Germ line mutation of the GCNF gene proves that the encoded orphan nuclear receptor is essential for embryonic survival and normal development. GCNF−/− embryos cannot survive beyond 10.5 dpc, probably due to cardiovascular failure. Prior to death, GCNF−/− embryos suffer significant defects in posterior development, which are probably due to the loss of GCNF expression within cells of the posterior. The defects observed are not due to the neo cassette, as a GCNF knockout line with the neo cassette deleted phenocopies the GCNF knockout described here (data not shown). Defects in GCNF−/− embryos may be related to the retardation and/or halt in somitogenesis after 13 somites in mutant embryos and the abnormal positioning of the tailbud. Indeed, alterations in expression of multiple marker genes were identified in the posterior of GCNF−/− embryos, including the primitive streak, node, and PSM. These results suggest that GCNF is required for proper anteroposterior development, somite formation, and posterior development and that it is essential for embryonic survival.
The results of this study highlight some interesting similarities and differences in how GCNF functions in amphibians and mammals (19
). First, GCNF is important for both amphibian and mammalian embryonic survival. In Xenopus
, the expression of a dominant-negative form of GCNF results in malformations of the head, eyes, ears, cement gland, and somites, resulting in embryonic lethality (19
). Second, when wild-type GCNF is overexpressed in Xenopus
embryos, it causes disturbances in somitogenesis and tail formation, with normal head development (19
). Finally, failure of neural tube closure was also observed in Xenopus
embryos after expression of the dominant-negative GCNF (19
). Together with the results from our study, the findings indicate that the presence of GCNF is critical for normal posterior development, somitogenesis, and neural tube closure. Unlike the results of our study, expression of the dominant-negative form of GCNF in more posterior regions of Xenopus
had no significant effect on posterior development. Alternatively, severe posterior defects in Xenopus
may have been overlooked, as only embryos exhibiting less severe phenotypes were analyzed (19
The results presented here suggest that the lack of trunk development in GCNF−/−
embryos is distinct from the effects of other genes that have previously been shown to cause truncations of caudal structures. For example, brachyury T−/−
embryos have little or no notochord and have somites only up to the region of forelimb buds, forming no trunk or tail (22
). The posterior truncation is due to a failure to form mesoderm in T−/−
embryos. Inactivation of the HNF3
β gene has demonstrated effects on notochord development (2
). Although the expression pattern of HNF3
β in GCNF−/−
embryos is somewhat different from the wild-type pattern, HNF3
β is expressed in the midline structures, such as the notochord and floor plate, of GCNF−/−
embryos. When Wnt-3a
was mutated (63
), the resulting truncation occurred caudal to the forelimb buds. Wnt-3a
was expressed in the ectopic tailbud of GCNF−/−
embryos, although forelimbs did not develop. In contrast to Wnt3a−/−
embryos, which do not express brachyury T, GCNF−/−
embryos do express brachyury T in their tailbuds, indicating that these embryos possess a tailbud structure similar to that of wild-type embryos. In addition, the development of the GCNF−/−
tailbud progresses to the expression of later-stage markers such as Hoxb-13. Hoxb-13 is not expressed in the normal tailbud until 9.0 dpc; thus, its appearance in GCNF−/−
embryos implies that the tailbud not only is present but has also sustained some degree of normal development. Other mouse models, such as paraxis
, lunatic fringe
, and Mesp2
, have less severe posterior defects (10
). Taken together, these data suggest that GCNF may regulate a unique signaling pathway distinct from the brachyury T, HNF3β, Wnt-3a, and Hoxb-13 pathways.
The initiation and differentiation of the first seven to nine somites are not affected by the absence of GCNF. Previous fate mapping studies suggest that by the late streak stage, the bulk of prospective cranial mesoderm has already exited the streak (64
). The cells in the anterior segment of the primitive streak at this stage are mainly destined for the first 6 to 10 somites and the PSM of the early-somite-stage embryo, a finding which was confirmed by ablation experiments (65
). Thus, GCNF may not be required for the initiation of primary body formation or participation in somite formation during the early stages of development. The expression of paraxis
at 8.5 dpc in these somites reinforces this speculation. A transition in cellular recruitment to the paraxial mesoderm from the primitive streak to the tailbud mesenchyme occurs at the stage of posterior neuropore closure at the end of the neural stage (65
). Since GCNF may be required in the PSM and/or tailbud, somitogenesis is affected after this stage in GCNF−/−
embryos. Ectopic expression of paraxis
in the posterior of GCNF−/−
embryos indicates there is inappropriate differentiation within the PSM, ultimately leading to the halt in somitogenesis and the posterior truncation. The altered differentiation of the PSM is probably due to the loss of GCNF within cells of the posterior; however, the ectopic location of the tailbud also likely contributes to the phenotype.
The tailbud develops ectopically outside the yolk sac after the late streak stage, which could be due to the failure of the embryos to turn and envelop correctly in their embryonic membranes. The failure of mutant embryos to turn has been described for many gene knockout models, yet the positioning of the tailbud outside the yolk sac suggests that this is a novel phenotype related to the loss of GCNF function and not merely due to a failure of the GCNF mutant embryos to turn (25
). The halt in somitogenesis does not occur until after the posterior of the GCNF−/−
embryo develops outside the yolk sac. Therefore, in addition to the loss of GCNF function contributing to the halt in somitogenesis and the posterior truncation, the ectopic location of the tailbud may play a role as well.
The exact mechanism that results in the formation of this protrusion remains to be investigated. Here we propose a model that accounts for its formation based on the data presented here (Fig. ). In both GCNF+/+ and GCNF−/− embryos, as the somites, which express paraxis, are generated from the primitive streak, the neural plate forms at 8.0 dpc (Fig. A and B). As GCNF+/+ embryos continue to grow at 8.5 dpc, the node, which expresses nodal, regresses caudally (Fig. C), with a resulting lengthening of the notochord that continues to induce the neural plate to form the neural folds. The edges of the neural plate begin to elevate, forming the neural groove. In GCNF−/− embryos, a neural groove has been observed histologically, but neural epithelium abnormally invaginates within the primitive streak region by 8.5 dpc (Fig. D; Fig. B and D). This invagination then pushes the primitive streak away from the original direction of posterior regression.
FIG. 9 Model of ectopic tailbud formation in GCNF−/− embryos. At 8.0 dpc in both GCNF+/+ (A) and GCNF−/− (B) embryos, the neural plate arises when the somites are generated from the primitive streak. Arrows indicate (more ...)
A factor contributing to ectopic development of the tailbud is the altered location of the posterior site of attachment of the yolk sac. The yolk sac is normally attached around the node, anterior to the primitive streak in GCNF+/+ embryos (Fig. C). In GCNF−/− embryos, however, the yolk sac is attached more caudally, at the posterior of the primitive streak close to the allantois (Fig. D). Consequently, the resulting PSM and undifferentiated somites are pushed out of the yolk sac by the invagination of the neuroepithelium and growth of the posterior. At 9.5 dpc, the posterior neuropore in GCNF+/+ embryos is closed by “zipping up” the neural groove caudally (Fig. E). In contrast, no posterior neuropore was observed in GCNF−/− embryos. In addition nodal expression is down-regulated in wild-type embryos at 9.5 dpc (Fig. C and E); however, the disorganized and persistent expression of nodal (Fig. F) is indicative of altered function of the node, the posterior organizer, which in turn may lead to the abnormalities observed in the posterior of GCNF−/− embryos. The neural tube elongates within the tailbud as it pushes through the yolk sac (Fig. F) and forms a new posterior that continues to produce somites until the abnormal differentiation observed in the PSM, i.e., ectopic expression of paraxis, eventually leads to a halt in somitogenesis.
In summary, inactivation of the GCNF gene leads to embryonic lethality with major disruption of normal anteroposterior development. GCNF is likely to be a receptor for a novel ligand signaling pathway that is involved in regulating various aspects of embryonic development and normal anteroposterior development. Further work will be required to elucidate the GCNF signaling pathway.