Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes.

A strategy was devised for identifying regions of the mouse genome that are transcriptionally active in a temporally and spatially restricted manner during development. The approach is based on the introduction into embryonic stem cells of two types of lacZ reporter constructs that can be activated by flanking mouse genomic sequences. Embryonic stem cells containing the lacZ constructs were used to produce chimaeric mice. Developmental regulation of lacZ expression occurred at a high frequency. Molecular cloning of the flanking endogenous genes and introduction of these potential insertional mutations into the mouse germ line should provide an efficient means of identifying and mutating novel genes important for the control of mammalian development.

Germ Line Deletion Reveals a Nonessential Role of Atypical Mitogen-Activated Protein Kinase 6/Extracellular Signal-Regulated Kinase 3.

Mitogen-activated protein kinase 6/extracellular signal-regulated kinase 3 (MAPK6/ERK3) is an atypical member of the MAPKs. An essential role has been suggested by the perinatal lethal phenotype of ERK3 knockout mice carrying a lacZ insertion in exon 2 due to pulmonary dysfunction and by defects in function, activation, and positive selection of T cells. To study the role of ERK3 in vivo, we generated mice carrying a conditional Erk3 allele with exon 3 flanked by loxP sites. Loss of ERK3 protein was validated after deletion of Erk3 in the female germ line using zona pellucida 3 (Zp3)-cre and a clear reduction of the protein kinase MK5 is detected, providing the first evidence for the existence of the ERK3/MK5 signaling complex in vivo In contrast to the previously reported Erk3 knockout phenotype, these mice are viable and fertile and do not display pulmonary hypoplasia, acute respiratory failure, abnormal T-cell development, reduction of thymocyte numbers, or altered T-cell selection. Hence, ERK3 is dispensable for pulmonary and T-cell functions. The perinatal lethality and lung and T-cell defects of the previous ERK3 knockout mice are likely due to ERK3-unrelated effects of the inserted lacZ-neomycin resistance cassette. The knockout mouse of the closely related atypical MAPK ERK4/MAPK4 is also normal, suggesting redundant functions of both protein kinases.

Interaction between Notch signalling and Lunatic fringe during somite boundary formation in the mouse.

BACKGROUND: The process of somitogenesis can be divided into three major events: the prepatterning of the mesoderm; the formation of boundaries between the prospective somites; and the cellular differentiation of the somites. Expression and functional studies have demonstrated the involvement of the murine Notch pathway in somitogenesis, although its precise role in this process is not yet well understood. We examined the effect of mutations in the Notch pathway elements Delta like 1 (Dll1), Notch1 and RBPJkappa on genes expressed in the presomitic mesoderm (PSM) and have defined the spatial relationships of Notch pathway gene expression in this region. RESULTS: We have shown that expression of Notch pathway genes in the PSM overlaps in the region where the boundary between the posterior and anterior halves of two consecutive somites will form. The Dll1, Notch1 and RBPJkappa mutations disrupt the expression of Lunatic fringe (L-fng), Jagged1, Mesp1, Mesp2 and Hes5 in the PSM. Furthermore, expression of EphA4, mCer 1 and uncx4.1, markers for the anterior-posterior subdivisions of the somites, is down-regulated to different extents in Notch pathway mutants, indicating a global alteration of pattern in the PSM. CONCLUSIONS: We propose a model for the mechanism of somite border formation in which the activity of Notch in the PSM is restricted by L-fng to a boundary-forming territory in the posterior half of the prospective somite. In this region, Notch function activates a set of genes that are involved in boundary formation and anterior-posterior somite identity.

Aberrant regulation of imprinted gene expression in Gtl2lacZ mice.

The imprinted region on mouse distal chromosome 12 covers about 1 Mb and contains at least three paternally expressed genes (Pegs: Peg9/Dlk1, Peg11/Rtl1, and Dio3) and four maternally expressed genes (Megs: Meg3/Gtl2, antiPeg11/antiRlt1, Meg8/Rian, and Meg9/Mirg). Gtl2(lacZ) (Gene trap locus 2) mice have a transgene (TG) insertion 2.3 kb upstream from the Meg3/Gtl2 promoter and show about 40% growth retardation when the TG-inserted allele is paternally derived. Quantitative RT-PCR experiments showed that the expression levels of Pegs in this region were reduced below 50%. These results are consistent with the observed phenotype in Gtl2lacZ mice, because at least two Pegs(Peg9/Dlk1 and Dio3) have growth-promoting effects. The aberrant induction of Megs from silent paternal alleles was also observed in association with changes in the DNA methylation level of a differentially methylated region (DMR) located around Meg3/Gtl2 exon 1. Interestingly, a 60 approximately 80% reduction in all Megs was observed when the TG was maternally derived, although the pups showed no apparent growth or morphological abnormalities. Therefore, the paternal or maternal inheritance of the TG results in the down-regulation in cis of either Pegs or Megs, respectively, suggesting that the TG insertion influences the mechanism regulating the entire imprinted region.

The mouse rib-vertebrae mutation disrupts anterior-posterior somite patterning and genetically interacts with a Delta1 null allele.

Rib-vertebrae (rv) is an autosomal recessive mutation in mouse that affects the morphogenesis of the vertebral column. Axial skeleton defects vary along the anterior-posterior body axis, and include split vertebrae and neural arches, and fusions of adjacent segments. Here, we show that defective somite patterning underlies the vertebral malformations and altered Notch signaling may contribute to the phenotype. Somites in affected regions are irregular in size and shape, epithelial morphology is disrupted, and anterior-posterior somite patterning is abnormal, reminiscent of somite defects obtained in loss-of-function alleles of Notch signaling pathway components. Expression of Dll1, Dll3, Lfng and Notch1 is altered in rv mutant embryos, and rv and Dll1(lacZ), a null allele of the Notch ligand Delta1, genetically interact. Mice double heterozygous for rv and Dll1(lacZ), show vertebral defects, and one copy of Dll1(lacZ) on the homozygous rv background enhances the mutant phenotype and is lethal in the majority of cases. However, fine genetic mapping places rv into an interval on chromosome seven that does not contain a gene encoding a known component of the Notch signaling pathway.

Skeletal dysplasias, growth retardation, reduced postnatal survival, and impaired fertility in mice lacking the SNF2/SWI2 family member ETL1.

The mouse Etl1 gene encodes a nuclear protein belonging to the rapidly growing SNF2/SWI2 family. Members of this family are related to helicases and nucleic-acid-dependent ATPases and have functions in essential cellular processes such as transcriptional regulation, maintenance of chromosome stability and various aspects of DNA repair. The ETL1 protein is expressed from the two-cell stage onwards, throughout embryogenesis in a dynamic pattern with particularly high levels in the thymus, epithelia and the nervous system and in most adult tissues. As a first step to address the role of ETL1 in cells and during development, we inactivated the gene by homologous recombination. ES cells and mice lacking detectable ETL1 protein were viable, indicating that ETL1 is not essential for cell survival or for embryonic development. However, mutant mice showed retarded growth, peri/post natal lethality, reduced fertility and various defects in the sternum and vertebral column. Expressivity and penetrance of all observed phenotypes were influenced by the genetic background. Isogenic 129Sv(Pas) mice lacking ETL1 had a severely reduced thoracic volume, which might lead to respiratory failure and could account for the high incidence of perinatal death on this genetic background.

Enhancer trap integrations in mouse embryonic stem cells give rise to staining patterns in chimaeric embryos with a high frequency and detect endogenous genes.

We have generated mouse embryonic stem cell lines that carry lacZ enhancer trap constructs integrated in their genome. Fifty-nine cell lines were analysed for lacZ expression in undifferentiated stem cells and at day 7.5, 8.5 and 12.5 of development in chimaeric embryos obtained after blastocyst injection. In 13 cell lines the lacZ reporter gene was expressed in undifferentiated stem cells ('blue', lines) as monitored by beta-galactosidase activity; 46 cell lines did not show detectable beta-galactosidase activity ('white', lines). In chimaeric embryos one-third of the analysed 59 embryonic stem cell lines gave rise to a variety of patterns. Six out of the 13 'blue' lines and 14 out of the 46 'white' lines showed spatially and temporally regulated patterns of beta-galactosidase expression and were additionally analysed on day 9.5. The majority of patterns showed staining exclusively or predominantly in structures of the developing nervous system, three patterns were observed only or predominantly in non-neuronal structures and five patterns were found exclusively in extraembryonic tissues. The analysis of DNA from cell lines that gave rise to staining patterns in chimaeric embryos showed that in 11 out of 15 cases simple integrations had occurred at a single site while in the remaining four cell lines multiple copies had integrated either at a single or at multiple sites. Flanking sequences from five reporter gene integrations have been cloned. At present, three integration sites have been analysed further and in all three cases we have identified transcribed sequences in the flanking DNA and isolated corresponding cDNA clones. The expression patterns of two of these genes were analysed by RNA in situ hybridisation. In both cases, expression of the endogenous genes was more widespread than the corresponding beta-galactosidase staining, suggesting that the reporter gene responded to only a subset of the regulatory elements of the endogenous gene. Our results demonstrate that enhancer trap integrations in embryonic stem cells can be used to efficiently identify transcriptional activation patterns during mouse embryogenesis and to isolate endogenous genes expressed in spatially and temporally regulated patterns.

The mouse Enhancer trap locus 1 (Etl-1): a novel mammalian gene related to Drosophila and yeast transcriptional regulator genes.

A novel mouse gene, Enhancer trap locus 1 (Etl-1), was identified in close proximity to a lacZ enhancer trap integration in the mouse genome showing a specific beta-galactosidase staining pattern during development. In situ analysis revealed a widespread but not ubiquitous expression of Etl-1 throughout development with particularly high levels in the central nervous system and epithelial cells. The amino acid sequence of the Etl-1 protein deduced from the cDNA shows strong similarity, over a stretch of 500 amino acids, to the Drosophila brahma protein involved in the regulation of homeotic genes and to the yeast transcriptional activator protein SNF2/SWI2 as well as to the RAD54 protein and the recently described helicase-related yeast proteins STH1 and MOT1. Etl-1 is the first mammalian member of this group of proteins that are implicated in gene regulation and/or influencing chromatin structure. The homology to the regulatory proteins SNF2/SWI2 and brahma and the expression pattern during embryogenesis suggest that Etl-1 protein might be involved in gene regulating pathways during mouse development.

Distinct regulatory elements direct delta1 expression in the nervous system and paraxial mesoderm of transgenic mice.

The Delta1 gene encodes one of the Notch ligands in mice. Delta1 is expressed during early embryogenesis in a complex and dynamic pattern in the paraxial mesoderm and neuroectoderm, and is essential for normal somitogenesis and neuronal differentiation. Molecular components thought to act in response to ligand binding and Notch activation have been identified in different species. In contrast, little is known about the transcriptional regulation of Notch receptors and their ligands. As a first step to identify upstream factors regulating Delta1 expression in different tissues, we searched for cis-regulatory regions in the Delta1 promoter able to direct heterologous gene expression in a tissue specific manner in transgenic mice. Our results show that a 4.3 kb genomic DNA fragment of the Delta1 gene is sufficient in a lacZ reporter transgene to reproduce most aspects of Delta1 expression from the primitive streak stage to early organogenesis. Using a minimal Delta1 promoter we also show that this upstream region contains distinct regulatory modules that individually direct tissue-specific transgene expression in subdomains of the endogenous expression pattern. It appears that expression in the paraxial mesoderm depends on the interaction of multiple positive and negative regulatory elements. We also find that at least some regulatory sequences required for transgene expression in subdomains of the neural tube have been maintained during the evolution of mammals and teleost fish, suggesting that part of the regulatory network that controls expression of Delta genes may be conserved.

Expression of the mouse Delta1 gene during organogenesis and fetal development.

Cell-to-cell communication mediated by the evolutionary conserved Notch signalling pathway regulates cell fate decisions and patterning in various tissues in diverse organisms (Artavanis-Tsakonas et al., 1995, Science 268, 225-232). Signalling between neighboring cells is transduced by binding of DSL and Notch proteins which interact as ligand (DSL) and receptor (Notch). Mouse Delta1 (delta-like 1; Dll1) encodes one of the four known mammalian DSL proteins and is essential for normal somitogenesis and neuronal differentiation. Here, we describe Delta1 expression during organogenesis and fetal development using the highly sensitive histochemical detection of the lacZ gene product expressed from a targeted Delta1:lacZ knock-in allele (Dll1(lacZ)). We find that Delta1 is expressed in epithelial ducts of several organs, skeletal and smooth muscles, the central nervous system, as well as some sensory epithelia.

Expression of Delta1 and Serrate1 (Jagged1) in the mouse inner ear.

The Notch signalling pathway is thought to play a key part in controlling the production of sensory hair cells in the vertebrate inner ear via lateral inhibition; but there is disagreement as to which Notch ligands are expressed in hair cells as they develop. We show, using a mouse Delta1:LacZ knock-in as a reporter, that nascent hair cells, but not their neighbours, express Delta1. Expression of Serrate1 (Jagged1), meanwhile becomes restricted to the supporting cells of each sensory patch. Delta1 is also expressed: (a) at early stages, at the site of otic neurogenesis; and (b) in scattered cells of the endolymphatic sac, as is Serrate1.

Use of coisogenic host blastocysts for efficient establishment of germline chimeras with C57BL/6J ES cell lines.

Gene targeting in embryonic stem (ES) cells allows the production of mice with specified genetic mutations. Currently, germline-competent ES cell lines are available from only a limited number of mouse strains, and inappropriate ES cell/host blastocyst combinations often restrict the efficient production of gene-targeted mice. Here, we describe the derivation of C57BL/6J (B6) ES lines and compare the effectiveness of two host blastocyst donors, FVB/NJ (FVB) and the coisogenic strain C57BL/6-Tyr(c)-2J (c2J), for the production of germline chimeras. We found that when B6 ES cells were injected into c2J host blastocysts, a high rate of coat-color chimerism was detected, and germline transmission could be obtained with few blastocyst injections. In all but one case, highly chimeric mice transmitted to 100% of their offspring. The injection of B6 ES cells into FVB blastocysts produced some chimeric mice. However; the proportion of coat-color chimerism was low, with many more blastocyst injections required to generate chimeras capable of germline transmission. Our data support the use of the coisogenic albino host strain, c2J, for the generation of germline-competent chimeric mice when using B6 ES cells.

Efficient isolation of novel mouse genes differentially expressed in early postimplantation embryos.

Most genes with regulatory functions in embryogenesis are expressed in highly specific patterns, suggesting that expression patterns can serve as criteria to define potential candidates for developmentally relevant genes. To isolate such genes, we selected and partially sequenced 80 cDNA clones from a 10.5-day mouse embryo library. Forty-one clones that represented novel mouse genes were analyzed for expression in embryos of the same stage by whole-mount in situ hybridization. A high proportion (24%) of these genes, including a homologue of the Drosophila Delta gene, were expressed in specific spatially restricted patterns, suggesting that selection based on expression patterns is a useful strategy to isolate novel genes that may play pivotal roles in mammalian development.

The molecular and genetic analysis of mouse development.

This review describes some recent advances in the molecular-genetic analysis of mouse development. Reversed genetics and gene assignment have been used to isolate genes affected in developmental mutations. The establishment of a high-density molecular-genetic map promises to facilitate cloning of additional genes with developmental functions. Based on molecular, biochemical or other biological criteria many mouse genes that code for transcriptional regulators, growth-factor-like molecules and their receptors have been isolated. The role of these genes during development can be analysed in vivo after producing targeted mutations. Mutations can be generated by homologous recombination in the genome of embryonic stem cells and can then be introduced into the mouse germ line by means of germ-line chimaeras. Additional approaches employing stem cells to identify and mutate putative developmental genes are coming into use.

The Danforth's short tail mutation acts cell autonomously in notochord cells and ventral hindgut endoderm.

Danforth's short tail (Sd) is a semidominant mutation in mouse affecting the axial skeleton and urogenital system. The notochord is the first visibly abnormal structure in mutant embryos, and disintegrates beginning around embryonic day 9.5 along its entire length, suggesting an essential role for Sd in notochord development and maintenance. Here, we report on the fate of Sd/+ and Sd/Sd cells in chimeric embryos. Up to day 9-9.5, Sd cells contributed efficiently to the notochord of chimeric embryos. In advanced day 9.5 embryos, Sd cells were less abundant in the posterior-most region of the notochord and in the notochordal plate. During subsequent development, Sd cells were specifically lost from the notochord and replaced by wild-type cells. In Sd/+<-->+/+ chimeras, the notochord appeared histologically and functionally normal, leading to a rescue of the mutant phenotype. However, strong Sd/Sd<-->+/+ chimeras showed malformations of the axial skeleton and urogenital system. All Sd/Sd<-->+/+ chimeras with malformations of the axial skeleton also had kidney defects, whereas chimeras without vertebral column defects had highly chimeric kidneys that appeared normal, suggesting that the urogenital malformations arise secondarily to impaired posterior development caused by the degenerating notochord. Sd mutant cells also were specifically absent from the ventral portion of the hindgut, whereas they contributed efficiently to the dorsal region, implying the existence of distinct cell populations in the dorsal and ventral hindgut. Our findings demonstrate that the Sd mutation acts cell autonomously in cells of the notochord and ventral hind gut. Sd leads to the degeneration of notochord cells and the number or allocation of notochord precursors from the tail bud to the notochordal plate seems impaired, whereas notochord formation from the node appears to be unaffected.

Somitogenesis.

We are still far from understanding "somitogenesis" as a whole, but there is an emerging picture of the tissue interactions and molecular mechanisms that underlie and govern various aspects of this essential multistep patterning process in vertebrates. The ability to form segmental units appears to be a property specific to the paraxial mesoderm (as opposed to lateral or limb mesoderm), and this ability is probably acquired during early development, when paraxial mesoderm is specified and emerges from the primitive streak. Signaling molecules expressed in the primitive streak and tail bud are prime candidates involved in specifying paraxial (as well as other mesodermal) fates. Increasing levels of signaling molecules may be required in posterior regions of the embryo, and combinatorial signals may be essential to specify the paraxial mesoderm along the entire anterior-posterior axis. However, most of the pivotal signals, and the ways in which they are integrated and interact, remain enigmatic. Once the paraxial mesoderm is formed, segmentation proceeds largely without the requirement for continuous interactions with surrounding tissues. Somitomeres represent a morphologic pattern in the mesenchymal presomitic mesoderm, but their significance for somite formation is unclear. Molecular patterns are established in the presomitic mesoderm and probably are of functional significance. Cell interactions within the paraxial mesoderm appear to be involved in forming segment borders and ensuring their maintenance during subsequent differentiation of somites. These interactions are, at least in part, mediated by components of the conserved Notch signaling pathway, which may have multiple functions during somitogenesis. Epithelial somites are clearly a result of segmentation, but epithelialization is not the mechanism to form segments, supporting the idea that the basic mechanisms that govern segmentation in the mesoderm of vertebrates are very similar in different species despite divergent types of resulting segments (i.e., epithelial somites versus rotated myotomes). Concomitantly with segmentation, segment polarity and positional specification are established. How these processes are linked to, and depend on, each other is unknown, as is how they are regulated and how segmentation is coordinated on both sides of the neural tube. In contrast to early patterning in the presomitic mesoderm, patterning of the mature somites during their subsequent differentiation is the result of extensive tissue interactions. Virtually all tissues in close proximity to somites provide signals that are involved in induction or inhibition of particular differentiation pathways, but how these pathways are initiated is less clear. Some of the molecules mediating inductive signals and tissue interactions are known, and a growing number of candidate genes are potentially involved in regulating various steps of somitogenesis. The roles of these genes have yet to be analyzed. In addition, the molecular genetic analysis of mutations affecting somitogenesis, which were collected in the mouse and more recently in the zebrafish (Driever et al., 1996; Haffter et al., 1996; van Eeden et al., 1996), promises to add important new insights into this process. Much remains to be done, but the tools are at hand to provide further understanding of the molecular mechanisms underlying somitogenesis.

Maternal IL-11Ralpha function is required for normal decidua and fetoplacental development in mice.

In eutherian mammals, implantation and establishment of the chorioallantoic placenta are essential for embryo development and survival. As a maternal response to implantation, uterine stromal cells proliferate, differentiate, and generate the decidua, which encapsulates the conceptus and forms the maternal part of the placenta. Little is known about decidual functions and the molecular interactions that regulate its development and maintenance. Here we show that the receptor for the cytokine interleukin-11 (IL-11Ralpha) is required specifically for normal establishment of the decidua. Females homozygous for a hypomorphic IL-11Ralpha allele are fertile and their blastocysts implant and elicit the decidual response. Because of reduced cell proliferation, however, only small deciduae form. Mutant deciduae degenerate progressively, and consequently embryo-derived trophoblast cells generate a network of trophoblast giant cells but fail to form a chorioallantoic placenta, indicating that the decidua is essential for normal fetoplacentation. IL-11Ralpha is expressed in the decidua as well as in numerous other tissues and cell types, including the ovary and lymphocytes. The differentiation state and proliferative responses of B and T-lymphocytes in mutant females were normal, and wild-type females carrying IL-11Ralpha mutant ovaries had normal deciduae, suggesting that the decidualization defects do not arise secondarily as a consequence of perturbed IL-11Ralpha signaling defects in lymphoid organs or in the ovary. Therefore, IL-11Ralpha signaling at the implantation site appears to be required for decidua development.

Genetic interactions suggest that Danforth's short tail (Sd) is a gain-of-function mutation.

Danforth's short tail (Sd) is a semidominant mutation on mouse chromosome 2 that acts cell autonomously in the notochord and leads to its distintegration, and thus causes severe defects in somite patterning and vertebral column development. The molecular nature of the Sd gene and mutation is unknown, and it is unclear whether Sd is a loss-of-function mutation and the semidominant inheritance of the Sd phenotype is due to haploinsufficiency, or whether Sd represents a gain-of-function mutation in a gene essential for notochord development and maintenance. Here, we report on the genetic interaction between Sd and an insertional mutation called Etl4lacZ, which provides genetic evidence that Sd is a gain-of-function mutation. Etl4lacZ is an enhancer trap insertion, which gives rise to lacZ expression in distinct cell types, including the notochord. In homozygosity, the lacZ insertion leads to abnormal vertebrae in the caudal part of the vertebral column. Etl4lacZ maps approximately 0.75 cM distal to Sd, and in double heterozy gotes modifies the Sd phenotype contrarily, depending on the chromosomal configuration of the Sd and Etl4lacZ mutations: when Etl4lacZ is present on the chromosome wild type for Sd (Sd+/+ Etl4lacZ; trans configuration), the Sd phenotype is enhanced, i.e., vertebral malformations extend to more anterior positions and the vertebral body of the axis is further reduced. Conversely, when Etl4lacZ is present on the same chromosome as Sd (Sd Etl4lacZ/+ +; cis configuration), the Sd phenotype is attenuated, i.e., vertebral malformations are confined to more posterior levels, and the dens axis, which is severely reduced or absent in Sd heterozygotes, is restored. The different effect of the Etl4lacZ insertion on Sd, depending on its presence in trans or cis, suggests a direct interaction of the transgene insertion with the Sd gene. Additionally, the attenuation of the Sd phenotype by Etl4lacZ in cis suggests that Sd is a gain-of-function mutation and lends support to the idea that Etl4lacZ is a new allele of Sd. Dev. Genet. 23:86-96, 1998.

Maintenance of somite borders in mice requires the Delta homologue DII1.

During vertebrate embryonic development, the paraxial mesoderm is subdivided into metameric subunits called somites. The arrangement and cranio-caudal polarity of the somites governs the metamerism of all somite-derived tissues and spinal ganglia. Little is known about the molecular mechanisms underlying somite formation, segment polarity, maintenance of segment borders, and the interdependency of these processes. The mouse Delta homologue Dll1, a member of the DSL gene family, is expressed in the presomitic mesoderm and posterior halves of somites. Here we report that, in Dll1-deficient mouse embryos, a primary metameric pattern is established in mesoderm, and cytodifferentiation is apparently normal, but the segments have no cranio-caudal polarity, and no epithelial somites form. Caudal sclerotome halves do not condense, and the pattern of spinal ganglia and nerves is perturbed, indicating loss of segment polarity. Myoblasts span segment borders, demonstrating that these borders are not maintained. These results show that Dll1 is involved in compartmentalization of somites, that dermomyotome and sclerotome differentiation are independent of formation of epithelia and subdivision of somites in cranial and caudal halves, and that compartmentalization is essential for the maintenance of segment borders in paraxial mesoderm-derived structures.