Inactivation of the Huntington's disease gene (Hdh) impairs anterior streak formation and early patterning of the mouse embryo
© Woda et al; licensee BioMed Central Ltd. 2005
Received: 21 May 2005
Accepted: 18 August 2005
Published: 18 August 2005
Huntingtin, the HD gene encoded protein mutated by polyglutamine expansion in Huntington's disease, is required in extraembryonic tissues for proper gastrulation, implicating its activities in nutrition or patterning of the developing embryo. To test these possibilities, we have used whole mount in situ hybridization to examine embryonic patterning and morphogenesis in homozygous Hdhex4/5huntingtin deficient embryos.
In the absence of huntingtin, expression of nutritive genes appears normal but E7.0–7.5 embryos exhibit a unique combination of patterning defects. Notable are a shortened primitive streak, absence of a proper node and diminished production of anterior streak derivatives. Reduced Wnt3a, Tbx6 and Dll1 expression signify decreased paraxial mesoderm and reduced Otx2 expression and lack of headfolds denote a failure of head development. In addition, genes initially broadly expressed are not properly restricted to the posterior, as evidenced by the ectopic expression of Nodal, Fgf8 and Gsc in the epiblast and T (Brachyury) and Evx1 in proximal mesoderm derivatives. Despite impaired posterior restriction and anterior streak deficits, overall anterior/posterior polarity is established. A single primitive streak forms and marker expression shows that the anterior epiblast and anterior visceral endoderm (AVE) are specified.
Huntingtin is essential in the early patterning of the embryo for formation of the anterior region of the primitive streak, and for down-regulation of a subset of dynamic growth and transcription factor genes. These findings provide fundamental starting points for identifying the novel cellular and molecular activities of huntingtin in the extraembryonic tissues that govern normal anterior streak development. This knowledge may prove to be important for understanding the mechanism by which the dominant polyglutamine expansion in huntingtin determines the loss of neurons in Huntington's disease.
Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder that is caused by CAG repeats in the HD locus that extend a polyglutamine tract in a ubiquitous HEAT domain protein called huntingtin . The molecular mechanism by which the new property that is conferred on huntingtin by the polyglutamine expansion leads to the hallmark loss of striatal neurons in HD is not known. However, polyglutamine expansions in unrelated proteins that target distinct neuronal cell populations cause distinct 'polyglutamine' neurodegenerative disorders. This observation strongly suggests that the striatal cell specificity of the polyglutamine expansion in the context of huntingtin must be determined by some aspect of huntingtin's structure, subcellular location or activities .
Huntingtin is postulated to function as a flexible ~350 kDa HEAT domain scaffold that may facilitate the assembly and possibly the subcellular location of large protein complexes [3–7]. Huntingtin's large number of diverse cytoplasmic and nuclear protein binding partners strongly suggest that huntingtin may participate in a variety of cellular processes that range from trafficking of growth factor complexes to gene transcription (reviewed in [5, 8, 9]. However, despite the potential importance of huntingtin's normal function to our understanding of how the dominant polyglutamine mutation causes HD pathology, huntingtin's precise molecular and cellular activities have not been defined.
Therefore, we, and others, set out to discover huntingtin's essential activities by studying the effects of huntingtin deficiency in the mouse. Inactivation of the mouse HD gene (Hdh) has shown that huntingtin is not required for cell viability, as evidenced by the survival of mouse embryonic stem cells and neurons that lack huntingtin [10–12]. However, huntingtin is needed at the level of the organism for proper mammalian embryonic development [10, 13, 14]. Complete lack of huntingtin results in developmental arrest during gastrulation, while severe reduction of huntingtin levels results in abnormal neurogenesis and perinatal lethality .
Analysis of huntingtin deficient Hdhex4/5/Hdhex4/5embryos reveals that homozygous inactivation of the mouse HD gene does not overtly affect development until E7.0. By E7.5, mutant embryos exhibit a shortened primitive streak, reduced size and, by morphology, lack a node and head folds. Mutants are rapidly resorbed by E8.0 . Importantly, the expression of huntingtin only in extraembryonic tissues in chimeras rescues this gastrulation phenotype, suggesting that huntingtin is required only in cells of the extraembryonic lineage and acts in a cell non-autonomous manner at this stage .
Extraembryonic tissues are essential for supplying nutrients and signals that direct anterior/posterior axis formation and patterning in the developing embryo (reviewed in ), implicating huntingtin in either or both of these processes. Of these possibilities, the nutritive role has been more extensively investigated. However, huntingtin deficient embryos do not display obvious visceral endoderm defects, with the notable exception of compromised iron transport in later stage mutants, although iron uptake is undisturbed  and endocytosis is not impaired in huntingtin deficient embryos or embryonic stem cells [16, 18].
By the same token, huntingtin shuttles through the nucleus, where it is required for proper nuclear localization of its transcription factor partners, suggesting that huntingtin may play a role in transcription cascades in extraembryonic tissues that pattern the embryo . Therefore, we have examined this hypothesis, by monitoring the expression of genes that determine normal embryonic patterning and morphogenesis in Hdhex4/5/Hdhex4/5huntingtin deficient embryos. Our results support and refine the hypothesis, indicating that huntingtin is required for proper mesoderm patterning and for normal regional restriction of the expression of a subset of growth and transcription factors.
Huntingtin-deficient embryos exhibit abnormal streak progression and paraxial mesoderm production
Since extraembryonic tissues supply nutrients to the developing embryo, we tested the possibility that huntingtin deficiency may perturb this function by performing RT-PCR analysis to examine the expression of a panel of 'nutritive' genes in E7.5 wild-type and Hdhex4/5/Hdhex4/5huntingtin deficient embryos. Consistent with a previous report , no obvious differences were found in the expression of "nutritive" genes (Hnf4, Afp, Tfn, ApoAI, Apo-AIV, and ApoB) or genes involved in yolk sac hematopoiesis or vasculogenesis (Ttr, Rbp, Flt1, Flk1, Tal1, Rbtn2, GATA1) (data not shown), suggesting that huntingtin is not essential for the proper expression of genes required for the nutritive function of the extraembryonic tissues.
To investigate huntingtin's developmental activities, we then analyzed the expression of genes which pattern the early embryo or mark morphogenic landmarks in wild-type and Hdhex4/5/Hdhex4/5embryos by whole mount and section in situ hybridization. The dissections confirmed previous morphologic data at E7.0–7.5 that all Hdhex4/5/Hdhex4/5homozygotes exhibit abnormal morphology, including shortened primitive streak and a lack of morphological head folds or node [10, 13]. The results of in situ hybridization analysis also confirmed that all three germ layers and extraembryonic tissue are formed in huntingtin deficient embryos.
To examine definitive endoderm formation, the expression of Hnf3β (FoxA2) in mutant and wild-type embryos was analyzed. In wild-type embryos, Hnf3β expression is confined to the node and anterior definitive endoderm (Fig. 1G,I). Mutant embryos exhibit Hnf3β-reactive definitive endoderm over the disorganized anterior streak region and proceeding rostrally around the distal tip (Fig. 1H,J). In both normal and mutant embryos, the AVE exhibits little Hnf3β expression. Therefore, huntingtin deficiency does not greatly affect Hnf3β regulation or the reorganization of the visceral endoderm.
The anterior streak generates paraxial mesoderm. Therefore we examined paraxial mesoderm formation in wild-type and mutant embryos, revealing deficits in mesoderm patterning. Starting at E.7.5, Wnt3A is expressed in the primitive streak in cells fated to become paraxial mesoderm. In huntingtin deficient mutants, Wnt3a is induced in the proximal streak (Fig. 2G,H), confirming stage appropriate posterior development, in contrast to the absence of anterior head folds. However, expression of Wnt3a is noticeably reduced in Hdhex4/5/Hdhex4/5embryos, suggesting a defect in paraxial mesoderm development (Fig. 2H). Reduced expression of Tbx6 in the mesoderm lateral to the primitive streak in mutant embryos confirms this interpretation (Fig. 2I,J). Furthermore, in mutant embryos at E7.5, the expression of Dll1 in the distal streak region and in only a narrow swath of cells located laterally confirms the paucity of paraxial mesoderm (Fig. 2K,L, ). These results strongly suggest that anterior primitive streak formation is impaired, resulting in reduced axial and paraxial mesoderm formation and impaired neural development.
Impaired regional restriction of growth factor expression in the absence of huntingtin
Fgf8 signaling is also essential for normal gastrulation in the mouse embryo. Fgf8 is required for cell migration away from the primitive streak . Expressed just prior to streak formation in the posterior epiblast and visceral endoderm, Fgf8 is restricted to the streak mesoderm at E7.5 in a decreasing proximal-distal gradient and is downregulated in cells shortly after they exit the streak (Fig. 3E,G). In Hdhex4/5/Hdhex4/5embryos, Fgf8 expression is strongly expressed in the posterior region in the primitive streak and ectopically in the endoderm overlying the entire epiblast (Fig. 3F,H). However, streak derivatives appear to migrate normally as evidenced by the proper anterior expression of markers such as Otx2, Hnf3β and Hesx1 anteriorly (Fig. 1). Therefore, mutant embryos exhibit normal migration of streak derivatives but display impaired Fgf8 repression in mutant endoderm.
Hdhex4/5/Hdhex4/5embryos also fail to restrict the expression of goosecoid (Gsc). Normally, Gsc is initially expressed in the visceral endoderm and proximal, posterior streak where the primitive streak will form prior to gastrulation. As the primitive streak forms and extends, Gsc is expressed in the distal streak, the node, and the axial mesoderm extending anteriorly from the node (Fig. 3I,K, [31, 32]). However, in the mutant Hdhex4/5/Hdhex4/5embryos, high levels of Gsc expression remain unrestricted in the endoderm overlying the entire embryo and ectopically in cells adjacent to the ectoplacental cone (Fig. 3J,L). These results suggest that, in contrast to proper Hnf3β regulation, Gsc remains inappropriately activated in mutant visceral and definitive endoderm, implicating huntingtin in the proper restriction of this homeodomain transcription factor.
Huntingtin is not required for expression of extraembryonic signaling molecules
Signals from the extraembryonic tissues, including the anterior visceral endoderm and extraembryonic ectoderm are required for proper formation and patterning of the epiblast . 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, ). 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/Hdhex4/5extraembryonic 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 . 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/Hdhex4/5embryos, 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/Hdhex4/5embryos, 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/Hdhex4/5embryos, 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/Hdhex4/5embryos. 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.
We have investigated the embryonic processes that require huntingtin in order to more precisely delineate huntingtin's essential molecular and cellular activities and to provide clues to the mechanism by which the dominant polyglutamine expansion mutation in huntingtin leads to HD pathogenesis. In pursuing the finding that huntingtin is needed only in extraembryonic tissues for normal gastrulation, our data fail to provide evidence of abnormal nutritive gene expression in Hdhex4/5/Hdhex4/5embryos. Instead, our results reveal that huntingtin is required for normal anterior streak formation and the consequent production of paraxial mesoderm, with a previously unrecognized role for huntingtin in the proper extinction of transiently and/or dynamically expressed genes.
Indeed, the hallmark of the huntingtin deficient molecular phenotype is the impaired down-regulation of a subset of dynamically expressed genes, after the proper onset of expression. This phenomenon does not reflect a lack of anterior/posterior axis formation, as evidenced by the formation of the AVE anteriorly and the primitive streak posteriorly. Nor can it be simply explained by delayed development, as stage-specific markers, such as Wnt3a and primordial germ cells, which are detectable at E7.0 in wild-type embryos, are induced appropriately. Furthermore, the expression of T and Evx1 in the extraembryonic mesoderm of mutant embryos is not a feature of wild-type embryos, even at earlier stages. This strongly suggests that in huntingtin deficient embryos, the migration of the distal streak derivatives to the extraembryonic mesoderm occurs normally but that the down-regulation of these genes is impaired. This impairment may also explain the failure of huntingtin deficient embryos to properly restrict the expression of Fgf8, Nodal and Gsc. Thus, huntingtin may play a direct role in the transcriptional regulation, or mRNA stability of these genes or it may act indirectly by intersecting with other pathways that regulate the expression of these genes.
The requirement for huntingtin in the extraembryonic tissues had prompted us to test whether impaired extraembryonic signals might be responsible for the dysregulation of gene expression within the epiblast that is observed in Hdhex4/5/Hdhex4/5embryos. Extraembryonic development in Hdhex4/5/Hdhex4/5embryos is associated with mildly elevated levels expression of Hnf4 in the primitive endoderm and Pem in the lopsided anterior chorion but the expression of other known signals, such as Bmp4 from the extraembryonic ectoderm, and Dkk1 and Lefty1 from the AVE, appear to be normal, although the slight increase in Dkk-1 expression in Hdhex4/5/Hdhex4/5embryos suggests that further investigation into Wnt signaling is warranted. Moreover, extraembryonic Bmp4 signaling is not impaired in the absence of huntingtin, as the induction of PCGs in mutant embryos is normal, implying proper transport and secretion of the appropriate extraembryonic signals. However, Nodal, Fgf8 and Gsc are expressed ectopically in the visceral endoderm of Hdhex4/5/Hdhex4/5embryos. Both Nodal and Fgf8, important growth factors required for normal development of the epiblast, are tightly regulated during gastrulation. Therefore, misexpression of either or both of these factors, or of goosecoid, in the visceral endoderm could contribute to the Hdhex4/5/Hdhex4/5mutant phenotype. In addition, it is possible that other extraembryonic signal(s) that we have not analyzed may also be affected by the lack of huntingtin activity in extraembryonic cells in mutant embryos.
Huntingtin deficient embryos also fail to form headfolds, and to undergo organogenesis, even after culturing in nutrient rich media. The absence of headfold formation in these embryos does not appear to be a result of a failure to induce neurectoderm or a failure to form the AVE, since mutant embryos express markers such as Otx2, Ddk1, Lefty1 and Hesx1. In addition, since node formation is not required for neural induction [44–46], the failure to form a node in huntingtin deficient embryos is also unlikely to explain the lack of headfolds. The apparent reduction of paraxial mesoderm in Hdhex4/5/Hdhex4/5embryos could explain the lack of headfolds since paraxial mesoderm is important for the full development of neuroectoderm, and consequently, headfolds. Alternatively, the inability to manifest headfolds could suggest that huntingtin is required at a very early stage for normal CNS development. This conclusion is consistent with the finding that severely reduced levels of huntingtin, from a hypomorphic Hdh allele, lead to abnormal brains later in embryonic development .
The cardinal features of complete Hdh inactivation that we observe are similar to the phenotypes that stem from the complete inactivation of the Polycomb group gene (Pc-g) Eed (embryonic ectoderm development). Indeed, complete deficiency for either huntingtin or the eed protein leads to abnormal streak development, lack of headfold formation, ectopic T, Evx1 and Nodal expression and disruption of anterior primitive streak mesoderm production . Interestingly, Eed protein is also required for proper trophoblast development and normal maintenance of imprinted X-inactivation and genomic imprinting [47–49], suggesting that these activities warrant investigation in huntingtin deficient embryos.
Thus, our observations provide unexpected starting-points in the search for huntingtin's precise molecular activities, which began with the discovery that this HEAT domain protein hosts the dominant polyglutamine property that is the fundamental basis of HD pathogenesis. In HD patients and in accurate genetic replicas, HD CAG knock-in mice, the dominant mutation specifically affects the major population of neurons in the striatum, without impairing huntingtin's essential activities in embryonic development [50–53]. Indeed, homozygous HD patients make no wild-type huntingtin, and, in the mouse, a single mutant Hdh allele's worth of mutant huntingtin can fully rescue huntingtin deficiency embryonic phenotypes [15, 51]. The quest to understand the HD mechanism, therefore, is aimed at delineating the huntingtin activity that may explain the striatal cell specificity of the polyglutamine mutant version of huntingtin. One hypothesis is that huntingtin is normally involved in gene transcription, as proposed for NRSF/REST mediated BDNF expression . Now, our finding that huntingtin can be absolutely necessary for the appropriate regulation of genes with dynamic expression patterns in vivo, provides a compelling reason to elucidate the cellular machinery that is necessary for huntingtin mediated gene regulation.
Our findings indicate that huntingtin is required for proper patterning of the epiblast during early embryogenesis, for proper anterior streak and node formation, primitive streak progression, paraxial mesoderm and head fold formation, as well as for the proper restriction of transiently expressed growth and transcription factor genes. Knowledge of the molecular basis of these changes in huntingtin deficient embryos should facilitate the identification of the cellular pathways that are dependent on huntingtin activities. These will be important for implicating candidates to be assessed in the extraembryonic signals that determine anterior streak progression in the developing embryo and in delineating the dominant activity of the polyglutamine tract in huntingtin that determines the striatal specificity of HD.
Mice and genotyping
The Hdhex4/5mice carrying a pGKneo insertion/replacement inactivating mutation in the mouse HD gene homologue have been described previously . The experiments were conducted in accordance with an IACUC approved protocol, through the MGH Subcommittee on Animal Research. Mutant Hdhex4/5/Hdhex4/5and normal littermates were obtained in timed pregnancies from mating of Hdhex4/5/Hdh+ heterozygotes, genotyped by PCR assay, as described . The day of plug was taken to be E0.5. Embryos that were morphologically normal were pooled separately from morphologically mutant embryos for analysis. Nodal expression was determined in embryos from matings of Hdhex4/5/Hdh+; Ndll acZ /Ndl+ compound heterozygotes genotyped by PCR assay as described in .
Whole mount and section in situhybridization and β-gal staining
After dissection in PBS, embryos were fixed overnight in 4% paraformaldehyde at 4°C. For sections, decidua fixed in 4% paraformaldehyde, were embedded in paraffin and sectioned at 7 microns. RNA in situ hybridizations were performed as described previously . Nodal.lacZ expression was assessed by β-galactosidase staining as reported , on embryos post fixed in 4% paraformaldehyde. Embryos were mounted in 80% glycerol before being photographed.
The huntingtin deficient phenotype is fully penetrant at each of the stages that were assessed . Three to six embryos were evaluated for each marker, with every embryo exhibiting the same mutant phenotype in each case.
Alkaline phosphatase staining of Primordial Germ Cells (PCGs)
After dissections, embryos were fixed in 4% paraformaldehyde briefly and washed and stored in 1 × PBS/0.1% TX-100 at 4°C. Embryos were washed once with Tris-Maleate Buffer (25 mM Tris-Maleate, pH = 9.0, 0.8 mM MgCl2) and were subsequently incubated in alkaline phosphatase staining solution (25 mM Tris-Maleate, pH = 9.0, 0.8 mM MgCl2, 0.4 mg/ml alpha-naphthyl phosphate, 1 mg/ml Fast-Red). Stained embryos were washed in 1 × PBS/0.1% TX-100.
Whole embryo culture
Embryos were dissected at E7.5 and washed in DMEM. Embryos were then cultured individually in 1 ml of culture media (75% immediately centrifuged rat serum and 25% DMEM ) for 24 hours while rotating in a 37°C incubator in 5% CO2. Embryos were then fixed in 4% paraformaldehyde for analysis.
anterior visceral endoderm
- HD :
Huntington's disease gene
- Hdh :
mouse HD gene homologue
primordial germ cells
We are grateful to Drs. A. Gossler, J. Darnell, Jr., J Rossant, G. Keller, S. Orkin, G. Martin, T. Yamaguchi, A. McMahon, R. Maas, K., Muneoka, A. Simeone, Hamada H. and C. Niehrs for the generous gifts of clones and antibody reagents and Dr. E. Robertson for NdllacZ mice. We would like to thank Kathy Molyneaux for her helpful suggestions and technical assistance. We also thank Vladimir Vrbanac, Janice Espinola and Edith Toral Lopez for assistance with animal husbandry. We also thank the members of the MacDonald lab for helpful discussions during the completion of this work. This work was supported by the NINDS grants NS32765 and NS16367, and grants from the Foundation for the Care and Cure of Huntington's disease and with the support of the Huntington's Disease Society of America Coalition for the Cure and the Hereditary Disease Foundation. Juliana M. Woda is the recipient of the Milton Wexler Postdoctoral Fellowship from the Hereditary Disease Foundation.
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