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Summary Inflammatory responses are known to facilitate tissue recovery following injury. However, the precise mechanisms that enhance lung alveolar regeneration remain unclear. Here, using an organoid-based screening assay, we find that interleukin-1 (IL-1) and tumor necrosis factor α (TNFα) enhance the proliferation of AEC2s while maintaining their differentiation capacity. Furthermore, we find that expression of IL-1β and TNFα are induced in the AEC2 niche following influenza-induced injury in vivo, and lineage tracing analysis revealed that surviving AEC2s around the damaged area contribute to alveolar regeneration. Through genetic and pharmacological modulation of multiple components of the IL-1-nuclear factor κB (NF-κB) signaling axis, we show that cell-intrinsic as well as stromal mediated IL-1 signaling are essential for AEC2 mediated lung regeneration. Taken together, we propose that the IL-1/TNFα-NF-κB signaling axis functions as a component of an inflammation-associated niche to regulate proliferation of surviving AEC2s and promote lung regeneration., Graphical Abstract, Highlights • IL-1/TNFα enhance the growth of lung alveolar stem cells (AEC2s) in organoid culture • AEC2s treated with IL-1 or TNFα maintain differentiation ability • AEC2s proliferate and contribute to lung repair after influenza virus infection • NF-κB pathway is activated in AEC2s treated with IL-1 or TNFα, The role of inflammatory responses in tissue repair and stem cell behavior is largely unexplored in the lung. Katsura et al. demonstrate that the cytokines interleukin-1 and tumor necrosis factor α, upregulated after influenza infection, enhance the proliferation of alveolar type 2 epithelial cells (AEC2s), endogenous lung stem cells. Moreover, these AEC2s generate AEC1s and contribute to alveolar regeneration.

SummaryTo directly test the contribution of Scgb1a1+ Clara cells to postnatal growth, homeostasis, and repair of lung epithelium, we generated a Scgb1a1-CreERTM “knockin” mouse for lineage-tracing these cells. Under all conditions tested, the majority of Clara cells in the bronchioles both self-renews and generates ciliated cells. In the trachea, Clara cells give rise to ciliated cells but do not self-renew extensively. Nevertheless, they can contribute to tracheal repair. In the postnatal mouse lung, it has been proposed that bronchioalveolar stem cells (BASCs) which coexpress Scgb1a1 (Secretoglobin1a1) and SftpC (Surfactant Protein C), contribute descendants to both bronchioles and alveoli. The putative BASCs were lineage labeled in our studies. However, we find no evidence for the function of a special BASC population during postnatal growth, adult homeostasis, or repair. Rather, our results support a model in which the trachea, bronchioles, and alveoli are maintained by distinct populations of epithelial progenitor cells.

Stem cell research is being driven forward at an intense pace by creative interactions among scientists working in different fields. These include developmental and reproductive biology, regeneration, genomics, live cell imaging, RNA biology, and cancer biology, to name a few. Numerous model systems and techniques are being exploited, and lab scientists are teaming up with bioengineers and clinicians. The ferment of ideas that makes the field so exciting was in full evidence throughout the Symposium. However, many challenges still need to be overcome to translate basic discoveries into therapeutic outcomes that will save lives and fulfill the promises that have been made. This chapter summarizes some of the highlights of the Symposium and indicates future directions that are being taken by leaders in the field.

The adult lung consists of a trachea leading into a system of branched airways ending in millions of alveolar sacs. It contains many different epithelial cell types arranged in precise patterns along the proximodistal axis. Each region of the lung has the capacity to repair through the proliferation of different epithelial cell types. However, the precise identity of the cells mediating repair is not fully resolved. To address this problem, we are using genetic lineage-labeling techniques in the mouse. The tools we have made will also be useful for understanding how progenitor cell behavior is regulated under normal and pathological conditions.

Craniofacial development is severely affected by null mutations in Foxc1, indicating a multifunctional role for Foxc1 in ocular, maxilla and mandible, skull and facial gland development. To delineate signaling pathways in which Foxc1 is involved we compared the transcriptomes of whole heads of Foxc1+/+ and Foxc1-/- embryos using a candidate cDNA array comprising genes expressed in the head mesenchyme and ocular region, and a 7K oligo array. Absence of Foxc1 led to downregulation of Stat1 and Galnt4, and upregulation of Tgf beta1i4 at embryonic day 13.5 in the developing head mesenchyme. Comparative analyses revealed differences in the expression pattern of Tgf beta1i4 in the head mesenchyme of Foxc1-/- and Foxc1+/+ embryos. In the ocular regions of Foxc1-/- embryos, Tgf beta1i4 was expressed in higher levels in the conjunctival epithelium and in the condensing mesenchyme on the nasal aspect of the developing eye while in wild-type embryos more intense expression was seen in the mesenchyme on the temporal aspect of the eye. Such data indicate that Foxc1 regulation of Tgf beta1i4 is complex and may be cell-type dependent. Analysis of the regulation of Tgf beta1i4 by Foxc1 in a more homogenous cell population, mesenchymal cells isolated from the periocular region revealed that, in these cells, Foxc1 negatively regulated Tgf beta1i4 expression, presumably via secreted factors such as TGF-beta1. Since Foxc1 expression is essential for normal craniofacial development, it is possible that its downstream targets play a role in the development of the phenotypes associated with null mutations in Foxc1.

The embryonic telencephalon is patterned into several areas that give rise to functionally distinct structures in the adult forebrain. Previous studies have shown that BMP4 and BMP2 can induce features characteristic of the telencephalic midline in cultured explants, suggesting that the normal role of BMP4 in the forebrain is to pattern the medial lateral axis of the telencephalon by promoting midline cell fates. To test this hypothesis directly in vivo, the Bmp4 gene was efficiently disrupted in the telencephalon using a CRE/loxP approach. Analysis of Bmp4-deficient telencephalons fails to reveal a defect in patterning, cell proliferation, differentiation, or apoptosis. The absence of a phenotype in the Bmp4-deficient telencephalon along with recent genetic studies establishing a role for a BMP4 receptor, BMPRIA, in telencephalic midline development, demonstrate that loss of Bmp4 function in the telencephalon can be compensated for by at least one other Bmp gene, the identity of which has not yet been determined.

Bone morphogenetic proteins (Bmps) are members of the transforming growth factor β (TGFβ) superfamily that play critical roles during mouse embryogenesis. Signaling by Bmp receptors is mediated mainly by Smad proteins. In this study, we show that a targeted null mutation of Ecsit, encoding a signaling intermediate of the Toll pathway, leads to reduced cell proliferation, altered epiblast patterning, impairment of mesoderm formation, and embryonic lethality at embryonic day 7.5 (E7.5), phenotypes that mimic the Bmp receptor type1a (Bmpr1a) null mutant. In addition, specific Bmp target gene expression is abolished in the absence of Ecsit. Biochemical analysis demonstrates that Ecsit associates constitutively with Smad4 and associates with Smad1 in a Bmp-inducible manner. Together with Smad1 and Smad4, Ecsit binds to the promoter of specific Bmp target genes. Finally, knock-down of Ecsit with Ecsit-specific short hairpin RNA inhibits both Bmp and Toll signaling. Therefore, these results show that Ecsit functions as an essential component in two important signal transduction pathways and establishes a novel role for Ecsit as a cofactor for Smad proteins in the Bmp signaling pathway.

Proper septation and valvulogenesis during cardiogenesis depend on interactions between the myocardium and the endocardium. By combining use of a hypomorphic Bone morphogenetic protein 4 (Bmp4) allele with conditional gene inactivation, we here identify Bmp4 as a signal from the myocardium directly mediating atrioventricular septation. Defects in this process cause one of the most common human congenital heart abnormalities, atrioventricular canal defect (AVCD). The spectrum of defects obtained through altering Bmp4 expression in the myocardium recapitulates the range of AVCDs diagnosed in patients, thus providing a useful genetic model with AVCD as the primary defect.

During the early development of the mouse lung a number of genes encoding signaling molecules are differentially expressed in the epithelium and mesenchyme of the distal buds. Evidence suggests they play a role in regulating the stereotypic processes of bud outgrowth and branching as well as proximal-distal patterning of both cell layers. To better understand the mechanisms underlying branching morphogenesis, a subtractive hybridization and differential screen was carried out for genes preferentially expressed in the epithelium at the tips of embryonic day 11.5 lung buds, versus more proximal regions. Twenty genes were identified, assigned to different categories based on sequence analysis, and their distal expression confirmed by whole-mount in situ hybridization.

As organisms have evolved in size and complexity, tubular systems have developed to enable the efficient transport of substances into and out of tissues. These tubular systems are generated using strategies that are based on common elements of cell behaviour, including cell polarization, tube migration to target sites, cell-fate diversification and localization of specialized cells to different regions of the tube system. Using examples from both invertebrate and vertebrate systems, this review highlights progress in understanding these basic principles and briefly discusses the possible evolution of strategies to regulate the morphogenesis of tubular systems.

Respiratory disease is the third leading cause of death in the industrialized world. Consequently, the trachea, lungs, and cardiopulmonary vasculature have been the focus of extensive investigations. Recent studies have provided new information about the mechanisms driving lung development and differentiation. However, there is still much to learn about the ability of the adult respiratory system to undergo repair and to replace cells lost in response to injury and disease. This Review highlights the multiple stem/progenitor populations in different regions of the adult lung, the plasticity of their behavior in injury models, and molecular pathways that support homeostasis and repair.

Evidence suggests that the specification of primordial germ cells (PGCs) in the mammalian embryo does not depend on maternal determinants. Rather, previous genetic analysis in the mouse has shown that bone morphogenetic protein 4 (Bmp4) is required for the formation of both PGCs and allantois. Bmp4 is expressed in both the trophoblast-derived extraembryonic ectoderm (ExE) and in the epiblast-derived extraembryonic mesoderm (ExM), in which the PGCs, allantois primordium, and angioblasts are first detected. We have shown that Bmp4 made in the ExE functions to induce precursors of PGCs and allantois in the adjacent epiblast, resulting in complete lack of both cell types in homozygous null mutants. However, the function of Bmp4 in the ExM is totally unknown. To address this question, we generated tetraploid (4N) chimeras by aggregating Bmp4 null ES cells with wild-type tetraploid embryos. In this combination, wild-type tetraploid cells contribute to the extraembryonic trophoblast and primitive endoderm lineages but are excluded from the epiblast and its derivatives, including the ExM. Our results clearly demonstrate that Bmp4 made in the ExM does not affect the establishment of either PGC or allantois lineages, but is required for PGC localization and survival and for the differentiation of the allantois. These findings suggest that Bmp4 expressed in epiblast-derived tissues plays vital roles in reproduction by regulating both the development of the germ line and the vascular connection between the embryo and the placenta.

The murine Foxc1/Mf1 and Foxc2/Mfh1 genes encode closely related forkhead/winged helix transcription factors with overlapping expression in the forming somites and head mesoderm and endothelial and mesenchymal cells of the developing heart and blood vessels. Embryos lacking either Foxc1 or Foxc2, and most compound heterozygotes, die pre- or perinatally with similar abnormal phenotypes, including defects in the axial skeleton and cardiovascular system. However, somites and major blood vessels do form. This suggested that the genes have similar, dose-dependent functions, and compensate for each other in the early development of the heart, blood vessels, and somites. In support of this hypothesis, we show here that compound Foxc1; Foxc2 homozygotes die earlier and with much more severe defects than single homozygotes alone. Significantly, they have profound abnormalities in the first and second branchial arches, and the early remodeling of blood vessels. Moreover, they show a complete absence of segmented paraxial mesoderm, including anterior somites. Analysis of compound homozygotes shows thatFoxc1 and Foxc2 are both required for transcription in the anterior presomitic mesoderm of paraxis, Mesp1,Mesp2, Hes5, and Notch1, and for the formation of sharp boundaries of Dll1, Lfng, and ephrinB2expression. We propose that the two genes interact with the Notch signaling pathway and are required for the prepatterning of anterior and posterior domains in the presumptive somites through a putative Notch/Delta/Mesp regulatory loop.

Factors controlling the differentiation of the multipotent embryonic lung endoderm and mesoderm are poorly understood. Recent evidence that Delta-like 1 (Dll1) and other genes in the Notch/Delta signaling pathway are expressed in the embryonic mouse lung suggests that this pathway is important for cell fate decisions and/or the differentiation of lung cell types. Here, we report the localization of transcripts of several genes encoding members of the Notch/Delta pathway in the early mouse lung. Most genes are expressed in specific populations and so may contribute to cell diversification.

Before gastrulation, the mouse embryo consists of three distinct cell lineages which were established in the blastocyst during the peri-implantation period, that is, epiblast, extraembryonic endoderm, and trophectoderm. The epiblast, from which the entire fetus will form, as well as the extraembryonic mesoderm and amnion ectoderm, is a cup-shaped epithelium apposed on its open end to the extraembryonic ectoderm, a trophectoderm derivative. Both epiblast and extraembryonic ectoderm are covered by visceral endoderm, which is part of the extraembryonic endoderm lineage (Hogan et al. 1994). The primordial germ cells (PGCs) of the mouse embryo are derived from part of the population of epiblast cells that will give rise mainly to the extraembryonic mesoderm. Precursors of the PGCs are located before gastrulation in the extreme proximal region of the epiblast adjacent to the extraembryonic ectoderm, and have descendants not only in the germ line, but also in extraembryonic structures, that is, the allantois, blood islands, and yolk sac mesoderm, as well as both layers of the amnion. At embryonic day (E) 6.0, these precursors lie scattered in a ring that extends up to three cell diameters from the junction with the extraembryonic ectoderm (Lawson and Hage 1994). Early in gastrulation, they converge toward the primitive streak in the posterior of the embryo and translocate through it. Allocation to the germ cell lineage is thought to occur in ∼45 cells around E7.2, after the precursors have passed through the streak and have come to reside in the extraembryonic mesoderm (Lawson and Hage 1994). This is about the time when the putative PGCs can first be identified morphologically in a cluster posterior to the primitive streak in a position that will later become the base of the allantois (Ginsburg et al. 1990). PGCs stain strongly in a characteristic pattern for alkaline phosphatase (AP) activity (Chiquoine 1954), which by this stage is due to tissue nonspecific AP (Hahnel et al. 1990; MacGregor et al. 1995). The PGCs continue to express AP during their proliferation in the developing hindgut and migration into the genital ridges (for review, see Buehr 1997). Transplantation studies have shown that genetically marked distal epiblast cells from pre- and early-primitive streak-stage embryos, which would normally contribute to neuroectoderm and never to the PGCs, can give rise to PGCs and extraembryonic mesoderm when grafted to the proximal epiblast (Tam and Zhou 1996). These results raise the possibility that PGC precursors are induced by extracellular factors and/or cell interactions present locally at the junction between the extraembryonic ectoderm and epiblast. Candidate genes encoding putative germ cell precursor inducing factors are predicted to be expressed in the mouse embryo before and during gastrulation. One such factor is Bone Morphogenetic Protein 4 (Bmp4), a member of the TGFβ superfamily of intercellular signaling proteins (Hogan 1996; Waldrip et al. 1998). Most mouse embryos homozygous for a null mutation in Bmp4 die around gastrulation (∼E6.5) (Winnier et al. 1995). On some genetic backgrounds, however, a proportion of the mutant embryos survive until the early somite stage and show severe defects, particularly in the extraembryonic mesoderm (Winnier et al. 1995). In this paper, we exploit this late phenotype to show that PGC formation absolutely requires Bmp4 signaling. In addition, the size of the founding population of PGCs is significantly reduced in heterozygous mutant embryos. By using a Bmp4–lacZ reporter allele, we have definitively localized Bmp4 expression before gastrulation in the extraembryonic ectoderm and in mid- to late- primitive streak stage embryos in the extraembryonic mesoderm. Thus, Bmp4 is expressed at the right time and in the right place to play a role both in the quantitative induction of PGC precursors in the proximal epiblast and in their allocation to the germ cell lineage in the extraembryonic mesoderm. Furthermore, by analyzing genetic chimeras, we have clearly established a role for Bmp4 in the induction of PGC precursors and demonstrate for the first time that a secreted signal from the extraembryonic ectoderm is required for the normal development of the epiblast.

SummaryGenetic linkage, genome mismatch scanning, and analysis of patients with alterations of chromosome 6 have indicated that a major locus for development of the anterior segment of the eye, IRID1, is located at 6p25. Abnormalities of this locus lead to glaucoma. FKHL7 (also called “FREAC3”), a member of the forkhead/winged-helix transcription-factor family, has also been mapped to 6p25. DNA sequencing of FKHL7 in five IRID1 families and 16 sporadic patients with anterior-segment defects revealed three mutations: a 10-bp deletion predicted to cause a frameshift and premature protein truncation prior to the FKHL7 forkhead DNA-binding domain, as well as two missense mutations of conserved amino acids within the FKHL7 forkhead domain. Mf1, the murine homologue of FKHL7, is expressed in the developing brain, skeletal system, and eye, consistent with FKHL7 having a role in ocular development. However, mutational screening and genetic-linkage analyses excluded FKHL7 from underlying the anterior-segment disorders in two IRID1 families with linkage to 6p25. Our findings demonstrate that, although mutations of FKHL7 result in anterior-segment defects and glaucoma in some patients, it is probable that at least one more locus involved in the regulation of eye development is also located at 6p25.

The establishment of branched tubular epithelial structures is critical for the viability of multicellular organisms: the tracheal system in Drosophila and the vertebrate lung being two such structures. Although there are obvious differences in the complexity of these branched organs, many of the underlying mechanisms and genes regulating their development appear to have been evolutionarily conserved.

Bmp6, a member of the 60A subgroup of bone morphogenetic proteins (BMPs), is expressed in diverse sites in the developing mouse embryo from preimplantation stages onwards. To evaluate roles for Bmp6 signaling in vivo, gene targeting was used to generate a null mutation at the Bmp6 locus. The resulting Bmp6 mutant mice are viable and fertile, and show no overt defects in tissues known to express Bmp6 mRNA. The skeletal elements of newborn and adult mutants are indistinguishable from wild-type. However, careful examination of skeletogenesis in late gestation embryos reveals a consistent delay in ossification strictly confined to the developing sternum. In situ hybridization studies in the developing long bones and sternum show that other BMP family members are expressed in overlapping domains. In particular we find that Bmp2 and Bmp6 are coexpressed in hypertrophic cartilage, suggesting that Bmp2 may functionally compensate in Bmp6 null mice. The defects in sternum development in Bmp6 null mice are likely to be associated with a transient early expression of Bmp6 in the sternal bands, prior to ossification. These sternal defects are slightly exacerbated in Bmp5/6 double mutant animals. Dev. Genet. 22:321–339, 1998. © 1998 Wiley-Liss, Inc.

Analysis of the skeletal phenotypes caused by the genetic inactivation of individual Bmps, along with the study of their expression patterns, suggest possible functional redundancy of these molecules. To investigate the effect on skeleton development of the combined absence of some Bmp genes expressed in the same areas, we have intercrossed heterozygous Bmp7 mice with Bmp2 1/2 , Bmp4 1/2 ,o rBmp5 1/2 animals. Bmp2/7 and Bmp5/7 double heterozygous animals do not present with any abnormalities. In contrast, Bmp4/7 double heterozygotes develop minor defects in two restricted areas of the skeleton, the rib cage, and the distal part of the limbs. In the ribs, Bmp4 and Bmp7 seem to act in the same pathway to assure proper guidance of mesenchymal condensations of the ribs extending to- ward the sternum. In the limbs, these molecules appear to play a similar role in controlling digit number, possibly through induction of apoptosis in the interdigital and anterior mesenchyme. Dev. Genet. 22:340-348, 1998. r 1998 Wiley-Liss, Inc.

We have taken advantage of an enhancer trap event in a line of transgenic mice to identify a unique developmentally regulated endothelial cell locus (Del1). The protein encoded in this locus contains three EGF-like repeats homologous to those in Notch and related proteins, including an EGF-like repeat that contains an RGD motif, and two discoidin I-like domains. Del1 is shown to be a matrix protein and to promote adhesion of endothelial cells through interaction with the αvβ3 integrin receptor. Embryonic endothelial-like yolk sac cells expressing recombinant Del1 protein, or grown on an extracellular matrix containing Del1 protein, are inhibited from forming vascular-like structures. Expression of Del1 protein in the chick chorioallantoic membrane leads to loss of vascular integrity and promotes vessel remodeling. Del1 is thus a new ligand for the αvβ3 integrin receptor and may function to regulate vascular morphogenesis or remodeling in embryonic development.

The bone morphogenetic proteins (BMPs) constitute a large family of cytokines related to members of the transforming growth factor-beta superfamily. Recent evidence, in particular from gene targeting experiments in the mouse, indicates that BMPs are required for mesoderm formation and for the development and patterning of many different organ systems. Significant progress has also been made in understanding the role of BMPs in gastrulation and neurulation in Xenopus and in identifying genes regulating BMP expression and components of the downstream signaling pathways. Extracellular modifiers of BMP activity may constitute an opposing morphogenetic system.

Bone morphogenetic protein 8B (BMP8B) is a member of the TGFbeta superfamily of growth factors. In the mouse, Bmp8b is expressed in male germ cells of the testis and trophoblast cells of the placenta, suggesting that it has a role in spermatogenesis and reproduction. To investigate these possibilities, we have generated mice with a targeted mutation in Bmp8b. Here, we show that homozygous Bmp8b(tm1blh) mutant males exhibit variable degrees of germ-cell deficiency and infertility. Detailed analysis reveals two separable defects in the homozygous mutant testes. First, during early puberty (2 weeks old or younger) the germ cells of all homozygous mutants either fail to proliferate or show a marked reduction in proliferation and a delayed differentiation. Second, in adults, there is a significant increase in programmed cell death (apoptosis) of spermatocytes, leading to germ-cell depletion and sterility. Sertoli cells and Leydig cells appear relatively unaffected in mutants. This study therefore provides the first genetic evidence that a murine germ cell-produced factor, BMP8B, is required for the resumption of male germ-cell proliferation in early puberty, and for germ-cell survival and fertility in the adult.

Secreted polypeptide signalling molecules of the TGF- superfamily play critical roles during mouse development. There are 20 known members of this diverse family in the mouse, including the TGF-s, inhibin/activins, BMPs, GDFs, and nodal. Mutations in genes for some of these ligands have been identified or created by targeted mutagenesis. A complex network of binding proteins, proteases, and receptors is involved in the processing, presentation and biological activity of TGF-β-related ligands. It is likely that variations in these modifying factors will explain how different genetic backgrounds can influence the severity and penetrance of mutant phenotypes.

Skin papillomas and squamous cell carcinomas (SCCs) are induced in mice by tumor initiation with a carcinogen followed by tumor promotion with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). These usually arise from preneoplastic lesions characterized by epidermal proliferation and hyperplasia, dermal edema, and inflammation. To evaluate the role of polypeptide growth factors in chemically induced skin carcinogenesis, we used transgenic mice carrying the cDNA for a TGF-β related molecule, bone morphogenetic protein-4 (BMP-4), under the control of the regulatory elements of the cytokeratin IV* gene in a skin carcinogenesis protocol. Control non-transgenic littermates and BMP-4 transgenic mice were treated with a single dose of a carcinogen, N-methyl-N′-nitrosoguanidine (MNNG), and biweekly with the tumor promoter TPA for 9 months. In control littermates TPA induced epidermal hyperproliferation, atypia with “dark” cells, and dermal inflammation, resulting in papillomas and SCCs in 13 of 26 animals tested. In BMP-4 transgenic mice, TPA treatment induced the expression of the BMP-4 transgene in interfollicular epidermis but only minimal epidermal thickening, hyperproliferation, and inflammation were noted after the initial dose of TPA. Furthermore, the mitotic indices in transgenic epidermis after 9 months of TPA treatment were significantly lower than the corresponding indices from untreated transgenic epidermis. Consequently, none of the 22 transgenic animals tested developed papillomas or SCCs. In conclusion, we have shown that the TPA induced expression of the BMP-4 transgene blocks proliferation and inflammation in skin, steps that are critical to the subsequent formation of papillomas and SCCs and we characterized an inducible promotersystem which expresses polypeptides in interfollicular epidermis after exogenous stimulation. © 1995 Wiley-Liss, Inc.

Delta-Notch signaling is essential for somitogenesis in vertebrate embryos. In a search for genes that control somite formation in zebrafish we have identified two paralogues encoding proteins related to Nrarp (Notch regulated ankyrin repeat protein). Zebrafish nrarp-a and-b encode small proteins with two ankyrin repeat domains. Here, we report the expression patterns of both genes in normal and mutant embryos.

Embryonic induction is the process by which signals from one cell population change the developmental fate of another. Polypeptides related to growth factors are one group of molecules mediating many inductive events. Recent data on the embryonic expression and function of signaling proteins related to transforming growth factor beta, in both vertebrate and invertebrate systems, have shown that these molecules play important roles in both pattern formation and tissue specification during 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 iden- tified as mammalian growth factors. The largest known family is related to Transforming Growth Factor-β (TGF-β related to Transforming Growth Factor-β and currently consists of at least 24 different members. Genetic studies in Drosophila on the TGF-β 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-β related gene, known as Bone Morphogenetic Protein-4 (BMP-4), in Xenopus and mouse point to a conserved role in specifying posteroventral mesoderm during gastrula- tion. 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.

cDNAs encoded by the mouse homolog (Lim-1) of the Xenopus LIM-class homeobox gene Xlim-1 have been isolated from an 8.5-day mouse embryo cDNA library. Nucleotide and deduced amino acid sequences show a high degree of identity with Xlim-1 in the LIM and homeodomains, and 85% identity over the whole protein. An interspecific back-cross has been used to show close linkage of Lim-1 to the endogenous proviral marker Mpmv-4 on mouse chromosome 11. Whole mount in situ hybridization studies have been carried out on mouse embryos between 6.5 and 10.5 days. In mid- to late-streak stage embryos, Lim-1 is expressed in a restricted region of mesoderm in the primitive streak, with the highest level of signal at the anterior. At 7.5 days, transcripts can be seen in a horseshoe-shaped pattern in the periphery of the node, as well as along both sides of the immediately adjacent notochord. In addition, transcripts are present in presumptive lateral and intermediate mesoderm. Later, expression becomes progressively restricted to intermediate mesoderm, the nephrogenic cords, and eventually mesonephric ducts and tubules. By 10.5 days Lim-1 transcripts also appear in restricted regions of the central nervous system (CNS) that are associated with sensory function. The lateral diencephalon, hindbrain, and presumed commissural neurons in the dorsal spinal cord all show Lim-1 expression. In the adult, Lim-1 is expressed in the cerebellum/medulla and kidney, and at very low levels in the cerebrum. These data suggest that in the mouse embryo Lim-1 plays a role in early mesoderm formation and later specification of a differentiated phenotype in subsets of cells of the mesonephros and sensory neurons of the CNS.

The transforming growth factors beta (TGFs-beta) are potent inhibitors of cell proliferation and are usually secreted in a latent form. TGF-beta 1, TGF-beta 2, and TGF-beta 3 are expressed in distinct but overlapping patterns in the developing mouse mammary gland. To study the role of transforming growth factor-beta 1 (TGF-beta 1) in normal mammary development and in mammary neoplasia, we have constructed three transgenic mouse lines that express a simian TGF-beta 1 s223/225 mutated to produce a constitutively active product under the control of the MMTV enhancer/promoter. Expression of the transgene, as confirmed by in situ hybridization, immunohistochemistry, and Northern blot analysis, was associated with marked suppression of the normal pattern of mammary ductal tree development in female transgenics. Reduction in total ductal tree volume was observed at 7 weeks, soon after estrous begins, and was most apparent at 13 weeks, as ductal growth in the normal mammary gland declines. This effect was seen in all three lines. However, during pregnancy, alveolar outgrowths developed from the hypoplastic ductal tree, and lactation occurred, therefore, all transgenic females could feed full litters. Unlike many other transgenic mouse models in which expression of growth factors or oncogenes under control of the MMTV promoter leads to mammary epithelial hyperplasia and increased tumor formation, the MMTV-TGF-beta 1S223/225 transgene causes conditional hypoplasia of the mammary ductal tree and no spontaneous tumors have been detected in the MMTV-TGF-beta 1S223/225 transgenic animals.

There is increasing evidence that members of the TGF-beta superfamily are important regulators of epithelial growth and differentiation in vivo. Here, transgenic mice have been used to study the role of the TGF-beta-related growth factors BMP-2 and BMP-4 in hair and whisker development. In the mature hair follicle, BMP-2 transcripts are normally seen only in precortex cells at the base of the hair shaft. In the transgenic mice reported here, BMP-4, a closely related molecule, has been ectopically expressed in the outer root sheath of hair and whisker follicles using an expression vector based on the bovine cytokeratin IV* promoter. In response to transgene expression, both outer root sheath cells below the stem cell compartment and hair matrix cells around the dermal papilla cease proliferation. In addition, the expression pattern of cytokeratin markers is disturbed in some transgenic hair follicles. These results support a model in which members of the TGF-beta superfamily play an active role in the inhibiton of cell proliferation and the onset of expression of trichocyte-specific genes that take place when cells leave the matrix of the follicle and differentiate into shaft cells.

Establishment of mesodermal tissues in the amphibian body involves a series of inductive interactions probably elicited by a variety of peptide growth factors. Results reported here suggest that mesodermal patterning involves an array of signalling molecules including DVR-4, a TGF-β-like molecule. We show that ectopic expression of DVR-4 causes embryos to develop with an overall posterior and/or ventral character, and that DVR-4 induces ventral types of mesoderm in animal cap explants. Moreover, DVR-4 overrides the dorsalizing effects of activin. DVR-4 is therefore the first molecule reported both to induce posteroventral mesoderm and to counteract dorsalizing signals such as activin. Possible interactions between these molecules resulting in establishment of the embryonic body plan are discussed.

Small eye (Sey) in mouse is a semidominant mutation which in the homozygous condition results in the complete lack of eyes and nasal primordia. On the basis of comparative mapping studies and on phenotypic similarities, Sey has been suggested to be homologous to congenital aniridia (lack of iris) in human. A candidate gene for the aniridia (AN) locus at 11p13 has been isolated by positional cloning and its sequence and that of the mouse homologue has been established (C.T., manuscript in preparation). This gene belongs to the paired-like class of developmental genes first described in Drosophila which contain two highly conserved motifs, the paired box and the homeobox. In vertebrates, genes which encode the single paired domain as well as those which express both motifs have been described as the Pax multigene family. A Pax gene recently described as Pax-6 is identical to the mouse homologue of the candidate aniridia gene. Here we report the analysis of three independent Sey alleles and show that indeed this gene is mutated and that the mutations would predictably interrupt gene function.

The DVR gene family consists of at least 15 members, including decapentaplegic from Drosophila, Xenopus Vg1 and the mammalian bone morphogenetic protein genes, encoding secreted proteins closely related to transforming growth factor β Genetic and biochemical evidence supports the idea that DVR proteins form part of a cascade of extracellular signalling molecules mediating inductive tissue interactions during development.

Angular head movements in vertebrates are detected by the three semicircular canals of the inner ear and their associated sensory tissues, the cristae. Bone morphogenetic protein 4 (Bmp4), a member of the Transforming growth factor family (TGF-β), is conservatively expressed in the developing cristae in several species, including zebrafish, frog, chicken, and mouse. Using mouse models in which Bmp4 is conditionally deleted within the inner ear, as well as chicken models in which Bmp signaling is knocked down specifically in the cristae, we show that Bmp4 is essential for the formation of all three cristae and their associated canals. Our results indicate that Bmp4 does not mediate the formation of sensory hair and supporting cells within the cristae by directly regulating genes required for prosensory development in the inner ear such as Serrate1 (Jagged1 in mouse), Fgf10, and Sox2. Instead, Bmp4 most likely mediates crista formation by regulating Lmo4 and Msx1 in the sensory region and Gata3, p75Ngfr, and Lmo4 in the non-sensory region of the crista, the septum cruciatum. In the canals, Bmp2 and Dlx5 are regulated by Bmp4, either directly or indirectly. Mechanisms involved in the formation of sensory organs of the vertebrate inner ear are thought to be analogous to those regulating sensory bristle formation in Drosophila. Our results suggest that, in comparison to sensory bristles, crista formation within the inner ear requires an additional step of sensory and non-sensory fate specification., Author Summary Disruption of the sense of balance is highly debilitating, causing vertigo and nausea. Maintenance of proper balance requires sensory inputs from many body parts, including the inner ears and the eyes. Within the inner ear, the vestibular apparatus plays a key role in the sense of balance and is responsible for detecting head orientation and movements. The portion of the vestibular apparatus that detects angular head movements consists of three fluid-filled, semicircular canals oriented at right angles to each other. At one end of each canal is an enlargement that houses the sensory tissue, crista ampullaris, consisting of sensory hair cells and supporting cells. Bone morphogenetic protein 4 (Bmp4), a secreted signaling molecule, is expressed in these sensory regions during development. However, the lack of Bmp4 in mice affects the formation of not only the sensory regions but also their associated canals. These results demonstrate for the first time that a single gene, Bmp4, is required for the formation of the entire sensory apparatus for detecting angular head movements.

Using and interspecific backcross, we have mapped the gene involved in the mouse Small eye mutation ( Sey MH ) relative to six cloned markers on chromosome 2 ( Hox-5.1, Cas-1, Fshb, Bmp-2a , and ld ) and the agouti locus. The results suggest that the Sey gene maps between Fshb and Cas-1 . Human mapping studies have shown that the aniridia ( AN2 ) gene, which is part of the Wilms tumor susceptibility, aniridia, genitourinary abnormalities, and mental retardation (WARG) complex, is also between FSHB and CAT on human chromosome 11. The conserved linkage of the cloned markers and the similarity of the Sey /+ and AN2 /+ phenotypes suggest that the gene involved in the Sey mutation is the mouse homolog of the human AN2 gene.

Molecular characterization of mutations in the mouse, particularly those involving agent-induced major structural alterations, is proving to be useful for correlating the structure and expression of individual genes with their function in the whole organism. Here we present the characterization of a radiation-induced mutation that simultaneously generated distinct alleles of both the limb deformity (ld) and agouti (a) loci, two developmentally important regions of chromosome 2 normally separated by 20 centimorgans. Cytogenetic analysis revealed that an interstitial segment of chromosome 17 (17B- 17C; or, possibly, 17A2-17B) had been translocated into the distal end of chromosome 2, resulting in a smaller-than-normal chromosome 17 (designated 17del) and a larger form of chromosome 2 (designated 2(17). Additionally, a large interstitial segment of the 2(17) chromosome, immediately adjacent and proximal to the insertion site, did not match bands 2E4-2H1 at corresponding positions on a normal chromosome 2. Molecular analysis detected a DNA rearrangement in which a portion of the ld locus was joined to sequences normally tightly linked to the a locus. This result, along with the genetic and cytogenetic data, suggests that the alleles of ld and a in this radiation-induced mutation, designated ldIn2 and ajIn2, were associated with DNA breaks caused by an inversion of an interstitial segment in the 2(17) chromosome.

The genes that encode the bone morphogenetic proteins and the Vg-related proteins are mammalian members of a group of TGF-beta-related genes, designated the DVR family, that includes the decapentaplegic gene of Drosophila and the Vg1 gene of Xenopus. Members of the DVR (decapentaplegic-Vg-related) family have been implicated in diverse processes during development, particularly in epithelial-mesenchymal interactions. The results of our in situ hybridization studies with postimplantation mouse embryos provide evidence for the involvement of DVR family members, particularly DVR-2, DVR-4 and DVR-6, in specific inductive interactions during the development of many organs, including the limb, the whisker follicle and the heart.

Sox2 is expressed in basal epithelial cells of the tongue, with high levels in taste bud placodes, fungiform papillae, and mature taste cells, and low levels in filiform papillae. High Sox2 expression appears to lie downstream from canonical Wnt signaling. In hypomorphic Sox2EGFP/LP embryos, placodes form but no mature taste buds develop. In contrast, transgenic overexpression of Sox2 in the basal cells inhibits differentiation of filiform keratinocytes. Together, our loss-of-function and gain-of-function studies suggest that Sox2 functions in a dose-dependent manner to regulate the differentiation of endodermal progenitor cells of the tongue into taste bud sensory cells versus keratinocytes.

Previous studies identified zebrafish foxc1a andfoxc1b as homologs of the mouse forkhead gene, Foxc1. Both genes are transcribed in the unsegmented presomitic mesoderm (PSM), newly formed somites, adaxial cells, and head mesoderm. Here, we show that inhibiting synthesis of Foxc1a (but not Foxc1b) protein with two different morpholino antisense oligonucleotides blocks formation of morphological somites, segment boundaries, and segmented expression of genes normally transcribed in anterior and posterior somites and expression of paraxis implicated in somite epithelialization. Patterning of the anterior PSM is also affected, as judged by the absence of mesp-b, ephrinB2, and ephA4expression, and the down-regulation of notch5 andnotch6. In contrast, the expression of other genes, includingmesp-a and papc, in the anterior of somite primordia, and the oscillating expression of deltaC and deltaD in the PSM appear normal. Nevertheless, this expression is apparently insufficient for the maturation of the presumptive somites to proceed to the stage when boundary formation occurs or for the maintenance of anterior/posterior patterning. Mouse embryos that are compound null mutants for Foxc1 and the closely related Foxc2 have no morphological somites and show abnormal expression of Notch signaling pathway genes in the anterior PSM. Therefore, zebrafish foxc1aplays an essential and conserved role in somite formation, regulating both the expression of paraxis and the A/P patterning of somite primordia.

Mesodermal signaling is critical for patterning the embryonic endoderm into different tissue domains. Classical tissue transplant experiments in the chick and recent studies in the mouse indicated that interactions with the cardiogenic mesoderm are necessary and sufficient to induce the liver in the ventral foregut endoderm. Using molecular markers and functional assays, we now show that septum transversum mesenchyme cells, a distinct mesoderm cell type, are closely apposed to the ventral endoderm and contribute to hepatic induction. Specifically, using a mouse Bmp4 null mutation and an inhibitor of BMPs, we find that BMP signaling from the septum transversum mesenchyme is necessary to induce liver genes in the endoderm and to exclude a pancreatic fate. BMPs apparently function, in part, by affecting the levels of the GATA4 transcription factor, and work in parallel to FGF signaling from the cardiac mesoderm. BMP signaling also appears critical for morphogenetic growth of the hepatic endoderm into a liver bud. Thus, the endodermal domain for the liver is specified by simultaneous signaling from distinct mesodermal sources.

Anterior segment developmental disorders, including Axenfeld-Rieger anomaly (ARA), variably associate with harmfully elevated intraocular pressure (IOP), which causes glaucoma. Clinically observed dysgenesis does not correlate with IOP, however, and the etiology of glaucoma development is not understood. The forkhead transcription factor genes Foxc1 (formerly Mf1 ) and Foxc2 (formerly Mfh1 ) are expressed in the mesenchyme from which the ocular drainage structures derive. Mutations in the human homolog of Foxc1, FKHL7, cause dominant anterior segment defects and glaucoma in various families. We show that Foxc1 (+/-)mice have anterior segment abnormalities similar to those reported in human patients. These abnormalities include small or absent Schlemm's canal, aberrantly developed trabecular meshwork, iris hypoplasia, severely eccentric pupils and displaced Schwalbe's line. The penetrance of clinically obvious abnormalities varies with genetic background. In some affected eyes, collagen bundles were half normal diameter, or collagen and elastic tissue were very sparse. Thus, abnormalities in extracellular matrix synthesis or organization may contribute to development of the ocular phenotypes. Despite the abnormalities in ocular drainage structures in Foxc1 (+/-)mice, IOP was normal in almost all mice analyzed, on all genetic backgrounds and at all ages. Similar abnormalities were found in Foxc2 (+/-)mice, but no disease-associated mutations were identified in the human homolog FKHL14 in 32 ARA patients. Foxc1 (+/-)and Foxc2 (+/-)mice are useful models for studying anterior segment development and its anomalies, and may allow identification of genes that interact with Foxc1 and Foxc2 (or FKHL7 and FKHL14 ) to produce a phenotype with elevated IOP and glaucoma.

Mammalian Tolloid-like 1 (mTLL-1) is an astacin-like metalloprotease, highly similar in domain structure to the morphogenetically important proteases bone morphogenetic protein-1 (BMP-1) and Drosophila Tolloid. To investigate possible roles for mTLL-1 in mammalian development, we have used gene targeting in ES cells to produce mice with a disrupted allele for the corresponding gene, Tll1. Homozygous mutants were embryonic lethal, with death at mid-gestation from cardiac failure and a unique constellation of developmental defects that were apparently confined solely to the heart. Constant features were incomplete formation of the muscular interventricular septum and an abnormal and novel positioning of the heart and aorta. Consistent with roles in cardiac development, Tll1 expression was specific to precardiac tissue and endocardium in 7.5 and 8.5 days p.c.embryos, respectively. Tll1 expression was also high in the developing interventricular septum, where expression of the BMP-1 gene, Bmp1, was not observed. Cardiac structures that were not affected in Tll1−/− embryos either showed no Tll1 expression (atrio-ventricular cushions) or showed overlapping expression of Tll1 and Bmp1 (aortico-pulmonary septum), suggesting that products of the Bmp1 gene may be capable of functionally substituting for mTLL-1 at sites in which they are co-expressed. Together, the various data show that mTLL-1 plays multiple roles in formation of the mammalian heart and is essential for formation of the interventricular septum.

Vertebrate eye development proceeds by a series of reciprocal tissue interactions between derivatives of the head surface ectoderm and the forebrain neuroectoderm. In particular, the process of lens induction has been studied as a model system to explore general mechanisms underlying embryonic induction (for review, see Jacobson and Sater 1988). Soon after the turn of this century, a few classical experiments using amphibian embryos led to the idea that the optic vesicle has an instructive role in inducing lens formation in the overlying surface ectoderm (Spemann 1901; Lewis 1904, 1907a,b). According to this original model, the optic vesicle is potentially able to induce a lens from ectoderm anywhere on the embryo. Subsequently, however, other studies using different species and experimental conditions raised doubts about this hypothesis, and rather supported the idea that the optic vesicle is not essential for lens formation (for review, see Saha et al. 1989). Furthermore, a key experiment using a host–donor cell-marking technique revealed that the optic vesicle is insufficient to induce lens in ectopic ectoderm taken from outside the lens field; the induced lens in the recombinants was derived from the optic rudiment, rather than from the grafted ectopic ectoderm (Grainger et al. 1988). The current model, based mainly on experiments using Xenopus embryos, proposes that lens induction proceeds through multiple intermediate states, starting early in development from the gastrula stage (for review, see Grainger 1992). Evidence from many in vivo and in vitro studies indicates that the lens can be formed in the absence of the optic vesicle in several vertebrate species (for review, see Jacobson and Sater 1988; Saha et al. 1989; Henry and Grainger 1990). The role of the optic vesicle during lens induction has, therefore, been considered minor—merely to establish the precise location of the lens within the head ectoderm (Grainger 1992). Several lines of evidence, however, suggest that, in vivo, there is apparent species specificity in the extent to which lens formation is dependent on the optic vesicle. Ablation of prospective retinal neuroectoderm in chick embryos abolishes lens formation (Li et al. 1994; Kamachi et al. 1998). Furthermore, lens formation is completely absent in mouse embryos lacking a functional Lhx2 gene, which encodes a LIM-homeodomain protein and is expressed in the forebrain, including the forming optic vesicle neuroectoderm. In these embryos, impairment of optic vesicle development results in failure of the optic vesicle to contact the surface ectoderm (Porter et al. 1997). Therefore, it appears that lens formation in higher vertebrates requires the presence of the optic vesicle in vivo. Like many other embryonic induction processes, secreted signaling molecules are likely to have critical roles during lens induction. In spite of the extensive experimental studies in the amphibian system described above, however, no actual signaling molecules have yet been identified. Likewise, although a number of genes have been implicated in mammalian eye development by molecular and genetic approaches (for review, see Graw 1996; Oliver and Gruss 1997), characterization of their precise in vivo function in many cases awaits further studies. One exception is the Small eye mutant in mouse (Pax6Sey) and rat (rSey), which have been studied extensively in relation to lens induction, as homozygous mutant embryos completely lack lens formation (Hill et al. 1991; Matsuo et al. 1993). Mutations in the Pax6 gene, which encodes a paired-type homeodomain protein, have also been associated with congenital eye defects in humans (Jordan et al. 1992; Glaser et al. 1994; Hanson et al. 1994). Tissue recombination experiments using rSey mutant embryos have revealed that homozygous mutant ectoderm does not form a lens when recombined with a wild-type optic vesicle, whereas the reciprocal recombination allows lens formation in wild-type ectoderm (Fujiwara et al. 1994). This clearly implies a requirement for PAX6 in the head ectoderm for competence to respond to the optic vesicle signal, and indicates that the optic vesicle retains lens inducing activity in the absence of PAX6 function. The underlying mechanisms of PAX6 function in the ectoderm and inductive signaling factors involved in lens induction are still unknown. Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily of secretory signaling molecules, have been implicated in many aspects of embryonic tissue interactions (for review, see Hogan 1996). Several BMP family members have been reported to be expressed during mouse eye development (Dudley and Robertson 1997). Furthermore, Bmp7 is required for normal embryonic eye development in the mouse (Dudley et al. 1995; Luo et al. 1995). Here, we report that another member of the Bmp gene family, Bmp4, has critical roles during the lens induction process. We show that the optic vesicle is the major source of BMP4 in the early developing eye, and that it is required for lens induction. A series of explant culture experiments suggest that BMP4 is an essential factor to manifest lens inducing activity of the optic vesicle. We also show that BMP4 regulates specific gene expression in the optic vesicle neuroectoderm. We discuss a model in which BMP4 has multiple roles during mammalian lens induction.

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.

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.

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.