Mixl1 is required for axial mesendoderm morphogenesis and patterning in the murine embryo

In Xenopus, the Mix/Bix family of homeobox genes has been implicated in mesendoderm development. Mixl1 is the only known murine member of this family. To examine the role of Mixl1 in murine embryogenesis, we used gene targeting to create mice bearing a null mutation of Mixl1. Homozygous Mixl1 mutant embryos can be distinguished from their littermates by a marked thickening of the primitive streak. By the early somite stage, embryonic development is arrested, with the formation of abnormal head folds, foreshortened body axis, absence of heart tube and gut, deficient paraxial mesoderm, and an enlarged midline tissue mass that replaces the notochord. Development of extra-embryonic structures is generally normal except that the allantois is often disproportionately large for the size of the mutant embryo. In chimeras, Mixl1(-/-) mutant cells can contribute to all embryonic structures, with the exception of the hindgut, suggesting that Mixl1 activity is most crucial for endodermal differentiation. Mixl1 is therefore required for the morphogenesis of axial mesoderm, the heart and the gut during embryogenesis.     

A conditional mutant allele for analysis of Mixl1 function in the mouse

Mixl1 is the only member of the Mix/Bix homeobox gene family identified in mammals. During mouse embryogenesis, Mixl1 is first expressed at embryonic day (E)5.5 in cells of the visceral endoderm (VE). At the time of gastrulation, Mixl1 expression is detected in the vicinity of the primitive streak. Mixl1 is expressed in cells located within the primitive streak, in nascent mesoderm cells exiting the primitive streak, and in posterior VE overlying the primitive streak. Genetic ablation of Mixl1 in mice has revealed its crucial role in mesoderm and endoderm cell specification and tissue morphogenesis during early embryonic development. However, the early lethality of the constitutive Mixl1^?/? mutant precludes the study of its role at later stages of embryogenesis and in adult mice. To circumvent this limitation, we have generated a conditional Mixl1 allele (Mixl1^cKO) that permits temporal as well as spatial control of gene ablation. Animals homozygous for the Mixl1^cKO conditional allele were viable and fertile. Mixl1^KO/KO embryos generated by crossing of Mixl1^cKO/cKO mice with Sox2‐Cre or EIIa‐Cre transgenic mice were embryonic lethal at early somite stages. By contrast to wild‐type embryos, Mixl1^KO/KO embryos contained no detectable Mixl1, validating the Mixl1^cKO as a protein null after Cre‐mediated excision. Mixl1^KO/KO embryos resembled the previously reported Mixl1^?/? mutant phenotype. Therefore, the Mixl1 cKO allele provides a tool for investigating the temporal and tissue‐specific requirements for Mixl1 in the mouse. genesis 52:417–423, 2014. © 2014 Wiley Periodicals, Inc.     

Foxh1 and Foxa2 are not required for formation of the midgut and hindgut definitive endoderm

The definitive endoderm forms during gastrulation and is rapidly transformed into the gut tube which is divided along the anterior-posterior axis into the foregut, midgut, and hindgut. Lineage tracing and genetic analysis have examined the origin of the definitive endoderm during gastrulation and demonstrated that the majority of definitive endoderm arises at the anterior end of the primitive streak (APS). Foxh1 and Foxa2 have been shown to play a role in specification of the APS and definitive endoderm. However, prior studies have focused on the role of these factors in specification of foregut definitive endoderm, while their role in the specification of midgut and hindgut definitive endoderm is less understood. Furthermore, previous analyses of these mutants have utilized definitive endoderm markers that are restricted to the anterior endoderm, expressed in extraembryonic endoderm, or present in other germ layers. Here, we characterized the expression of several novel definitive and visceral endoderm markers in Foxh1 and Foxa2 null embryos. In accordance with previous studies, we observed a deficiency of foregut definitive endoderm resulting in incorporation of visceral endoderm into the foregut. Interestingly, this analysis revealed that formation of midgut and hindgut definitive endoderm is unaffected by loss of Foxh1 or Foxa2. This finding represents a significant insight into specification and regionalization of mouse definitive endoderm.     

Sequential allocation and global pattern of movement of the definitive endoderm in the mouse embryo during gastrulation

During mouse gastrulation, endoderm cells of the dorsal foregut are recruited ahead of the ventral foregut and move to the anterior region of the embryo via different routes. Precursors of the anterior-most part of the foregut and those of the mid- and hind-gut are allocated to the endoderm of the mid-streak-stage embryo, whereas the precursors of the rest of the foregut are recruited at later stages of gastrulation. Loss of Mixl1 function results in reduced recruitment of the definitive endoderm, and causes cells in the endoderm to remain stationary during gastrulation. The observation that the endoderm cells are inherently unable to move despite the expansion of the mesoderm in the Mixl1-null mutant suggests that the movement of the endoderm and the mesoderm is driven independently of one another.