Mixl1 localizes to putative axial stem cell reservoirs and their posterior descendants in the mouse embryo

• Mixl1 protein has been localized to the mouse gastrula from ∼E6.5–E9.5.
• Mixl1 may identify putative mesendodermal progenitors within the axial midline.
• Mixl1 localizes to early hemogenic/vasculogenic sites in the posterior conceptus.
• Mixl1 localizes to the invaginating hindgut.
• Results support Mixl1 in mesendodermal stem cell niches in early organogenesis.

Mixl1 is thought to play important roles in formation of mesoderm and endoderm. Previously, Mixl1 expression was reported in the posterior primitive streak and allantois, but the precise spatiotemporal whereabouts of Mixl1 protein throughout gastrulation have not been elucidated. To localize Mixl1 protein, immunohistochemistry was carried out at 2–4 h intervals on mouse gastrulae between primitive streak and 16-somite pair (s) stages (∼E6.5–9.5). Mixl1 localized to the entire primitive streak early in gastrulation. However, by headfold stages (∼E7.75–8.0), Mixl1 diminished within the mid-streak but remained concentrated at either end of the streak, and localized throughout midline posterior visceral endoderm. At the streak’s anterior end, Mixl1 was confined to the posterior crown cells of Hensen’s node, which contribute to dorsal hindgut endoderm, and the posterior notochord. In the posterior streak, Mixl1 localized to the Allantoic Core Domain (ACD), which is the source of most of the allantois and contributes to the posterior embryonic–extraembryonic interface. In addition, Mix1 co-localized with the early hematopoietic marker, Runx1, in the allantois and visceral yolk sac blood islands. During hindgut invagination (4–16s, ∼E8.5–9.5), Mixl1 localized to the hindgut lip, becoming concentrated within the midline anastomosis of the splanchnopleure, which appears to create the ventral component of the hindgut and omphalomesenteric artery. Surrounding the distal hindgut, Mixl1 identified midline cells within tailbud mesoderm. Mixl1 was also found in the posterior notochord. These findings provide a critical systematic, and tissue-level understanding of embryonic Mixl1 localization, and support its role in regulation of crucial posterior axial mesendodermal stem cell niches during embryogenesis.