Transgenic expression of oncogenic BRAF induces loss of stem cells in the mouse intestine, which is antagonized by ß-catenin activity.

Colon cancer cells frequently carry mutations that activate the ß-catenin and mitogen-activated protein kinase (MAPK) signaling cascades. Yet how oncogenic alterations interact to control cellular hierarchies during tumor initiation and progression is largely unknown. We found that oncogenic BRAF modulates gene expression associated with cell differentiation in colon cancer cells. We therefore engineered a mouse with an inducible oncogenic BRAF transgene, and analyzed BRAF effects on cellular hierarchies in the intestinal epithelium in vivo and in primary organotypic culture. We demonstrate that transgenic expression of oncogenic BRAF in the mouse strongly activated MAPK signal transduction, resulted in the rapid development of generalized serrated dysplasia, but unexpectedly also induced depletion of the intestinal stem cell (ISC) pool. Histological and gene expression analyses indicate that ISCs collectively converted to short-lived progenitor cells after BRAF activation. As Wnt/ß-catenin signals encourage ISC identity, we asked whether ß-catenin activity could counteract oncogenic BRAF. Indeed, we found that intestinal organoids could be partially protected from deleterious oncogenic BRAF effects by Wnt3a or by small-molecule inhibition of GSK3ß. Similarly, transgenic expression of stabilized ß-catenin in addition to oncogenic BRAF partially prevented loss of stem cells in the mouse intestine. We also used BRAF(V637E) knock-in mice to follow changes in the stem cell pool during serrated tumor progression and found ISC marker expression reduced in serrated hyperplasia forming after BRAF activation, but intensified in progressive dysplastic foci characterized by additional mutations that activate the Wnt/ß-catenin pathway. Our study suggests that oncogenic alterations activating the MAPK and Wnt/ß-catenin pathways must be consecutively and coordinately selected to assure stem cell maintenance during colon cancer initiation and progression. Notably, loss of stem cell identity upon induction of BRAF/MAPK activity may represent a novel fail-safe mechanism protecting intestinal tissue from oncogene activation.

Isolation and characterization of a downstream target of Pax6 in the mammalian retinal primordium.

The transcription factor Pax6 is required for eye morphogenesis in humans, mice and insects, and can induce ectopic eye formation in vertebrate and invertebrate organisms. Although the role of Pax6 has intensively been studied, only a limited number of genes have been identified that depend on Pax6 activity for their expression in the mammalian visual system. Using a large-scale in situ hybridization screen approach, we have identified a novel gene expressed in the mouse optic vesicle. This gene, Necab, encodes a putative cytoplasmic Ca(2+)-binding protein and coincides with Pax6 expression pattern in the neural ectoderm of the optic vesicle and in the forebrain pretectum. Remarkably, Necab expression is absent in both structures in Pax6 mutant embryos. By contrast, the optic vesicle-expressed homeobox genes Rx, Six3, Otx2 and Lhx2 do not exhibit an altered expression pattern. Using gain-of-function experiments, we show that Pax6 can induce ectopic expression of Necab, suggesting that Necab is a direct or indirect transcriptional target of Pax6. In addition, we have found that Necab misexpression can induce ectopic expression of the homeobox gene Chx10, a transcription factor implicated in retina development. Taken together, our results provide evidence that Necab is genetically downstream of Pax6 and that it is a part of a signal transduction pathway in retina development.

Large-scale screen for genes involved in gonad development.

The vertebrate gonad develops from the intermediate mesoderm as an initially bipotential organ anlage, the genital ridge. In mammals, Sry acts as a genetic switch towards testis development. Sox9 has been shown to act downstream of Sry in testis development, while Dax1 appears to counteract Sry. Few more genes have been implicated in early gonad development. However, the genetic networks controlling early differentiation events in testis and ovary are still far from being understood. In order to provide a broader basis for the molecular analysis of gonad development, high-throughput gene expression analysis was utilized to identify genes specifically expressed in the gonad. In total, among 138 genes isolated which showed tissue specific expression in the embryo, 79 were detected in the developing gonad or sex ducts. Twenty-seven have not been functionally described before, while 40 represent known genes and 12 are putative mouse orthologues. Forty-five of the latter two groups (86%) have not been described previously in the fetal gonad. In addition, 21 of the gonad specific genes showed sex-dimorphic expression suggesting a role in sex determination and/or gonad differentiation. Eighteen of the latter (86%) have not been described previously in the fetal gonad. In total we provide new data on 72 genes which may play a role in gonad or sex duct development and/or sex determination. Thus we have generated a large gene resource for the investigation of these processes, and demonstrate the suitability of high-throughput gene expression screening for the genetic analysis of organogenesis.

Large-scale screen for genes controlling mammalian embryogenesis, using high-throughput gene expression analysis in mouse embryos.

We have adapted the whole-mount in situ hybridization technique to perform high-throughput gene expression analysis in mouse embryos. A large-scale screen for genes showing specific expression patterns in the mid-gestation embryo was carried out, and a large number of genes controlling development were isolated. From 35760 clones of a 9.5 d.p.c. cDNA library, a total of 5348 cDNAs, enriched for rare transcripts, were selected and analyzed by whole-mount in situ hybridization. Four hundred and twenty-eight clones revealed specific expression patterns in the 9.5 d.p.c. embryo. Of 361 tag-sequenced clones, 198 (55%) represent 154 known mouse genes. Thirty-nine (25%) of the known genes are involved in transcriptional regulation and 33 (21%) in inter- or intracellular signaling. A large number of these genes have been shown to play an important role in embryogenesis. Furthermore, 24 (16%) of the known genes are implicated in human disorders and three others altered in classical mouse mutations. Similar proportions of regulators of embryonic development and candidates for human disorders or mouse mutations are expected among the 163 new mouse genes isolated. Thus, high-throughput gene expression analysis is suitable for isolating regulators of embryonic development on a large-scale, and in the long term, for determining the molecular anatomy of the mouse embryo. This knowledge will provide a basis for the systematic investigation of pattern formation, tissue differentiation and organogenesis in mammals.

The paired homeobox gene Uncx4.1 specifies pedicles, transverse processes and proximal ribs of the vertebral column.

The axial skeleton develops from the sclerotome, a mesenchymal cell mass derived from the ventral halves of the somites, segmentally repeated units located on either side of the neural tube. Cells from the medial part of the sclerotome form the axial perichondral tube, which gives rise to vertebral bodies and intervertebral discs; the lateral regions of the sclerotome will form the vertebral arches and ribs. Mesenchymal sclerotome cells condense and differentiate into chondrocytes to form a cartilaginous pre-skeleton that is later replaced by bone tissue. Uncx4.1 is a paired type homeodomain transcription factor expressed in a dynamic pattern in the somite and sclerotome. Here we show that mice homozygous for a targeted mutation of the Uncx4.1 gene die perinatally and exhibit severe malformations of the axial skeleton. Pedicles, transverse processes and proximal ribs, elements derived from the lateral sclerotome, are lacking along the entire length of the vertebral column. The mesenchymal anlagen for these elements are formed initially, but condensation and chondrogenesis do not occur. Hence, Uncx4.1 is required for the maintenance and differentiation of particular elements of the axial skeleton.

Brachyury is a target gene of the Wnt/beta-catenin signaling pathway.

To identify target genes of the Wnt/beta-catenin signaling pathway in early mouse embryonic development we have established a co-culture system consisting of NIH3T3 fibroblasts expressing different Wnts as feeder layer cells and embryonic stem (ES) cells expressing a green fluorescent protein (GFP) reporter gene transcriptionally regulated by the TCF/beta-catenin complex. ES cells specifically respond to Wnt signal as monitored by GFP expression. In GFP-positive ES cells we observe expression of Brachyury. Two TCF binding sites located in a 500 bp Brachyury promoter fragment bind the LEF-1/beta-catenin complex and respond specifically to beta-catenin-dependent transactivation. From these results we conclude that Brachyury is a target gene for Wnt/beta-catenin signaling.

Kidney-specific cadherin (cdh16) is expressed in embryonic kidney, lung, and sex ducts.

Cdh16 was initially described as a truncated cadherin expressed in the adult rabbit kidney. We have analyzed the expression pattern of cdh-16 during mouse embryogenesis, and show that cdh-16 transcripts are present in ureter-derived epithelia of the metanephric kidney. In addition, we demonstrate that cdh-16 is also transiently expressed in the epithelia of embryonic sex ducts and the lung of the embryo.

The mouse Rsk3 gene maps to the Leh66 elements carrying the t-complex responder Tcr.

A variant form of mouse Chromosome (Chr) 17, the t-haplotype, contains several loci responsible for transmission ratio distortion in males. Sperm carrying the responder locus (Tcr) have a high probability of fertilizing eggs at the expense of wild-type sperm, provided that distorter loci (Tcd-1 to Tcd-5) are expressed during spermatogenesis. Tcr has been mapped to the Leh66b region within a maximum of 155 kb. In the search for genes in the genomic region Leh66EI, we have identified the mouse homolog of human ribosome S6 kinase 3 (RSK3) on cosmid DNA. The complete mouse Rsk3 gene is encoded in the region Leh66a of t-haplotypes and Leh66EI of the wild-type chromosome. It consists of at least 13 exons spanning over more than 120 kb. Rsk3 is expressed in embryos and in several adult organs including testis. Cosmids covering 100 kb of the Leh66b region or 120 kb of the Leh66a region were isolated. Rsk3 covers about 65 kb of the Leh66b region and appears to be incomplete at its 5'-end. A correlation of the physical map provided here with the genetic mapping of Tcr reported previously suggests that Tcr is most likely encoded within a fragment of 30 kb upstream or 20 kb downstream of Rsk3. These data will facilitate the isolation of Tcr, a prerequisite for understanding transmission ratio distortion in mouse.

A protein kinase encoded by the t complex responder gene causes non-mendelian inheritance.

Males heterozygous for the t-haplotype form of mouse chromosome 17 preferentially transmit the t-chromosome to their progeny. Several distorter/sterility loci carried on the t-haplotype together impair flagellar function in all spermatozoa whereas the responder, Tcr, rescues t-sperm but not wild-type sperm. Thus, t-sperm have an advantage over wild-type sperm in fertilizing egg cells. We have isolated Tcr by positional cloning and show that it is a member of a novel protein kinase gene family, designated Smok, which is expressed late during spermiogenesis. Smok kinases are components of a signal cascade which may control sperm motility. Tcr has a reduced kinase activity, which may allow it to counterbalance a signalling impairment caused by the distorter/sterility loci. Tcr transgene constructs cause non-mendelian transmission of chromosomes on which they are carried, which leads to sex-ratio distortion when Tcr cosegregates with the Y chromosome.

Crystallographic structure of the T domain-DNA complex of the Brachyury transcription factor.

The mouse Brachyury (T) gene is the prototype of a growing family of so-called T-box genes which encode transcriptional regulators and have been identified in a variety of invertebrates and vertebrates, including humans. Mutations in Brachyury and other T-box genes result in drastic embryonic phenotypes, indicating that T-box gene products are essential in tissue specification, morphogenesis and organogenesis. The T-box encodes a DNA-binding domain of about 180 amino-acid residues, the T domain. Here we report the X-ray structure of the T domain from Xenopus laevis in complex with a 24-nucleotide palindromic DNA duplex. We show that the protein is bound as a dimer, interacting with the major and the minor grooves of the DNA. A new type of specific DNA contact is seen, in which a carboxy-terminal helix is deeply embedded into an enlarged minor groove without bending the DNA. Hydrophobic interactions and an unusual main-chain carbonyl contact to a guanine account for sequence-specific recognition in the minor groove by this helix. Thus the structure of this T domain complex with DNA reveals a new way in which a protein can recognize DNA.

A mouse gene of the paired-related homeobox class expressed in the caudal somite compartment and in the developing vertebral column, kidney and nervous system.

During vertebrate embryonic development, pairs of metameric units, the somites, bud off at the cranial end of the paraxial mesoderm. The somites soon obtain cranio-caudal and dorso-ventral polarity. Establishment of dorso-ventral and medio-lateral polarity depends on multiple signals from the notochord, neural tube surface ectoderm and lateral mesoderm. The establishment of cranio-caudal polarity in the somite is less well understood. One molecule involved is the Dll1 gene product, a transmembrane protein expressed in the unsegmented paraxial mesoderm and in the caudal half of the somites. We have identified a gene, Uncx4.1, expressed in the caudal half of newly formed somites. It encodes a protein belonging to the paired-related class of homeodomain transcription factors. Uncx4.1 expression is first detected in the entire caudal half of the somites, is later down-regulated in the myotome and dermatome, and is maintained in the caudal sclerotome and its derivatives from which part of the vertebral column will form. Thus, Uncx4.1 may be involved in the establishment and maintenance of segment polarity and in vertebral column formation. Uncx4.1 is also expressed in the first branchial arch, the meso- and metanephric kidney, the central nervous system and the first digit of the forelimb, suggesting control functions of Uncx4.1 in multiple processes of embryogenesis.

Nuclear localization of beta-catenin by interaction with transcription factor LEF-1.

Vertebrate beta-catenin and Drosophila Armadillo share structural similarities suggesting that beta-catenin, like Armadillo, has a developmental signaling function. Both proteins are present as components of cell adherens junctions, but accumulate in the cytoplasm upon Wingless/Wnt signaling. beta-Catenin has axis-inducing properties like Wnt when injected into Xenopus blastomeres, providing evidence for participation of beta-catenin in the Wnt-pathway, but until now no downstream targets for beta-catenin have been identified. Here we demonstrate that beta-catenin binds to the HMG-type transcription factor lymphoid enhancer factor-1 (LEF-1), resulting in a nuclear translocation of beta-catenin both in cultured mouse cells and after ectopic expression of LEF-1 in two-cell mouse embryos. LEF-1/beta-catenin complexes bind to the promoter region of the E-cadherin gene in vitro, suggesting that this interaction could regulate E-cadherin transcription. As shown for beta-catenin, ectopic expression of LEF-1 in Xenopus embryos caused duplication of the body axis, indicating a regulatory role for a LEF-1-like molecule in dorsal mesoderm formation.

Distinct regulatory control of the Brachyury gene in axial and non-axial mesoderm suggests separation of mesoderm lineages early in mouse gastrulation.

Brachyury is required for the normal extension of the anteroposterior axis during mouse embryogenesis. A transgene comprising sequences from -500 to +150 relative to the start of Brachyury transcription, and the reporter gene lacZ, recapitulates some, but not all elements of Brachyury expression. Beta-Galactosidase expression is seen in the primitive streak from 6.5 d.p.c. but there is no detectable reporter expression in the node or notochord. Thus, the regulatory sequences required for the expression of Brachyury in the cells traversing the primitive streak are distinct from those required for the initiation of expression in the node. This suggests that different or additional signals are involved in activation of Brachyury in the node and notochord than those inducing Brachyury in the primitive streak. Additionally, the data suggest the possibility that axial and non-axial mesoderm are distinct from the earliest stages of Brachyury expression.

Genomic organization of M line titin and its tissue-specific expression in two distinct isoforms.

Titin is a 3000 kDa large protein of vertebrate striated muscle which extends from Z discs to M lines. Within the segment of titin that locates in the I band, tissue-specific isoforms are expressed by differential splicing in correlation to the sarcomeric ultrastructure. We have now searched the M-line region of titin for differential expression. The 20 kb section from the 3' end of the gene has been sequenced and contains 23 exons. Exon/intron organization is correlated to the modular organization of the titin protein. The six exons at the 3' end of the gene encode the M-line section of titin and are referred to as Mex1 to Mex6. Analysis of the RNAs expressed in different rabbit striated muscles reveals that the exon Mex5 is either included or excluded in the titin mRNA during splicing. The levels of inclusion of Mex5 vary between different types of striated muscles. Heart expresses (Mex5+)-titin, skeletal muscles co-express tissue-specifically distinct ratios of (Mex5+) and (Mex5-)-titins. In situ hybridization of whole-mount mouse embryos with Mex5 antisense RNA provide no evidence for the exclusion of Mex5 during embryonic development. We speculate that the establishment of differential splicing pathways of M-line titin late during development may correlate with and explain the postnatal development of different M-line fine structures in the different muscles. Comparison of titin gene sequences from different vertebrates reveals that the intron sequences located upstream of Mex3 and Mex5, referred to as Min-2 and Min-4, respectively, have remained strongly conserved during evolution. While the conservation of Min-4 may be explained by its participation in the regulation of the differential skipping of Mex5, the functional significance of the conservation of the Min-2 intron located upstream of Mex3 is yet unknown.

Conservation of Brachyury (T) genes in amphioxus and vertebrates: developmental and evolutionary implications.

Homologues of the murine Brachyury (T) gene have been cloned from several vertebrates, and are implicated in mesoderm formation and in differentiation of the notochord. In contrast, the roles of the ascidian Brachyury gene may be restricted to presumptive notochord. To understand the evolution of Brachyury genes and their developmental roles, we have searched for homologues in amphioxus, representing the third chordate subphylum and the probable closest relative of the vertebrates. We report the isolation of two amphioxus cDNA clones with clear homology to Brachyury genes, and demonstrate that these derive from separate loci resultant from a recent gene duplication. This finding represents an exception to the emerging consensus of an archetypal prevertebrate genome in amphioxus. The spatial and temporal distribution of Brachyury transcripts during amphioxus development is remarkably similar to vertebrate Brachyury, in presumptive mesoderm, posterior mesoderm and the notochord. Gene expression extends throughout the anteroposterior axis of the notochord, despite the most rostral regions being a more recent specialization; it also persists into larval stages, despite differentiation into contractile tissue. We propose that roles of Brachyury in notochord differentiation are more ancient than roles in mesoderm formation, and that the latter are shared by cephalochordates and all vertebrates.

The T protein encoded by Brachyury is a tissue-specific transcription factor.

The mouse Brachyury (T) gene is required for differentiation of the notochord and formation of mesoderm during posterior development. Homozygous embryos lacking T activity do not develop a trunk and tail and die in utero. The T gene is specifically expressed in notochord and early mesoderm cells in the embryo. recent data have demonstrated that the T protein is localized in the cell nucleus and specifically binds to a palindrome of 20 bp (the T site) in vitro. We show that the T protein activates expression of a reporter gene in HeLa cells through binding to the T site. Thus T is a novel tissue-specific transcription factor. It consists of a large N-terminal DNA binding domain (amino acids 1-229) and two pairs of transactivation and repression domains in the C-terminal protein half. T can also transactivate transcription through variously oriented and spaced T sites, a fact that may be relevant in the search for genes controlled by T protein and important in mesoderm development.

Promotion of gastrulation by maternal growth factor in cultured rabbit blastocysts.

Rabbit blastocysts of day 6 post coitus were cultured in a chemically defined, protein-free medium for 24 h. Although the trophoblast continued to grow, the embryonic disc degenerated. Addition of basic fibroblast growth factor (FGF-2, of human recombinant or bovine origin, 10 ng/ml) to the culture medium resulted in significant developmental progress. The embryonic disc became pear-shaped showing a round anterior edge and a posterior node. The primitive streak and Hensen's node indicated that gastrulation had begun. Mesoderm formation was confirmed from histological sections and by localization of the expression of T-gene transcripts in whole-mount preparations. FGF-2 mRNA was detected in both day-6 endometrium and day 6-blastocysts using in vitro translation followed by immunoprecipitation with a monoclonal antibody to FGF-2. In the uterine secretions of day-6 pregnant and pseudopregnant animals, several proteins exhibiting FGF-2 antigenicity were detected on Western blots following two-dimensional gel electrophoresis. As day-6 blastocysts required exogenous FGF-2 in vitro and as FGF-2 of uterine origin is present in the uterine secretion, the maternal growth factor can promote gastrulation in vivo.

The chick Brachyury gene: developmental expression pattern and response to axial induction by localized activin.

The mouse Brachyury gene (T) is required in notochord differentiation and posterior mesoderm formation during axial development. We have isolated the chick homologue of T(Ch-T) and determined its putative protein sequence and expression pattern during embryogenesis. Ch-T is expressed in the epiblast close to and within the primitive streak, in early migrating mesoderm and in the notochord. In later stages Ch-T expression is found in the tail bud and in the entire notochord. The notochord expression ceases in an anterior-posterior wave when the formation of the body anlage is completed. This pattern is consistent with those reported for the expression of the mouse T gene and the T homologues of Xenopus laevis and zebrafish, suggesting that the mechanisms of embryonic pattern formation are highly conserved in all vertebrates. The N-terminal half of Ch-T shows a very high degree of sequence identity with the corresponding region of mouse T which has DNA-binding activity, and with the N-terminal half of Xenopus (Xbra) and zebrafish (Ntl) T protein. Finally, we have analyzed the effects of activin A on Ch-T induction and axis formation. Localized activin A treatment of prestreak blastoderms results in ectopic Ch-T expression that correlates with formation of second primitive streaks or with repositioning of the site of single streak origin (Cooke et al., 1994). These results strengthen the previous evidence that Brachyury activation is an early response to axis-inducing signals in vivo.

Homologs of the mouse Brachyury gene are involved in the specification of posterior terminal structures in Drosophila, Tribolium, and Locusta.

The Brachyury (T) gene is required for notochord differentiation in vertebrates. We have identified a Drosophila gene, the T-related gene (Trg), with high similarity to T within a stretch of approximately 200 amino acids, the DNA-binding domain of T. Trg is expressed throughout embryogenesis, first at the blastoderm stage in the hindgut primordium under the control of the terminal gap genes tll and hkb, and then until the end of embryogenesis in the differentiating hindgut. Drosophila embryos deficient for Trg do not form the hindgut, a phenotype that can be rescued by a Trg transgene. Thus, a common feature of T and Trg is their requirement in specifying the development of a single embryonic structure. Homologs of Trg are also expressed in the developing hindgut of Tribolium and Locusta embryos suggesting a highly conserved function of Trg in insects. This conservation and the high similarity of T and Trg raise the question of a common evolutionary origin of the hindgut of insects and the notochord of chordates.

The T genes in embryogenesis.

Since its identification in 1927, the mouse T (Brachyury) locus has been implicated in mesoderm formation and notochord differentiation. Recent work has demonstrated that this gene encodes a putative transcription factor expressed specifically in nascent mesoderm and in the differentiating notochord. Homologous genes have been cloned from the frog Xenopus laevis, the zebrafish Brachydanio rerio and the ascidian Halocynthia roretzi. The T gene is an important tool for elucidating mesoderman and embryonic pattern formation.