Hidden Blood Factory Discovered at the Border Between Mother and Embryo

In mammals, the first blood cells of a new organism were thought to arise exclusively in the extraembryonic yolk sac, before hematopoietic stem cells later emerge in the aorta–gonad–mesonephros region. A new study published in Nature Immunology expands that map. Researchers led by Rohit Jain and Wolfgang Weninger have identified a previously unrecognized hematopoietic site at the maternal–fetal interface in mice, a tissue they have named MIXER, short for Maternally Interfaced eXtraEmbRyonic tissue.

Far from being a passive boundary zone, MIXER develops contemporaneously with the yolk sac and harbors robust hematopoietic activity, including a distinct class of blood progenitors not found in the yolk sac at all. The discovery began with a macroscopic observation. When the team collected mouse embryos at embryonic day 8.5, or E8.5, with their yolk sacs intact, they consistently noticed a densely vascularized red zone at the site of embryonic implantation.

This region, where embryo-derived trophoblasts invade the maternal uterine wall, is well known to contain rich maternal vasculature and immune cells. But the investigators wondered whether it might also harbor blood cells of embryonic origin, analogous to those in the yolk sac. Answering that question required solving a long-standing technical problem: distinguishing embryonic cells from maternal cells at the implantation site, something that has historically limited detailed characterization of this tissue.

To overcome this barrier, the researchers deployed a genetic labeling strategy. They paired β-actin-Cre male mice with mT/mG female reporter mice for timed mating. In this system, embryos express membrane-targeted green fluorescent protein in all embryonic tissues, while maternal tissues glow red with membrane-targeted Tomato.

When the team sectioned the entire gravid uterus and examined it by confocal laser scanning microscopy, they observed a clear separation between maternal and embryonic compartments. At the implantation site, the mesometrial pole where the placenta develops, embryonic cells actively intermingled with and invaded maternal tissue. At the anti-mesometrial pole, further from implantation, the two cell populations segregated into distinct layers.

The researchers designated this entire interface region MIXER, and notably, they found no maternal cells within it. Flow cytometric analysis of microdissected MIXER tissue revealed a striking diversity of embryonic cell types. The team detected embryonic erythrocytes marked by the Ter119 protein, leukocytes expressing CD45, endothelial cells carrying CD31, and epithelial cells bearing EpCAM, all of embryonic rather than maternal origin.

This was particularly notable because, at E8.5, fully functional vascular connections between the embryo and its extraembryonic tissues have not yet been established. Immunostaining followed by high-resolution confocal microscopy confirmed that embryonic erythrocytes and leukocytes were dispersed throughout MIXER rather than confined to a single subanatomical location. The embryonic leukocytes in MIXER were also visibly smaller than their maternal counterparts, underscoring that these were genuinely fetal cells in a maternal environment.

But the presence of mature blood cells does not necessarily mean the tissue is producing them. To test MIXER's hematopoietic capacity directly, the researchers sorted embryonic cells from MIXER and yolk sac tissues and cultured them on OP9 stromal cells in medium supplemented with stem cell factor, interleukin-3, interleukin-6, erythropoietin, thrombopoietin, granulocyte–macrophage colony-stimulating factor, and macrophage colony-stimulating factor. Both tissue sources generated robust erythroid, myeloid, and megakaryocytic lineages.

Most leukocytes derived from MIXER cultures expressed CD31, similar to those produced from yolk sac progenitors, suggesting a potential origin from hemogenic endothelium, the specialized vascular lining that gives rise to blood cells. These results established MIXER as an active site of embryonic hematopoiesis alongside the yolk sac, not merely a storage depot. The most surprising discovery came when the team examined the specific progenitor populations within MIXER using a different mouse strain called DPE-GFP, in which CD4 regulatory elements drive green fluorescent protein expression in T cells, perivascular macrophages, and plasmacytoid dendritic cells.

In an accompanying study, the same group had identified a population of erythro-myeloid progenitors arising from the CD4 lineage in adult bone marrow, which they called EMPROR cells. Now, in the E8.5 embryo, they found the embryonic equivalent. MIXER contained not only classical CSF1R-positive erythro-myeloid progenitors, which were also present in the yolk sac and were markedly more abundant in MIXER, but also a unique population of GFP-positive cells that expressed CD4 but lacked CSF1R.

These cells, which the researchers named eEMPROR cells, were found exclusively in MIXER and were entirely absent from yolk sac preparations at the same developmental stage. The hematopoietic character of MIXER extends even earlier in development. At E7.5, when yolk sac hematopoiesis is just beginning, the team detected embryonic endothelial cells expressing CD31 within MIXER, along with cells expressing CD309, also known as VEGFR2 or FLK1, a marker associated with early hemogenic endothelium and hemangioblastic populations.

Embryonic leukocytes marked by CD45 and abundant erythroid cells marked by Ter119 were already present throughout the tissue. Critically, CD4-positive cells were also detectable in MIXER at E7.5, suggesting that eEMPROR cells emerge during this earliest developmental window. Flow cytometric analyses of E7.5 tissues confirmed that GFP-positive eEMPROR cells were selectively present in MIXER while corresponding yolk sac preparations lacked this population entirely.

As development progressed, eEMPROR cells did not remain confined to the maternal–fetal interface. By E14.5, the team detected them at their highest frequency in the dorsal aorta, with additional populations in the fetal liver and embryo heads. Interestingly, the placental labyrinth, one of the major sites of hematopoiesis at this stage, harbored very few of these cells, and the vitelline arteries, veins, and yolk sacs were devoid of them.

In vitro colony-forming assays demonstrated that sorted eEMPROR cells from E14.5 fetal liver could give rise to colonies containing macrophages, neutrophils, megakaryocytes, and erythroid cells, confirming their broad erythro-myeloid potential. The researchers also found GFP-positive macrophages in the perivascular spaces of the embryo head and skin, suggesting that eEMPROR cells migrate to peripheral tissues where they differentiate into perivascular macrophages. To formally establish that eEMPROR cells represent a genuine developmental lineage, the researchers used CD4-Cre lineage-tracing systems that irreversibly label cells with a history of CD4 promoter activity.

At E14.5, they found tdTomato-positive progenitor cells matching the eEMPROR profile in the dorsal aorta and fetal liver, while conventional monocyte progenitors were uniformly unlabeled. TdTomato-positive macrophages were detected throughout the embryo, including in the head, meninges, fetal liver, skin, and yolk sac. In young adult mice, CD4-lineage descendants persisted as a small subset of liver macrophages and were found almost exclusively within the meningeal perivascular macrophage compartment in the brain, while the microglial population was entirely devoid of labeling.

An inducible pulse-chase approach, in which a single pulse of tamoxifen was administered at E7.5, E8.5, or E9.5, confirmed that CD4-expressing progenitors arising during the earliest phases of embryogenesis generate substantial fractions of fetal macrophages, with the strongest contribution following E7.5 and E8.5 induction. These labeled macrophages persisted into postnatal life, detectable in the meninges and liver at postnatal day 14. The researchers analyzed first-trimester human placental tissue from pregnancies at approximately 5 weeks of gestation, corresponding developmentally to a 3-week-old human embryo and predating the emergence of functional hematopoietic stem cells.

Immunostaining revealed CD4-positive cells confined to discrete clusters within the villous core, specifically inside CD31-positive endothelial-lined structures. These CD4-positive cells did not express CD45 or CD14, ruling out leukocytes and Hofbauer cells, the placenta's resident macrophages. Critically, the team identified both CD4-positive and CD4-negative nucleated erythrocytes expressing CD235a within villous vessels, confirming that active erythropoiesis is occurring in early human placental tissue and suggesting the existence of a CD4-marked precursor population that mirrors the mouse eEMPROR lineage.

The authors propose that MIXER may represent a transient preplacental interface that supplies progenitors before placental perfusion is fully established, and that its unique exposure to maternal factors, including uterine blood flow, decidual health, and maternal inflammation, could mean that disruption of this earliest hematopoietic environment has lasting consequences for macrophage ontogeny and developmental immune homeostasis. The findings substantially broaden the prevailing yolk sac-centered model of primitive hematopoiesis, suggesting instead that the earliest waves of blood progenitors arise across anatomically distinct extraembryonic niches shaped by different local microenvironments.

Subject of Research: Discovery of a novel embryonic hematopoietic niche at the maternal–fetal interface containing distinct erythro-myeloid progenitors

Article Title: The maternal–fetal interface is an early hematopoietic niche enriched in distinct erythro-myeloid progenitors

Article References: Jain, R., Tikoo, S., Márquez-Burgoa, C., Gerhofer, S. A., Martinez, B., Barresi, M., Symmank, D., Ghannadan, M., Öhler, B., Höbler, A.-L., e Silva, R. D. F., Baumjohann, D., Feil, S., Feil, R., Knapp, S., Starkl, P., Ellmeier, W., Jung, S., Pollheimer, J., … Weninger, W. (2026). The maternal–fetal interface is an early hematopoietic niche enriched in distinct erythro-myeloid progenitors. Nature Immunology. https://doi.org/10.1038/s41590-026-02683-4

Image Credits: AI Generated

DOI: 10.1038/s41590-026-02683-4

Keywords: MIXER tissue, maternal-fetal interface, embryonic hematopoiesis, erythro-myeloid progenitors, eEMPROR cells, yolk sac, placenta, perivascular macrophages, CD4 lineage tracing, hemogenic endothelium, primitive hematopoiesis, fetal development