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The Winged Helix Transcription Factor Mfhl Is Required for Proliferation and Patterning of Paraxial Mesoderm in the Mouse Embryo

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The Winged Helix Transcription Factor Mfhl Is Required for Proliferation and Patterning of Paraxial Mesoderm in the Mouse Embryo Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press The winged helix transcription factor MFHl is required for proliferation and patterning of paraxial mesoderm in the mouse embryo Glenn E. Winnier/ Linda Hargett/'^ and Brigid L.M. Hogan 1-3 ^Department of Cell Biology and ^ Howard Hughes Medical Institute, Vanderbilt University Medical School, Nashville, Tennessee 37232-2175 USA The gene mfhl, 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 mfbl 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, mfl, scleraxis, and al(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 pi 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 mfhl in regulating the proliferation and differentiation of cell lineages giving rise to the axial skeleton and skull. [Key Words: Mouse embryogenesis; winged helix, transcription factor; proliferation; patterning; paraxial mesoderm] Received December 11, 1996; revised version accepted February 28, 1997. A major challenge in vertebrate embryology is to eluci­ the presomitic mesoderm, a population of multipotent date the mechanisms by which mesodermal cells acquire cells generated from either the primitive streak or the and achieve their wide-ranging developmental fates. Me­ tail bud mesenchyme. Fate mapping and orthotopic soderm specification is tightly linked with the process of transplantation studies in the mouse have shown that gastrulation, in which epiblast cells enter the primitive cells in the primitive streak of 7.5 and 9.5 days post streak and become allocated to different mesoderm coitum (dpc) embryos tend to colonize the more anterior populations: Midline cells give rise to the prechordal somites (up to about somite 21), whereas tail bud mes­ plate and notochord, paraxial mesoderm cells generate enchyme normally contributes to posterior somites (Bed- the unsegmented mesoderm of the head and the paired dington 1981, 1982; Tarn and Trainor 1994; for review, somites on either side of the neural tube, and the lateral see Smith et al. 1994). In either case, newly formed mesoderm forms the splanchnopleure and somatopleure. somites are generated by the compaction and epithelial- The paraxial mesoderm contributes extensively to many ization of loosely associated mesenchymal units known adult tissues, including most of the axial skeleton, the as somitomeres, and become patterned along their an­ muscles of the trunk, and the dermis of the skin, as well teroposterior, dorsoventral, and mediolateral axes. The as to specific skull bones and muscles of the head and ventromedial region of the somite forms the sclerotome, neck. Mutations that affect paraxial mesoderm develop­ whereas the dorsolateral domain gives rise to the epithe­ ment are therefore likely to have considerable effects on lial dermomyotome. Recent in vitro studies have pro­ body form (for review, see Tarn and Trainor 1994). vided evidence that dorsoventral and mediolateral pat­ The formation of somites begins at the rostral end of terning are under the influence of local signaling mol­ ecules from the notochord, surface ectoderm, neural tube, and lateral mesoderm (Fan and Tessier-Lavigne 1994; Fan et al. 1995; Pourquie et al. 1995, 1996). ^Corresponding author. E-MAIL [email protected]; FAX (615) 343-2033. Following induction by signals from the notochord 926 GENES & DEVELOPMENT 11:926-940 © 1997 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/97 $5.00 Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press mftil winged helix gene in mouse development and ventral neural tube, sclerotome cells proliferate and development proceeds, mfhl transcripts are localized to migrate toward the notochord, giving rise to a medioven- condensing mesenchymal lineages of the vertebral col­ tral domain that will form the vertebral body and inter­ umn, nasal process, limbs, and the developing kidney vertebral discs, a ventrolateral domain that forms the (Miura et al. 1993; Kaestner et al. 1996a). ribs, and a ventromedial domain that gives rise to the To investigate the role of mfhl, we have generated a neural arches and the pedicles (Verbout 1985; Johnson null allele by homologous recombination in embryonic 1986; Christ and Ordahl 1995; Pourquie et al. 1996). Lin­ stem (ES) cells. Most mfhl homozygous null embryos eage tracing and orthotopical transplantation experiments die prenatally, beginning -13.5 dpc. However, mutants in chick embryos have also shown that within each devel­ that survive to later stages exhibit multiple craniofacial oping somite, sclerotomal cells segregate into rostral and and vertebral defects that result from the absence or mal­ caudal domains, expressing different genes and with looser formation of skeletal elements primarily derived from and more compact cell densities, respectively (Stem and cephalic and somitic mesoderm. Analysis of early mfhl Keynes 1987; Norris et al. 1989; Ranscht and Bronner- mutant embryos reveals a reduction in the proliferation Fraser 1991). Moreover, cell labeling studies have shown and possibly also the differentiation of sclerotome-de- that each vertebra is derived from the caudal half of one rived cell lineages. These findings suggest that mfhl en­ somite, and the rostral half of the posteriorly adjacent codes a transcription factor that is required for the pro­ somite (Bagnall 1992), a finding consistent with the liferation of a subset of paraxial mesoderm precursor theory of sclerotomal resegmentation proposed by Re- cells involved in the formation of the axial skeleton and mak in 1855 (Verbout 1985; Bagnall 1992). skull. Intense genetic and molecular analysis has led to the identification of many genes expressed in subpopula- tions of the dermomyotome of the somite and required Results for the development of specific muscle groups derived Localization of mfhl RNA during mouse from them (Bober et al. 1994; Buffinger and Stackdale embryogenesis 1994; Olson and Klein 1994; Christ and Ordahl 1995; Pourquie et al. 1995, 1996). However, much less is mfhl expression is first detected by whole-mount in situ known about the genes regulating the patterning of the hybridization at 7.0 dpc in non-notochordal mesoderm sclerotome, and the proliferation and the differentiation surrounding the node and notochord (data not shown). of the various cell types derived from it. The large num­ Figure 1A shows expression of mfhl in these locations at ber of mouse mutants with vertebral abnormalities 7.5 dpc. By 8.5 dpc, mfhl RNA is detected in the anterior promises to be an invaluable resource for investigating presomitic mesoderm adjacent to the youngest somites, this problem (Johnson 1986; Theiler 1989; Balling et al. in the somites, and in the cephalic mesoderm (Fig. IB; 1992; Dietrich et al. 1993). One of the first mutants to be data not shown). Later, at 9.5-10.5 dpc, mfhl is still ex­ studied at the molecular level was undulated [Un], pressed in the presomitic mesoderm and epithelial caused by a point mutation in the paxl gene, encoding a somites. However, as the somite differentiates, overall paired domain/homeodomain transcription factor. The mfhl expression levels decrease, so that transcripts are mutant phenotype is characterized by malformation of always highest in the most caudal or youngest somites vertebral bodies and intervertebral discs and the proxi­ and the anterior presomitic mesoderm (Fig. 1C,D). More­ mal ribs (Koseki et al. 1993; Wallin et al. 1994; Dietrich over, the localization of transcripts is very dynamic; ini­ and Gruss 1995). This phenotype, and in vitro studies in tially, expression is detected throughout the epithelial with embryonic tissue, have provided strong evidence somites, but later becomes progressively restricted, first that paxl is required for ventral sclerotome differentia­ to the dermomyotome, then to the dorsomedial and dor­ tion during mouse development (Fan and Tessier-La- solateral myotomal precursors and sclerotome, and fi­ vigne 1994; Fan et al. 1995). The functional analysis of nally to the sclerotome of differentiated somites (Fig. 1 other genes expressed in the sclerotome, including sclei- G-I; summarized in Fig. 9). axis and paiaxis, which encode basic helix-loop-helix By 10.5 dpc, mfhl expression is detected in the bran­ (bHLH) transcription factors (Burgess et al. 1995; Cserjes chial arches and mesenchymal cells surrounding the eye et al. 1995), will add greatly
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