A gene mapping to the sex-determining region of the mouse Y chromosome is deleted in a line of XY female mice mutant for Tdy, and is expressed at a stage during male gonadal development consistent with its having a role in testis determination. This gene is a member of a new family of at least five mouse genes, related by an amino-acid motif showing homology to other known or putative DNA-binding domains.

Growth factors related to TGF-beta provide important signals for patterning the vertebrate body plan. One such family member, nodal, is required for formation of the primitive streak during mouse gastrulation. Here we have used a nodal-lacZ reporter allele to demonstrate asymmetric nodal expression in the mouse node, a structure thought to be the functional equivalent of the frog and chick 'organizer', and in lateral place mesoderm cells. We have also identified two additional genes acting with nodal in a pathway determining the left-right body axis. Thus we observe in inv mutant embryos that the sidedness of nodal expression correlates with the direction of heart looping and embryonic turning. In contrast, HNF3-beta(+/-) nodal(lacZ/+) double-heterozygous embryos display LacZ staining on both left and right sides, and frequently exhibit defects in body situs. Taken together, these experiments, along with similar findings in chick, demonstrate that elements of the genetic pathway that establish the left-right body axis are conserved in vertebrates.

The establishment of the anteroposterior (AP) axis is a crucial step during animal embryo development. In mammals, genetic studies have shown that this process relies on signals spatiotemporally deployed in the extra-embryonic tissues that locate the position of the head and the onset of gastrulation, marked by T/Brachyury (T/Bra) at the posterior of the embryo. Here, we use gastruloids, mESC-based organoids, as a model system with which to study this process. We find that gastruloids localise T/Bra expression to one end and undergo elongation similar to the posterior region of the embryo, suggesting that they develop an AP axis. This process relies on precisely timed interactions between Wnt/β-catenin and Nodal signalling, whereas BMP signalling is dispensable. Additionally, polarised T/Bra expression occurs in the absence of extra-embryonic tissues or localised sources of signals. We suggest that the role of extra-embryonic tissues in the mammalian embryo might not be to induce the axes but to bias an intrinsic ability of the embryo to initially break symmetry. Furthermore, we suggest that Wnt signalling has a separable activity involved in the elongation of the axis.

There are five members of the RFX family of transcription factors in mammals. While RFX5 plays a well-defined role in the immune system, the functions of RFX1 to RFX4 remain largely unknown. We have generated mice with a deletion of the Rfx3 gene. RFX3-deficient mice exhibit frequent left-right (LR) asymmetry defects leading to a high rate of embryonic lethality and situs inversus in surviving adults. In vertebrates, specification of the LR body axis is controlled by monocilia in the embryonic node, and defects in nodal cilia consequently result in abnormal LR patterning. Consistent with this, Rfx3 is expressed in ciliated cells of the node and RFX3-deficient mice exhibit a pronounced defect in nodal cilia. In contrast to the case for wild-type embryos, for which we document for the first time a twofold increase in the length of nodal cilia during development, the cilia are present but remain markedly stunted in mutant embryos. Finally, we show that RFX3 regulates the expression of D2lic, the mouse orthologue of a Caenorhabditis elegans gene that is implicated in intraflagellar transport, a process required for the assembly and maintenance of cilia. In conclusion, RFX3 is essential for the differentiation of nodal monocilia and hence for LR body axis determination.

We describe a simple method of chick whole-embryo culture, which uses a filter paper carrier to hold the early blastoderm and vitelline membranes under tension while the embryo grows on a substratum of agar-albumen. This is a quick and efficient means of setting up cultures of chick embryos beginning at pre-primitive streak stages to stage 10 (stages X--XIV, Eyal-Giladi and Kochav [1976] Dev Biol 49:321-337; stages 1--10, Hamburger and Hamilton [1951] J Morphol 88:49--92). This is an improvement on the original method of New, which used a glass ring and watch glass (New [1955] Exp Morphol 3:320--331). Our modification of New's method, which we call EC (Early Chick, pronounced EASY) culture, facilitates several manipulations in early chick embryos, including microsurgery, grafting, bead implantation, microinjection, and electroporation. Using the EC method, embryos at stage 8 and older can be readily cultured either dorsal-side up (in contrast to New's method) or ventral-side up, as desired; embryos younger than stage 8 can be culture only ventral-side up (as with New's method). We also discuss some alternative methods for setting up these cultures.

After implantation, mouse embryos deficient for the activity of the transforming growth factor-beta member Nodal fail to form both the mesoderm and the definitive endoderm. They also fail to specify the anterior visceral endoderm, a specialized signaling center which has been shown to be required for the establishment of anterior identity in the epiblast. Our study reveals that Nodal-/- epiblast cells nevertheless express prematurely and ectopically molecular markers specific of anterior fate. Our analysis shows that neural specification occurs and regional identities characteristic of the forebrain are established precociously in the Nodal-/- mutant with a sequential progression equivalent to that of wild-type embryo. When explanted and cultured in vitro, Nodal-/- epiblast cells readily differentiate into neurons. Genes normally transcribed in organizer-derived tissues, such as Gsc and Foxa2, are also expressed in Nodal-/- epiblast. The analysis of Nodal-/-;Gsc-/- compound mutant embryos shows that Gsc activity plays no critical role in the acquisition of forebrain characters by Nodal-deficient cells. This study suggests that the initial steps of neural specification and forebrain development may take place well before gastrulation in the mouse and highlights a possible role for Nodal, at pregastrula stages, in the inhibition of anterior and neural fate determination.

The mammalian genome contains a family of genes that are related to SRY, the mammalian sex determining gene. The homology is restricted to the region of SRY that encodes a DNA binding motif of the HMG-box class. These genes have been named SOX genes (SRY-related HMG-box genes). We have cloned and characterised SOX3, a member of the human SOX gene family. SOX3 maps to the X chromosome in the region Xq26-27. A mentally retarded male patient with haemophilia B is deleted for both the Factor IX gene and SOX3. This suggests that SOX3 is not essential for testis formation. The phenotype of the patient and the expression of SOX3 gene in neuronal tissues raises the possibility that this gene is a candidate gene for Borjeson-Forssman-Lehmann, an X-linked mental retardation syndrome.

The Y chromosome determines maleness in mammals. A Y chromosome-linked gene diverts the indifferent embryonic gonad from the default ovarian pathway in favour of testis differentiation, initiating male development. Study of this basic developmental switch requires the isolation of the testis-determining gene, termed TDF in humans and Tdy in mice. ZFY, a candidate gene for TDF, potentially encodes a zinc-finger protein, and has two Y-linked homologues, Zfy-1 and Zfy-2, in mice. Although ZFY, Zfy-1 and Zfy-2 seem to map to the sex-determining regions of the human and mouse Y chromosomes, there is no direct evidence that these genes are involved in testis determination. We report here that Zfy-1 but not Zfy-2 is expressed in differentiating embryonic mouse testes. Neither gene, however, is expressed in We/We mutant embryonic testes which lack germ cells. These observations exclude both Zfy-1 and Zfy-2 as candidates for the mouse testis-determining gene.