1 The Walter and Eliza Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, Vic 3050, Australia
2 Embryology Unit, Childrens Medical Research Institute, Wentworthville, NSW 2145, Australia
* These authors contributed equally to this work
Present address: Centre for Early Human Development, Monash Institute of Reproduction and Development, 27-31 Wright St, Clayton, Vic 3168, Australia
Author for correspondence (e-mail: robb{at}wehi.edu.au)
Accepted 30 April 2002
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SUMMARY |
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Key words: Mix, Bix, Homeobox, Gastrulation, Mesendoderm, Notochord, Node, Endoderm, Mouse
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INTRODUCTION |
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Studies of MIX/BIX homeoprotein function in Xenopus have shown that they are downstream transcriptional targets in the TGFß superfamily pathway that regulates mesendodermal patterning. For Mix.2, the pathway of activin-induced transcriptional activation has been determined. Signals from TGFß family members are transduced to the nucleus by intracellular SMAD proteins. SMAD proteins are recruited to DNA by other DNA-binding proteins, the prototype of which is FOXH1 (FAST1). FOXH1 forms a complex with activated SMAD2/SMAD4 dimers to bind to the activin-responsive element of the Mix.2 promoter (reviewed by Massague and Wotton, 2000). Other Xenopus MIX/BIX proteins, MIXER, BIX2/MILK and BIX3, recruit active SMAD2/SMAD4 complexes directly to an activin-response element found in mesendodermal genes such as Gsc (Germain et al., 2000
).
Mix.1 has been implicated in endoderm development (Hudson et al., 1997; Lemaire et al., 1998
; Latinkic and Smith, 1999
). Expression of dominant negative Mix.1 constructs prevents endoderm formation and, while overexpression of Mix.1 does not induce endoderm formation in animal cap explants, it can act synergistically with the Paired-like homeobox genes Gsc or Siamois to upregulate transcription of endodermal markers (Henry and Melton, 1998
; Lemaire et al., 1998
; Latinkic and Smith, 1999
). Moreover, Mix.1 has been shown to repress the expression of the mesodermal marker Xbra, the Xenopus homolog of Brachyury (Latinkic et al., 1997
; Latinkic and Smith, 1999
). Bix2/Milk, Mix.3/Mixer and Bix4 have also been shown to induce endoderm formation (Ecochard et al., 1998
; Henry and Melton, 1998
; Casey et al., 1999
). Depending on the level of overexpression, Bix1/Mix4 can induce the expression of endoderm or ventral mesoderm markers in animal cap explants (Tada et al., 1998
). However, the roles of individual Mix/Bix genes in the regulation of mesendodermal patterning in Xenopus remain unclear, especially as homeobox proteins are known to homo- or heterodimerize with DNA (Wilson et al., 1993
; Wilson et al., 1995a
; Mead et al., 1998
).
In the chick, a Mix-like gene (Cmix) is expressed in epiblast and endoderm of the posterior marginal zone prior to formation of the primitive streak. Thereafter, expression is seen in epiblast and nascent mesoderm of the primitive streak but not in Hensens node. Cmix transcripts are no longer detectable after formation of the head process (Peale et al., 1998; Stein et al., 1998
). In zebrafish, the gene associated with the bonnie and clyde (Bon) mutation is a Mix-type gene with similarity to Xenopus Mixer (Kikuchi et al., 2000
). Bon is induced in the margin of late blastula stage embryos by NODAL signaling and is required for early endoderm formation and, like Mixer in Xenopus, regulates expression of the endodermal gene Sox17 (Henry and Melton, 1998
; Alexander and Stainer, 1999
). Bon mutants exhibit cardia bifida and have fewer Sox17-expressing endodermal cells (Alexander et al., 1999
; Kikuchi et al., 2000
). Genetic studies have shown that Bon lies downstream of the NODAL-related proteins encoded by the Cyclops and Squint genes (Kikuchi et al., 2000
).
To date, one murine Mix-like gene, Mixl1 (Mml, Mix) (Pearce and Evans, 1999; Robb et al., 2000
) and one human homolog (Robb et al., 2000
) have been identified. The amino acid sequence of the homeodomain and a conserved C-terminal acidic region of the murine MIXL1 is similar to MIX.1 and other proteins of Xenopus MIX/BIX family (Pearce and Evans, 1999
; Robb et al., 2000
). Mixl1 RNA was first detected in the visceral endoderm of the pre-gastrula embryo. By 6.5 days post coitum (dpc), expression was seen in nascent primitive streak and emerging mesoderm. Mixl1 expression was maintained in emerging mesoderm in mid- to late-streak embryos, becoming restricted to the posterior primitive streak by the head-fold stage. Expression was not seen in the node or in endoderm flanking the node. In early somite stage embryos, expression was detectable in the crown cells of the caudal notochord and in primitive streak (Pearce and Evans, 1999
; Robb et al., 2000
). A single human Mixl1 homolog has been identified (Robb et al., 2000
).
To establish the role of Mixl1 in murine development, we created a mouse strain bearing a targeted mutation of the Mixl1 gene. Gastrulation stage Mixl1-null embryos had abnormalities in primitive streak and node formation. Post-gastrulation, Mixl1 mutants exhibited complex defects in axial mesodermal and endodermal structures, suggesting a role for Mixl1 in the regulation of morphogenetic cell movements associated with gastrulation. Despite the defects in early morphology and terminal differentiation of trunk organizer cells, gene marker studies indicated that their inductive properties were maintained. Moreover, in chimera studies, Mixl1-null embryonic stem cells were found to contribute poorly to gut endoderm, thereby implicating Mixl1 in endoderm patterning.
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MATERIALS AND METHODS |
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Fluorescence imaging
Embryos obtained from timed matings of Mixl1+/ or Mixl1+/neo pairs were harvested and maintained at room temperature in Hepes-buffered Eagles Medium without Phenol Red during imaging with a Leica TCS-SP2 Confocal Imaging System connected to a Leitz Arisoplan microscope. Thick sections for confocal imaging were cut using disposable steel blades (Probing and Structure).
In situ hybridization and histology
For histology, embryos were fixed in 4% paraformaldehyde, dehydrated through an ethanol series and embedded in paraffin wax. Blocks were sectioned at 6 µm, dewaxed and stained in Hematoxylin and Eosin. Embryos for whole-mount in situ hybridization were fixed in 4% paraformaldehyde and stored at 4°C. Hybridization was performed as described (Belo et al., 1997). After hybridization, stained embryos were refixed in 4% paraformaldehyde. Selected embryos were dehydrated, embedded in paraffin wax and sectioned at 10 µm.
Mixl1-null ES cell derivation and generation of chimeric embryos
Wild-type R26.1 ES cells, which are derived from a mouse carrying the ROSA-26 lacZ gene trap (Varlet et al., 1997), were injected into blastocysts obtained by mating Mixl1+/ and Mixl1+/
neo animals. Chimeras derived from Mixl1/
neo blastocysts were identified by PCR of yolk sac DNA, using the primers pairs described above. PCR of Mixl1/
neo yolk sac DNA gave rise to products specific for the Mixl1 allele and the Mixl1
neo allele, in addition to the wild-type (maternally derived) allele. Mixl1/
neo ES cell lines were generated by first using CRE-mediated recombination to remove the loxP-flanked neomycin resistance cassette from a Mixl1+/ ES cell line, thereby creating Mixl1+/
neo cell lines. The second allele was then replaced by a further round of gene targeting with the original targeting construct. Two Mixl1/ ES cell lines were identified by Southern analysis of HindIII-digested DNA using the 3' probe to detect the 4.7 kb and 3.7 kb mutant alleles. The correct targeting of the locus was confirmed by further Southern analysis. Chimeric embryos were generated by injection of Mixl1/ ES cells into blastocysts carrying the ROSA-26 lacZ transgene. ß-Galactosidase activity in chimeric embryos was detected by X-gal histochemistry (Elefanty et al., 1999
). Stained embryos were post-fixed and sectioned at 10 µm.
Nodal expression analysis
Nodal+/lacZ mice, generously provided by Liz Robertson, were bred with Mixl1+/ mice. The offspring were genotyped for Mixl1 as above and for Nodal as described (Collignon et al., 1996). Compound heterozygotes were interbred and offspring genotyped by PCR of yolk sac DNA. ß-galactosidase activity was detected as described elsewhere (Elefanty et al., 1999
).
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RESULTS |
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After homologous recombination, the GFP reporter and the neomycin selection cassette replaced most of Mixl1 exon 1, leaving potential 5', 3' or intronic regulatory sequences intact. In keeping with pattern of Mixl1 RNA expression, the visceral endoderm, primitive streak and nascent mesoderm of gastrulation stage Mixl1+/ embryos expressed GFP. Fluorescence was first detected at 6.5 dpc, 1 day after Mixl1 RNA was detectable by whole-mount in situ hybridization (Pearce and Evans, 1999; Robb et al., 2000
) and GFP signal in Mixl1+/ embryos persisted in visceral endoderm up to the late bud stage. Co-incident with the Mixl1 mRNA expression at 7.5 dpc, maximal fluorescence was seen in the primitive streak and mesodermal wings at this time (Fig. 1C,F). At 8.25 dpc, fluorescence was confined to the posterior primitive streak of Mixl1+/ embryos, and by 9.0 dpc was completely absent, mirroring the Mixl1 mRNA expression pattern (data not shown). In Mixl1/ embryos, the fluorescence was brighter than in Mixl1+/ embryos and remained detectable until 9.5 dpc (Fig. 1D,F and not shown).
At 6.5 dpc, Mixl1/ embryos are morphologically indistinguishable from their littermates. However by 7 dpc, at the mid-streak stage, Mixl1/ embryos display a thickened primitive streak and lack a morphologically recognizable node at the anterior end of the primitive streak (Fig. 2A,F). An excess of mesoderm-like cells accumulates in the anterior streak and in the region immediately lateral to the primitive streak (Fig. 2B-E,G-J). The early-neural plate stage (7.75 dpc) Mixl1/ embryos are retarded in the elevation of neural folds and develop a shallow anterior intestinal portal (data not shown). At the early somite-stage (8.5 dpc), the embryos adopt a flattened disc morphology instead of a lordotic shape and the anteroposterior (AP) axis is markedly foreshortened (Fig. 3A-C). The neural folds are small and disorganized and very little head mesenchyme is found underneath the head folds (Fig. 3I-M). The heart tube is absent (Fig. 3I,J). The foregut invagination is rudimentary and no hindgut portal is formed (Fig. 3I,L,M). From two to five pairs of somites are formed in the paraxial mesoderm flanking a thick axial mass of flattened neurectoderm overlying a condensed core of mesodermal tissues that replaced the notochord.
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Loss of Mixl1 function results in enhanced Brachyury and Nodal expression
Brachyury (T), which is expressed in the primitive streak and axial mesoderm of the wild-type 7.5 dpc embryo (Wilkinson et al., 1990), is expressed in an expanded domain in both the epiblast and mesenchymal components of the thickened primitive streak and in the adjacent mesodermal cells in the Mixl1/ embryos (Fig. 5A-D). In 8.25 dpc mutant embryos, Brachyury is expressed in an irregular and broad strip of mesodermal cells underlying the midline of the head fold and neural plate along the AP axis. (Fig. 5E-G). Histological examination reveals that Brachury is expressed in ventral tissues of the open neural plate that resemble the floor plate of the neural tube (Fig. 5H). In the posterior region of the mutant embryo, Brachyury is expressed in the superficial layer of cells of the primitive streak and in the ventral-most tissues that appear to have organized into a notochordal plate-like structure (Fig. 5I). In contrast to the uniform expression in the primitive streak of wild-type embryo, expression is excluded from the core of the primitive streak (Fig. 5I).
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Tissues in the midline display the molecular properties of axial mesendoderm
In the wild-type embryo, Foxa2 (HNF3ß) expression is localized to the anterior primitive streak, the node and the anterior mesendoderm at 7.5 dpc and at early somite stages marks axial midline structures such as notochord, floor plate and gut endoderm (Ang et al., 1993; Monaghan et al., 1993
; Ruiz i Altaba et al., 1993
; Sasaki and Hogan, 1993
). In 7.5 dpc Mixl1/ embryos, an expanded domain of expression of Foxa2 is found in the mesendodermal tissues in distal region of the gastrula-stage embryo at the anterior end of the primitive streak (Fig. 6A,B). At 8.25 dpc, Foxa2 is present in the axial tissues over most of the length of the embryonic axis of the mutant embryos (Fig. 6C,F). Sonic hedgehog (Shh) is first detected at the late streak stage in the anterior mesendoderm, which extends rostrally from the primitive streak, and during organogenesis, in the node, head process, notochord, prechordal plate, floorplate and endoderm in the dorsolateral region of the embryonic gut (Echelard et al., 1993
; Roelink et al., 1994
). In the late-streak stage Mixl1/embryos, Shh expression is restricted to cells clustered near the anterior end of the streak (Fig. 6G,H). In 8.25 dpc mutants, weak, poorly localized Shh expression is seen in the ventral part of the condensed axial tissue that resembles the notochordal plate (Fig. 6I-L). Gsc, which marks the prechordal plate in late head-fold-stage embryos, is expressed in the mesendoderm in the anterior region of the mutants (data not shown). Overall, the marker studies showed that tissues displaying the molecular characteristics of axial mesendoderm are specified in the Mixl1/ embryos, despite proper axial structures not being formed.
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Mixl1 activity in epiblast is required for normal axial patterning and the expression of endodermal potency
As Mixl1 is expressed in both the epiblast and extra-embryonic tissues during gastrulation (Pearce and Evans, 1999; Robb et al., 2000
), it is possible that the proper inductive interactions do not exist between these tissues in the Mixl1/ embryos. In order to identify the tissues in which Mixl1 is required for axial mesoderm patterning, we generated mouse chimeras containing wild-type epiblast cells and Mixl1-deficient visceral endoderm and trophectodermal derivatives. Descendants of the wild-type R26.1 ES cells in the epiblast can be identified by the constitutive expression of a ROSA-26 lacZ transgene (Friedrich and Soriano, 1991
; Varlet et al., 1997
). These ES cells were injected into Mixl1/
neo blastocysts obtained by mating Mixl1+/ and Mixl1+/
neo mice. (This mating strategy was employed to facilitate genotyping of the chimeras see Materials and Methods.) In the chimeras, wild-type ES cells contribute efficiently to embryonic, but rarely to extra-embryonic, lineages (Beddington and Robertson, 1989
; Varlet et al., 1997
). If Mixl1 expression in visceral endoderm is essential, wild-type ES cells will not rescue the defects of Mixl1/ host blastocysts. Five 8.5 dpc Mixl1/ blastocyst-derived chimeras that contained a greater than 50% contribution from wild-type (lacZ-positive) ES cells were phenotypically indistinguishable from chimeric embryos derived from wild-type or Mixl1+/ host blastocysts and wild-type ES cells. The rescue of the mutant phenotype by the presence of wild-type cells in the embryo containing Mixl1-deficient extra-embryonic ectoderm and visceral endoderm indicates that loss of Mixl1 function in the extra-embryonic tissues is unlikely to have caused the disruption in embryogenesis.
The requirement for Mixl1 in embryonic development was studied in a reciprocal experiment. Two independent Mixl1/neo ES cell lines were derived by a second round of targeted mutation of the normal Mixl1 allele of a Mixl1+/
neo ES cell line using the original targeting construct (see Materials and Methods). Chimeras were produced by introducing Mixl1/
neo ES cells (identified by the lack of lacZ expression) into Mixl1+/+ ROSA-26 transgenic blastocysts. A proportion of the chimeric embryos with a high contribution of Mixl1+/
neo ES cells phenocopied the defective development of the Mixl1/ mutants, demonstrating that normal Mixl1 activity in visceral endoderm is not sufficient to compensate for the deficiency in the embryonic tissues (Fig. 9A). Chimeras with 20% or more wild-type cells showed partial to nearly complete rescue of developmental defects. Histological examination of these chimeras reveals that Mixl1/ cells can colonize most embryonic tissues, including the neural tube and the notochord. However Mixl1/ cells consistently display low level of contribution to the heart and foregut and are almost completely excluded from the endoderm of the midgut and hindgut (Fig. 9B-F)
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DISCUSSION |
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Mixl1 function is essential for endoderm differentiation
The defective morphogenesis of the embryonic gut in the Mixl1/ embryo and the low contribution of the Mixl1/ cells to the gut endoderm of the chimeras indicate that Mixl1 function is essential for gut morphogenesis and the potency of the embryonic cells for endoderm differentiation. Members of the Mix/Bix family have been implicated in endoderm formation in both Xenopus and zebrafish. In the mouse, the definitive endoderm is formed by recruitment from the epiblast to the endodermal layer and replaces the visceral endoderm during gastrulation (Lawson et al., 1987; Lawson et al., 1991
; Tremblay et al., 2000
). In Mixl1/ embryos, progenitors of definitive endoderm are properly specified at gastrulation. However, by the early somite stage, only a small population of presumptive definitive endodermal cells characterized by Sox17 and Cer1 expression is found in the poorly developed foregut portal. The reduction in definitive endoderm is consistent with the finding that fewer Mixl1/ cells colonize the foregut and almost none are found in the hindgut of the chimera. Mixl1/ cells can, however, colonize all other types of embryonic tissues, including the axial mesendoderm, neural tube and heart of the chimera. These results suggest that the abnormalities of gastrulation and organogenesis in the mutant embryo are probably caused by the disruption of the non-autonomous functions of Mixl1, but Mixl1 plays a tissue-specific and autonomous role in the differentiation of embryonic cells into definitive endoderm. As Mixl1 expression ceases in the definitive endoderm after its allocation to the embryonic gut, it is likely that Mixl1 activity impacts on the endodermal progenitors while they are in the anterior primitive streak.
The Mixl1 mutant phenotype points to a perturbation of signaling pathways regulating morphogenesis of mesendodermal tissues
Studies in Xenopus and zebrafish have shown that Mix/Bix genes are downstream transcriptional targets in the TGFß superfamily pathway that regulates mesendodermal patterning. In the mouse embryo, the TGFß family member Nodal is a key regulator of the formation of the anterior primitive streak, node, midline structures and definitive endoderm (reviewed by Schier and Shen, 2000). LEFTY2 (EGFB Mouse Genome Informatics) is an atypical member of the TGFß superfamily that antagonizes NODAL signaling and restricts the range and duration of NODAL activity (Meno et al., 1999
; Meno et al., 2001
). At gastrulation, Lefty2 mutants, like Mixl1 mutants, have an expanded primitive streak with an excess of axial mesoderm leading to a variety of associated patterning defects (Meno et al., 1999
). In 7.5 dpc Mixl1/ embryos, Lefty2 expression in the primitive streak was unaltered (data not shown).
Nodal expression is expanded in the Mixl1/ embryo, raising the possibility that Mixl1 may to regulate NODAL activity. Ectopic expression studies in Xenopus and zebrafish embryos have shown that NODAL and related factors can induce the formation of mesoderm and axis duplication (Jones et al., 1995; Toyama et al., 1995
; Joseph and Melton, 1997
). In mouse chimeras, Nodal/ cells are impaired in their ability to contribute to midline structures, indicating Nodal is required for midline morphogenesis (Varlet et al., 1997
). Furthermore, Smad2, which is activated by Nodal, is required for definitive endoderm formation and embryos with a null mutation of the forkhead DNA-binding protein Foxh1, which binds Smad2 (and Smad3), exhibit abnormal patterning of the node, prechordal mesoderm, notochord and definitive endoderm (Tremblay et al., 2000
; Hoodless et al., 2001
; Yamamoto et al., 2001
). Taken together, these observations showing disruption of the morphogenesis of mesendodermal structures following alteration of NODAL signaling suggest that Mixl1, like its Xenopus homologs, may influence the spatial or temporal pattern of NODAL signaling in the mouse embryo.
Potential impact of Mixl1 function on Brachyury activity
A role for the T-box gene Brachyury in notochord maintenance, axis elongation and specification of posterior mesoderm has been identified in many vertebrate species (Schulte-Merker et al., 1994; Wilson et al., 1995b
; Conlon and Smith, 1999
). In Xenopus, members of the Mix/Bix family have been shown to regulate expression of the Brachyury homolog Xbra (Latinkic et al., 1997
; Tada et al., 1998
; Casey et al., 1999
; Latinkic and Smith, 1999
). At the early gastrula stage, Xbra is expressed throughout the marginal zone of the embryo. In response to activin signaling, as gastrulation proceeds, transcripts are lost from involuting mesoderm, but persist in the notochord (Smith et al., 1991
). Mix.1, which is induced by activin, downregulates Xbra transcription, in part through activation of Gsc (Artinger et al., 1997
; Latinkic et al., 1997
; Latinkic and Smith, 1999
). In the Mixl1/ embryos, the Brachyury expression domain is strikingly expanded in the primitive streak and also in the abnormal midline structures present in older mutants, with weaker activity in the condensed core cells of the midline structure and of the primitive streak. This suggests that, like Mix.1 in Xenopus, Mixl1 may be acting as a repressor of Brachyury expression in the murine embryo.
Functional convergence of zebrafish and mammalian Mix/Bix family members
In zebrafish, the Mix-like gene, Bon, is responsible for the bonnie and clyde mutation (Kikuchi et al., 2000). Like Mixl1/ embryos, Bon mutants have cardiac bifida and a reduction in Sox17-expressing endodermal cells. However, they do not show the drastic disruption in axial morphogenesis seen in Mixl1-null embryos. Within the homeodomain, MIXL1 is more closely related to BON than any member of the Xenopus MIX/BIX family. Nevertheless, given that the overall the amino acid identity of BON and MIXL1 is only 25% and, taken together with the differences in phenotype of Bon and Mixl1 mutants, it is likely that the zebrafish homolog of Mixl1 remains to be discovered. Mixl1 is the only known mammalian member of the Mix/Bix family, and searching of the mouse and human genomes has not uncovered other potential homologs (A. H. H., A. G. E. and L. R., unpublished). By contrast, in Xenopus, seven Mix-like genes may be involved in the transcriptional regulation of mesoderm and endoderm development. Our study shows that, in the mouse, Mixl1 serves many functions of the Xenopus Mix/Bix genes to control the morphogenesis of mesendodermal structures, especially the node, notochord, axial mesoderm and the gut, and the differentiation of the definitive endoderm. We postulate that Mixl1 is the functional mammalian homolog of the members of the Xenopus Mix/Bix homeodomain family.
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ACKNOWLEDGMENTS |
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