trophectoderm defects and indicated that in the embryonic cell lineages Fgfr2 activity manifests in limb and lung development. Highly similar lung and limb phenotypes were detected recently in the loss of function mutation of Fgf10, a ligand of Fgfr2. It is likely, therefore, that whereas during early development Fgfr2 interacts with Fgf4, in limb and lung development interactions between Fgf10 and Fgfr2 may be required. Possible epithelial-mesenchymal interactions between the splicing alternatives of Fgfr2 and their specific ligands will be discussed.

Expression of Fgfr2 in the early mouse embryo indicates its involvement in preimplantation development.

We report that the IIIc transcriptional alternative of Fgfr2 is transcribed in the unfertilized egg and that during early zygotic transcription, messages encoded by both Fgfr2 alternatives (IIIc and IIIb) are present. The Fgfr2 protein was first detected in peripheral blastomeres of compacted morulae. Trophectoderm specificity of Fgfr2 became obvious in the early blastocyst and with maturation its localization underwent further specification, Fgfr2 concentration increased at the abembryonic pole and decreased at the embryonic pole. Moreover Fgfr2 expression became markedly asymmetrical along the animal-vegetal axis of the mature blastocyst. Our observations indicate a role for Fgfr2 in trophectoderm growth and specification and in the orientation and polarity of the preimplantation conceptus.

A mouse model for achondroplasia produced by targeting fibroblast growth factor receptor 3.

Achondroplasia, the most common form of dwarfism in man, is a dominant genetic disorder caused by a point mutation (G380R) in the transmembrane region of fibroblast growth factor receptor 3 (FGFR3). We used gene targeting to introduce the human achondroplasia mutation into the murine FGFR3 gene. Heterozygotes for this point mutation that carried the neo cassette were normal whereas neo+ homozygotes had a phenotype similar to FGFR3-deficient mice, exhibiting bone overgrowth. This was because of interference with mRNA processing in the presence of the neo cassette. Removal of the neo selection marker by Cre/loxP recombination yielded a dominant dwarf phenotype. These mice are distinguished by their small size, shortened craniofacial area, hypoplasia of the midface with protruding incisors, distorted brain case with anteriorly shifted foramen magnum, kyphosis, and narrowed and distorted growth plates in the long bones, vertebrae, and ribs. These experiments demonstrate that achondroplasia results from a gain-of-FGFR3-function leading to inhibition of chondrocyte proliferation. These achondroplastic dwarf mice represent a reliable and useful model for developing drugs for potential treatment of the human disease.

Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally.

Homozygous targeted disruption of the mouse Caspase 8 (Casp8) gene was found to be lethal in utero. The Caspase 8 null embryos exhibited impaired heart muscle development and congested accumulation of erythrocytes. Recovery of hematopoietic colony-forming cells from the embryos was very low. In fibroblast strains derived from these embryos, the TNF receptors, Fas/Apo1, and DR3 were able to activate the Jun N-terminal kinase and to trigger IkappaB alpha phosphorylation and degradation. They failed, however, to induce cell death, while doing so effectively in wild-type fibroblasts. These findings indicate that Caspase 8 plays a necessary and nonredundant role in death induction by several receptors of the TNF/NGF family and serves a vital role in embryonal development.

Complex regulation of multiple cytohesin-like genes in murine tissues and cells.

Three cytohesin-like cDNA molecules were isolated from murine ES cell derived embryoid bodies. The genomic structure of one of the three, CLM2, has been determined and transcriptional variants of each were isolated from a mouse brain cDNA library. The relative expression patterns of CLM1, 2, 3 and their transcriptional alternatives were determined by RT-PCR, nucleotide sequencing and RNA blotting. Their broad distribution and cell and tissue specific expression patterns suggest complex regulation during development and in the adult.

Maternally expressed PGK-Cre transgene as a tool for early and uniform activation of the Cre site-specific recombinase.

A transgenic mouse strain with early and uniform expression of the Cre site-specific recombinase is described. In this strain, PGK-Crem, Cre is driven by the early acting PGK-1 promoter, but, probably due to cis effects at the integration site, the recombinase is under dominant maternal control. When Cre is transmitted by PGK-Crem females mated to males that carry a reporter transgene flanked by loxP sites, even offspring that do not inherit PGK-Cre delete the target gene. It follows that in the PGK-Crem female Cre activity commences in the diploid phase of oogenesis. In PGK-Crem crosses complete recombination was observed in all organs, including testis and ovary. We prepared a mouse stock that is homozygous for PGK-Crem and at the albino (c) locus. This strain will be useful for the early and uniform induction of ectopic and dominant negative mutations, for the in vivo removal of selective elements from targeted mutations and in connection with the manipulation of targeted loci in 'knock in' and related technologies.

Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development.

We disrupted the fibroblast growth factor (FGF) receptor 2 (FGFR2) gene by introducing a neo cassette into the IIIc ligand binding exon and by deleting a genomic DNA fragment encoding its transmembrane domain and part of its kinase I domain. A recessive embryonic lethal mutation was obtained. Preimplantation development was normal until the blastocyst stage. Homozygous mutant embryos died a few hours after implantation at a random position in the uterine crypt, with collapsed yolk cavity. Mutant blastocysts hatched, adhered, and formed a layer of trophoblast giant cells in vitro, but after prolonged culture, the growth of the inner cell mass stopped, no visceral endoderm formed, and finally the egg cylinder disintegrated. It follows that FGFR2 is required for early postimplantation development between implantation and the formation of the egg cylinder. We suggest that FGFR2 contributes to the outgrowth, differentiation, and maintenance of the inner cell mass and raise the possibility that this activity is mediated by FGF4 signals transmitted by FGFR2. The role of early FGF signaling in pregastrulation development as a possible adaptation to mammalian (amniote) embryogenesis is discussed.

Transgenic mouse model for studying the transcriptional activity of the p53 protein: age- and tissue-dependent changes in radiation-induced activation during embryogenesis.

The p53 tumor suppressor protein is a sequence-specific transcriptional activator of target genes. Exposure of cells to DNA damage results in accumulation of biochemically active p53, with consequent activation of p53-responsive promoters. In order to study how the transcriptional activity of the p53 protein is regulated in vivo, a transgenic mouse strain was generated. These mice harbor the p53-dependent promoter of the mdm2 gene, fused to a lacZ reporter gene. Induction of lacZ activity by DNA damage (ionizing radiation) was monitored in embryos of different p53 genotypes. The transgenic promoter was substantially activated in vivo following irradiation; activation required functional p53. The activation pattern became more restricted with increasing embryo age, as well as with the state of differentiation of a given tissue. Generally, maximal p53 activation occurred in rapidly proliferating, relatively less differentiated cells. A striking extent of haploinsufficiency was revealed-induction of promoter activity was far less efficient in mice carrying only one wild-type p53 allele. This suggests that normal levels of cellular p53 are limiting, and any further reduction already compromises the p53 response significantly. Thus, the activation potential of p53 is tightly controlled in vivo, both spatially and temporally, and an important element in this control is the presence of limiting basal levels of activatable p53.

Differential expression of NDF/neuregulin receptors ErbB-3 and ErbB-4 and involvement in inhibition of neuronal differentiation.

Two receptor tyrosine kinases, ErB-3 and ErbB-4, mediate signaling by Neu differentiation factors (NDFs, also called neuregulins), while ErbB-1 and ErbB-2 serve as co-receptors. We show that the two NDF/neuregulin receptors differ in spatial and temporal expression patterns: The kinase-defective receptor, ErbB-3, is expressed primarily in epithelial layers of various organs, in the peripheral nervous system, and in adult brain, whereas ErbB-4 is restricted to the developing central nervous system and to the embryonic heart. An example of alternating expression of the two receptors is provided by the developing cerebellum: During postnatal cerebellar development, ErbB-4 expression slightly decreases along with a decline in NDF transcription, whereas ErbB-3 expression commences after the peak of neurogenesis. To study functional differences, we established primary brain cultures and found that ErbB-3 was expressed only in oligodendrocytes, whereas ErbB-4 expression was shared by oligodendrocytes, astrocytes and neurons. Blocking the action of endogenous NDF in vitro, by using a soluble form of ErbB-4, accelerated neurite outgrowth in both primary cultures and in neuronal-type cultures of the P19 teratocarcinoma, suggesting an inhibitory effect of NDF on neural differentiation. Apparently, ErbB-3 is associated with proliferation of P19 cells, whereas ErbB-4 correlates with a differentiated phenotype. We conclude that the two NDF receptors play distinct, rather than redundant, developmental and physiological roles.

Early expression of D3 dopamine receptors in murine embryonic development.

In order to determine whether the D2 and D3 dopamine receptors may have a role in prenatal development, we have studied the mRNA expression and distribution of these receptors during murine embryonic development. Using RT-PCR on RNA from embryos taken at progressive stages of development, we have shown that the D3 receptor is expressed significantly earlier than the D2 receptor, being detectable at day 9.5 post-conception (p.c.) compared with day 13.5 p.c. for the D2 subtype. We have also examined the mRNA distribution of the two receptors by whole mount in situ hybridisation. In agreement with the PCR assays, the D3 receptor was expressed earlier than the D2 subtype. D3 receptor transcripts were first detected at day 9.5 p.c. in the ventral aspect of the anterior neural tube, whereas D2 receptor transcripts first appeared a day later. By day 10.5-11.5 p.c. both D3 and D2 receptor transcripts were present in the developing forebrain, and later also in the branchial arches and along the prospective vertebral column. The early appearance of the D3 subtype in murine development and its predominance over the D2 subtype suggest that the D3 receptor may have a functional role in prenatal development.

Ectopic expression of Fgf-4 in chimeric mouse embryos induces the expression of early markers of limb development in the lateral ridge.

The biological consequences of constitutive fibroblast growth factor-4 (fgf-4) expression were investigated in chimeric embryos prepared between wild-type host embryos and murine ES cells transfected with a construct in which expression of the murine fgf-4 gene was directed by the phosphoglycerate kinase (PGK-1) promoter. The embryos exhibit abnormalities of the limbs and the anterior central nervous system (CNS). The limb phenotype comprised the induction of outgrowth along the lateral ridge between the definitive fore and hind limbs resembling the early phases of limb development. The CNS defects comprised a complete absence, or marked reduction in forebrain and midbrain structures and rudimentary or absent eye development. Constitutive expression of fgf-4 was also accompanied by ectopic expression of the sonic hedgehog (shh) and msx-1 genes in the lateral ridge. These findings indicate that FGF exhibits multiple activities in early development which include the ability to induce the expression of early markers of limb development in the lateral ridge.

Analysis of the Hoxd-3 gene: structure and localization of its sense and natural antisense transcripts.

This study set out to investigate the structure and localized expression of the mouse homeobox-containing gene Hoxd-3. In addition to identifying a transcript of the type known from other Antennapedia (Antp)-like mammalian homeobox cDNAs, an antisense transcript was also detected. The antisense form of Hoxd-3 overlaps with 603 bp of the sense transcript including the homeobox. Active antisense transcription has been confirmed by RNA blot analysis with single-stranded probes and by the direction of splicing of an intron in the antisense transcript. The localized expression of sense and antisense transcripts was compared by in situ hybridization. Hoxd-3 expression was observed from 8.5 days p.c., in the neural tube with a sharp border in the hind brain at the level of rhombomeres 4-5. In contrast, the earliest antisense expression was detected at 10.5 days p.c. in cDNA libraries. At 12.5 days p.c., sense and antisense transcripts colocalized in the liver. The possible role of antisense homeobox transcripts during liver and the hematopoietic development is discussed.

Developmental localization of the splicing alternatives of fibroblast growth factor receptor-2 (FGFR2).

The gene for fibroblast growth factor receptor-2 (FGFR2) encodes two splice variants designated here as keratinocyte growth factor (KGFR) and bek. Their ligand-binding specificity is markedly different due to mutually exclusive alternative splicing. We asked whether alternative exon usage, in addition to influencing receptor specificity, could be correlated with transcriptional localization. This problem was studied by in situ hybridization and PCR, using probes and primers specific for the alternative exons of FGFR2. Transcripts of both variants were detected in all three germ layers within the embryonic and the extraembryonic areas of the primitive-streak embryo. The overall level of KGFR expression surpassed that of bek. The localized expression of both variant receptors was, however, more diffuse in the gastrula than later during organogenesis, when KGFR transcripts were evident mainly in epithelia, whereas bek was present in the corresponding mesenchymes. Our findings show the following: (1) Expression of both FGFR2 variants is concordant with their involvement in murine gastrulation. They may endow competence to multiple areas, which may be restricted by their more confined ligands. (2) KGFR and bek seem to have unique roles in the development of the skin and its derivatives, whereas bek is preferentially expressed during osteogenesis. The two variants share potential regions of trans regulation in the genome; hence, we suggest that alternative splicing is jointly responsible for ligand binding and spatial specificity. (3) Finally, we defined the binding specificity of KGFR and bek to various FGF. The possibility of identifying specific functional areas for certain ligand-receptor pairs is discussed.

Murine prosaposin: expression in the reproductive system of a gene implicated in human genetic diseases.

Expression of the prosaposin gene, implicated in human genetic diseases, was studied in the mouse by Northern blot analysis and in situ hybridization. Using a cloned mouse prosaposin cDNA as a probe, we observed high expression of the prosaposin gene in the adult and embryonic nervous and reproductive systems while lower levels of prosaposin mRNA were detected in the heart, kidneys, liver, adrenals and lymphoid organs. Ubiquitous low level of prosaposin mRNA were detected by in situ hybridization in 12.5 days-old embryos. However, high expression was restricted to the hindbrain, the dorsal ganglia and to the genital ridge, the primordium for both male and female gonads. RNA transcription in the testis was detected in Sertoli cells, Leydig cells and peritubular cells but not in germ cells of adult mice. Similar observations were obtained with the atrichosis mutant mouse known to lack normal germ cells, indicating that prosaposin gene expression was not dependent on the presence of germ cells in the testis. Prosaposin was found to be expressed in the mature female gonads at various stages of the corpus luteum development. These results strongly suggest that prosaposin may play an important role in the reproductive system as well as the nervous system.

Neural expression and chromosomal mapping of Neu differentiation factor to 8p12-p21.

Neu differentiation factor (NDF/heregulin) is a 44-kDa glycoprotein that interacts with the Neu/ErbB-2 receptor tyrosine kinase to increase its phosphorylation on tyrosine residues. In vitro NDF promotes differentiation of certain mammary tumor cell lines to milk-producing cells. As a first step toward understanding the physiological role of NDF, we performed in situ hybridization analyses to determine mRNA distribution in the mouse embryo and to map the gene to human karyotypes. In 14.5-day-postcoitum mouse embryos, NDF expression is confined predominantly to the central and peripheral nervous system, including the neuroepithelium that lines the lateral ventricles of the brain, the ventral horn of the spinal cord, and the intestinal as well as dorsal root ganglia. Other tissues that contain NDF transcripts are the adrenal gland, liver, and distinct cell layers of the dermis and germinal ridge. In situ hybridization of a 3H-labeled probe to human metaphase spreads localized the NDF gene to the short arm of chromosome 8 at bands p12-p21.