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.

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.

Gtl2lacZ, an insertional mutation on mouse chromosome 12 with parental origin-dependent phenotype.

We have produced a transgenic mouse line, Gtl2lacZ (Gene trap locus 2), that carries an insertional mutation with a dominant modified pattern of inheritance:heterozygous Gtl2lacZ mice that inherited the transgene from the father show a proportionate dwarfism phenotype, whereas the penetrance and expressivity of the phenotype is strongly reduced in Gtl2lacZ mice that inherited the transgene from the mother. On a mixed genetic background this pattern of inheritance was reversible upon transmission of the transgene through the germ line of the opposite sex. On a predominantly 129/Sv genetic background, however, transgene passage through the female germ line modified the transgene effect, such that the penetrance of the mutation was drastically reduced and the phenotype was no longer obvious after subsequent male germ line transmission. Expression of the transgene, however, was neither affected by genetic background nor by parental legacy. Gtl2lacZ maps to mouse Chromosome 12 in a region that displays imprinting effects associated with maternal and paternal disomy. Our results suggest that the transgene insertion in Gtl2lacZ mice affects an endogenous gene(s) required for fetal and postnatal growth and that this gene(s) is predominantly paternally expressed.

Fine genetic mapping of the proximal part of mouse chromosome 2 excludes Pax-8 as a candidate gene for Danforth's short tail (Sd).

Danforth's short tail (Sd) is a semidominant mutation of the mouse with effects on the skeleton and the urogenital system. In view of its phenotype and its position in the proximal part of Chromosome (Chr) 2, three genes qualified as possible candidates: Pax-8, a paired box-containing gene; Midkine (Mdk), a retinoic acid-responsive gene; and a new locus (Etl-4) identified by enhancer trapping with a lacZ reporter gene which showed expression in the notochord, the mesonephric mesenchyme, and the apical ectodermal ridge. Three different backcrosses involving all three genes in different combinations were set up and analyzed. From our results we conclude that Sd, Etl-4, Pax-8, and Mdk are independent loci, with Etl-4 being the closest genetic marker (1.1 +/- 1.4 cM) to the Danforth's short tail (Sd) gene.

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.