Huntingtin is not required for expression of extraembryonic signaling molecules
Previous studies of chimeric embryos suggest that huntingtin is required only in the extraembryonic tissue for proper development [16]. Signals from the extraembryonic tissue are critical for the induction of embryonic signals and for patterning the epiblast. Consequently, we examined extraembryonic development in huntingtin deficient embryos. Hnf4 is a transcription factor expressed in the primitive endoderm as soon as this tissue becomes distinct and is a key regulator of visceral endoderm secreted factors such as alphafetoprotein, apolipoproteins, and transferrin. Inactivation of Hnf4 results in impaired gastrulation [33,34]. At E7.5, Hnf4 is expressed in the columnar visceral endoderm cells at the extraembryonic-ectoderm junction (Fig. 4A, [33]). In Hdhex4/5/Hdhex4/5 embryos, consistent with normal primitive and visceral endoderm differentiation, Hnf4 expression appears normal, although the signal is stronger in mutant embryos compared to wild-type embryos (Fig. 4B). Similarly, Pem, a transcription factor expressed in proximal visceral endoderm and ectoplacental cone in wild-type embryos at E7.5, also is expressed in these tissues in the mutant embryos (Fig. 4C,D[35]). However, Pem expressing visceral endoderm hangs over the anterior of the mutant embryos, revealing abnormal location despite grossly normal differentiation.
Figure 4  Normal expression of extraembryonic markers in huntingtin deficient embryos. Whole mount in situ hybridization analysis at E7.5 of markers of the extraembryonic tissues reveals grossly normal expression in the absence of huntingtin. Hnf4, expressed in the visceral endoderm at the junction of embryonic-ectoderm junction (A), is normal in mutant embryos, although the signal is slightly higher (B). Similarly, the expression of Pem transcripts is maintained in mutant embryos (D) similar to normal embryos (C), although Pem is expressed in the abnormal lopsided overhang of visceral endoderm over the anterior of the mutant embryos. Expression of extraembryonic signaling molecules is unaffected by the loss of huntingtin, as evidenced by the expression of Bmp4 (E,F) in the extraembryonic ectoderm, and Lefty1 and Dkk1 (I-L) in the AVE in mutant embryos. Bmp4 is not localized, however, to a ring of extraembryonic ectoderm in mutant embryos (F) as in normal embryos (E). Primitive germ cells (PCGs) are induced normally in both wild-type (G) and mutant embryos (H), suggesting the Bmp4 signaling from the extraembryonic ectoderm to the epiblast is normal. Lefty1 expression appears disorganized in mutant embryos (I) compared to wild-type embryos (J). In contrast, the anterior expression of Dkk1 in the AVE in mutant embryos (L) matches the wild-type expression pattern (K). Despite normal AVE formation, head folds fail to form in mutant embryos, even when cultured in nutrient rich media for 24 hours. Wild-type E7.5 embryos, when cultured in 75% rat serum, develop somites (M), heart (white arrow, N) and head folds (blue arrow head, N) in culture. In contrast, huntingtin deficient embryos continue to live in culture but do not form headfolds, heart or somites (O). Embryos are shown in a lateral view (A-F, I-J) with anterior oriented to the left. Embryos in (G,H,K,L) are shown in an anterior view with proximal oriented up. Signals from the extraembryonic tissues, including the anterior visceral endoderm and extraembryonic ectoderm are required for proper formation and patterning of the epiblast [17]. Bmp4 is a signaling molecule that is first expressed uniformly throughout the extraembryonic ectoderm and subsequently is localized to a ring of extraembryonic ectoderm adjacent to the epiblast (Fig. 4E, [36]). A key factor in regulating the formation of the node and primitive streak, Bmp4 is required for patterning the embryo along the proximodistal axis [37-40]. In the absence of huntingtin, Bmp4 expression is properly maintained in the Hdhex4/5/Hdhex4/5 extraembryonic ectoderm but is also expressed throughout the extraembryonic ectoderm (Fig. 4F) in a pattern that is similar to early Bmp4 expression rather than being restricted to a ring of extraembryonic ectoderm as seen in the wild-type embryos To assess Bmp4 signaling from the extraembryonic ectoderm, we evaluated primordial germ cells (PGCs), which require Bmp4 for their induction [37]. PGCs can first be detected at E7.0 and subsequently underlie the posterior portion of the primitive streak. Whole mount staining of E7.5 mutant and wild-type embryos for alkaline phosphatase activity reveals that PCGs form in Hdhex4/5/Hdhex4/5 embryos, suggesting that Bmp4 signaling is functional in the absence of huntingtin (Fig. 4G,H).
The anterior visceral endoderm (AVE) is also an extraembryonic source of signals that are critical for early patterning. Wnt and nodal antagonists, Dkk1 (mdkk-1) and Lefty1 respectively, are expressed in the AVE and are important in limiting the posteriorization of the anterior embryo by restricting Nodal and Wnt signaling [41-43]. In Hdhex4/5/Hdhex4/5 embryos, both Dkk1 (Fig. 4I,J) and Lefty1 (Fig. 4K,L) are expressed normally in the AVE as compared with wild-type embryos. However, Dkk-1 levels appear to be slightly increased in Hdhex4/5/Hdhex4/5 embryos, although the pattern of Dkk-1 expression remains unchanged and this increase may just reflect the same amount of expression in a smaller area. Therefore, the ectopic expression of Nodal (Fig. 3A–D) and the decreased Wnt3a expression (Fig. 2H) in mutant embryos do not appear to be result of changes in the expression pattern of Lefty1 or Dkk1.
Despite normal AVE formation and neuroectoderm induction, head folds do not form in Hdhex4/5/Hdhex4/5 embryos. Therefore, to determine whether mutant embryos are inherently capable of forming head folds, embryos harvested at stage E7.5 were allowed to progress in rich culture medium in vitro for 24 hours. Wild-type embryos continued to develop head folds, somites and hearts (Fig. 4M,N). In contrast, mutant stage 7.5 embryos did not develop headfolds, hearts or somites, although these embryos continued to live (Fig. 4O). These results strongly suggest that in the absence of huntingtin, embryos are unable to undergo organogenesis, even if they continue to live past E7.5 in a nutrient rich environment.