A mouse homologue of FAST-1 transduces TGF beta superfamily signals and is expressed during early embryogenesis.

The transcription factor FAST-1 has recently been shown to play a key role in the specification of mesoderm by TGF beta superfamily signals in the early Xenopus embryo. We have cloned Fast1, a mouse homologue of Xenopus FAST-1, and characterized its expression during embryogenesis and function in activin/TGF beta signal transduction. In vitro, Fast1 associates with Smads in response to an activin/TGF beta signal to form a complex that recognizes the Xenopus activin responsive element (ARE) targeted by Xenopus FAST-1. In intact cells, introduction of Fast1 confers activin/TGF beta regulation of an ARE-luciferase reporter. In embryos, Fast1 is expressed predominantly throughout the epiblast before gastrulation and declines as development progresses. We propose that mouse Fast1, like Xenopus FAST-1, mediates TGF beta superfamily signals specifying developmental fate during early embryogenesis.

Mouse Mesenchyme forkhead 2 (Mf2): expression, DNA binding and induction by sonic hedgehog during somitogenesis.

Cloning and sequencing of mouse Mf2 (mesoderm/mesenchyme forkhead 2) cDNAs revealed an open reading frame encoding a putative protein of 492 amino acids which, after in vitro translation, binds to a DNA consensus sequence. Mf2 is expressed at high levels in the ventral region of newly formed somites, in sclerotomal derivatives, in lateral plate and cephalic mesoderm and in the first and second branchial arches. Other regions of mesodermal expression include the developing tongue, meninges, nose, whiskers, kidney, genital tubercule and limb joints. In the nervous system Mf2 is transcribed in restricted regions of the mid- and forebrain. In several tissues, including the early somite, Mf2 is expressed in cell populations adjacent to regions expressing sonic hedgehog (Shh) and in explant cultures of presomitic mesoderm Mf2 is induced by Shh secreted by COS cells. These results suggest that Mf2, like other murine forkhead genes, has multiple roles in embryogenesis, possibly mediating the response of cells to signaling molecules such as SHH.

The winged helix transcription factor MFH1 is required for proliferation and patterning of paraxial mesoderm in the mouse embryo.

The gene mfh1, encoding a winged helix/forkhead domain transcription factor, is expressed in a dynamic pattern in paraxial and presomitic mesoderm and developing somites during mouse embryogenesis. Expression later becomes restricted to condensing mesenchyme of the vertebrae, head, limbs, and kidney. A targeted disruption of the gene was generated by homologous recombination in embryonic stem cells. Most homozygous mfh1 null embryos die prenatally but some survive to birth, with multiple craniofacial and vertebral column defects. Using molecular markers, we show that the initial formation and patterning of somites occurs normally in mutants. Differentiation of sclerotome-derived cells also appears unaffected, although a reduction of the level of some markers [e.g., mtwist, mf1, scleraxis, and alpha1(II) collagen] is seen in the anterior of homozygous mutants. The most significant difference, however, is a marked reduction in the proliferation of sclerotome-derived cells, as judged by BrdU incorporation. This proliferation defect was also seen in micromass cultures of somite-derived cells treated with transforming growth factor beta1 and fibroblast growth factors. Our findings establish a requirement for a winged helix/forkhead domain transcription factor in the development of the paraxial mesoderm. A model is proposed for the role of mfh1 in regulating the proliferation and differentiation of cell lineages giving rise to the axial skeleton and skull.

Growth factors in development: the role of TGF-beta related polypeptide signalling molecules in embryogenesis.

Embryonic induction, the process by which signals from one cell population influence the fate of another, plays an essential role in the development of all organisms so far studied. In many cases, the signalling molecules belong to large families of highly conserved proteins, originally identified as mammalian growth factors. The largest known family is related to Transforming Growth Factor-beta (TGF-beta) and currently consists of at least 24 different members. Genetic studies in Drosophila on the TGF-beta related gene, decapentaplegic (dpp), reveal the existence of conserved mechanisms regulating both the expression of the protein during development and the way in which it interacts with other signalling molecules to generate pattern within embryonic tissues. Comparative studies on another TGF-beta related gene, known as Bone Morphogenetic Protein-4 (BMP-4), in Xenopus and mouse point to a conserved role in specifying posteroventral mesoderm during gastrulation. Analysis of other polypeptide signalling molecules during gastrulation suggests that their interaction in the generation of the overall body plan has also been conserved during vertebrate evolution.

The chromosomal mapping of four genes encoding winged helix proteins expressed early in mouse development.

Members of the winged helix family of transcription factors are required for the normal embryonic development of the mouse. Using the interspecific backcross panel from The Jackson Laboratory, we have determined the chromosomal locations of four genes that encode winged helix containing proteins. Mf1 was assigned to mouse Chromosome 8, Mf2 to Chromosome 4, Mf3 to Chromosome 9, and Mf4 to Chromosome 13. Since Mf3 is located in a region of Chromosome 9 containing many well-characterized mouse mutations such as short ear (se), ashen (ash), and dilute (d), we have analyzed deletion mutants to determine the location of Mf3 more precisely.

Haploinsufficient phenotypes in Bmp4 heterozygous null mice and modification by mutations in Gli3 and Alx4.

Bone morphogenetic protein 4 (Bmp4), a vertebrate homolog of Drosophila decapentaplegic (dpp), encodes a signaling protein with multiple functions during embryogenesis. Most mouse embryos homozygous for the Bmp4(tm1blh) null allele die around the time of gastrulation, with little or no mesoderm. Two independently derived Bmp4(tm1) mutations were backcrossed onto the C57BL/6 genetic background. Several independently expressed, incompletely penetrant abnormalities were observed in heterozygotes, including cystic kidney, craniofacial malformations, microphthalmia, and preaxial polydactyly of the right hindlimb. In addition, heterozygotes were consistently underrepresented at weaning. These results indicate that Bmp4 gene dosage is essential for the normal development of a variety of organs and for neonatal viability. Two mutations that enhance the penetrance and expressivity of the polydactylous phenotype were identified: Gli3(XtJ), a deletion mutation involving a gene encoding a zinc-finger protein related to Drosophila cubitus interruptus, and Alx4(tm1rwm), a targeted null mutation in a gene encoding a paired class homeoprotein related to Drosophila aristaless. All double Bmp4(tm1); Gli3(XtJ) heterozygotes have extensive anterior digit abnormalities of both fore- and hindlimbs, while all double Bmp4(tm1); Alx4(tm1) heterozygotes display ectopic anterior digits only on the hindlimbs. These genetic interactions suggest a model for the multigenic control of anterior digit patterning during vertebrate limb development.

Essential role of the tyrosine kinase substrate phospholipase C-gamma1 in mammalian growth and development.

The activation of many tyrosine kinases leads to the phosphorylation and activation of phospholipase C-gamma1 (PLC-gamma1). To examine the biological function of this protein, homologous recombination has been used to selectively disrupt the Plcg1 gene in mice. Homozygous disruption of Plcg1 results in embryonic lethality at approximately embryonic day (E) 9.0. Histological analysis indicates that Plcg1 (-/-) embryos appear normal at E 8.5 but fail to continue normal development and growth beyond E 8.5-E9.0. These results clearly demonstrate that PLC-gamma1 with, by inference, its capacity to mobilize second messenger molecules is an essential signal transducing molecule whose absence is not compensated by other signaling pathways or other genes encoding PLC isozymes.

The bZIP transcription factor LCR-F1 is essential for mesoderm formation in mouse development.

LCR-F1 is a mammalian bZIP transcription factor containing a basic amino acid domain highly homologous to a domain in the Drosophila Cap 'N' Collar and Caenorhabditis elegans SKN-1 proteins. LCR-F1 binds to AP1-like sequences in the human beta-globin locus control region and activates high-level expression of beta-globin genes. To assess the role of LCR-F1 in mammalian development, the mouse Lcrf1 gene was deleted in embryonic stem (ES) cells, and mice derived from these cells were mated to produce Lcrf1 null animals. Homozygous mutant embryos progressed normally to the late egg cylinder stage at approximately 6.5 days post coitus (dpc), but development was arrested before 7.5 dpc. Lcrf1 mutant embryos failed to form a primitive streak and lacked detectable mesoderm. These results demonstrate that LCR-F1 is essential for gastrulation in the mouse and suggest that this transcription factor controls expression of genes critical for the earliest events in mesoderm formation. Interestingly, Lcrf1 null ES cells injected into wild-type blastocysts contributed to all mesodermally derived tissues examined, including erythroid cells producing hemoglobin. These results demonstrate that the Lcrf1 mutation is not cell autonomous and suggest that LCR-F1 regulates expression of signaling molecules essential for gastrulation. The synthesis of normal hemoglobin levels in erythroid cells of chimeras derived from Lcrf1 null cells suggests that LCR-F1 is not essential for globin gene expression. LCR-F1 and the related bZIP transcription factors NF-E2 p45 and NRF2 must compensate for each other in globin gene regulation.

Roles for the winged helix transcription factors MF1 and MFH1 in cardiovascular development revealed by nonallelic noncomplementation of null alleles.

The murine Mf1 and Mfh1 genes have overlapping patterns of expression in the embryo and encode forkhead/winged helix transcription factors with virtually identical DNA binding domains. Previous studies have shown that Mfh1 null mutants have severe cardiovascular defects, including interruptions and coarctations of the aortic arch and ventricular septal defects (Iida et al., Development 124, 4627-4638, 1997). Here, we show that Mf1(lacZ) homozygous null mutants also have a similar spectrum of cardiovascular abnormalities. Moreover, most embryos doubly heterozygous for Mfh1(tm1) and Mf1(lacZ) die before birth with interruptions and coarctations of the aortic arch, dysgenesis of the aortic and pulmonary valves, ventricular septal defects, and other cardiac anomalies. This nonallelic noncomplementation and the similar patterns of expression of the two genes in the mesenchyme and endothelial cells of the branchial arches, outflow tract, and heart suggest that Mf1 and Mfh1 play interactive roles in the morphogenesis of the cardiovascular system. Implications for the development of human congenital heart defects are discussed.

The winged helix gene, Mf3, is required for normal development of the diencephalon and midbrain, postnatal growth and the milk-ejection reflex.

The mouse Mf3 gene, also known as Fkh5 and HFH-e5.1, encodes a winged helix/forkhead transcription factor. In the early embryo, transcripts for Mf3 are restricted to the presomitic mesoderm and anterior neurectoderm and mesoderm. By 9.5 days post coitum, expression in the nervous system is predominantly in the diencephalon, midbrain and neural tube. After midgestation, the highest level of mRNA is in the mammillary bodies, the posterior-most part of the hypothalamus. Mice homozygous for a deletion of the mf3 locus on a [129 x Black Swiss] background display variable phenotypes consistent with a requirement for the gene at several stages of embryonic and postnatal development. Approximately six percent of the mf3-/- embryos show an open neural tube in the diencephalon and midbrain region, and another five percent show a severe reduction of the posterior body axis; both these classes of affected embryos die in utero. Surviving homozygotes have an apparently normal phenotype at birth. Postnatally, however, mf3-/- pups are severely growth retarded and approximately one third die before weaning. This growth defect is not a direct result of lack of circulating growth hormone or thyrotropin. Mice that survive to weaning are healthy, but they show an abnormal clasping of the hindfeet when suspended by the tail. Although much smaller than normal, the mice are fertile. However, mf3-/- females cannot eject their milk supply to feed their pups. This nursing defect can be corrected with interperitoneal injections of oxytocin. These results provide evidence that Mf3 is required for normal hypothalamus development and suggest that Mf3 may play a role in postnatal growth and lactation.