BMP Signaling Via the Cdx-Hox Pathway Allocates Mesoderm to Hematopoietic vs Cardiac Fates.

Studies in model organisms have implicated the BMP pathway in allocating mesodermal tissues to blood fates. BMP receptors are widely expressed in the AGM region, implying BMP signaling in formation of definitive hematopoietic stem cells (HSCs). To dissect the role of the BMP pathway in murine hematopoietic development, we have exploited embryonic stem (ES) cells as a model system allowing experimental modulation of BMP4 protein and the BMP inhibitor Noggin during in vitro differentiation. Distinct mesodermal progenitors formed in response to a BMP gradient: progressively higher concentrations promoted cells expressing transcription factors of the posterior (caudal) primitive streak (e.g. Evx1, Cdx1, Mesp1), at the expense of anterior (rostral) markers (e.g. Cerberus, Goosecoid, FoxA2). Noggin addition during d0–2.5 was sufficient to inhibit hematopoiesis, data consistent with the observation that hemangioblasts form almost exclusively from the posterior primitive streak in vivo (Huber et al, 2004). Noggin addition after d2.5 abrogated hematopoietic fate, demonstrating a subsequent requirement for BMPs. At a molecular level, BMP4 added at d2.5 induced posterior Hox genes of the A (A7, A9, A10), and B cluster (B7, B8), while Noggin promoted an anterior Hox profile (A2, A4, B1). BMP4 patterns populations within the posterior primitive streak to blood by activating specific Hox genes.

Cdx4 and Cdx1 are homeodomain-containing transcriptional regulators implicated in blood development in the zebrafish (Davidson et al, 2004Davidson et al, 2006), and hypothesized to convey positional information from morphogens to Hox genes. We have observed that Cdx genes show enhanced expression after BMP4, and suppression after Noggin treatment. Moreover, Cdx4 expression could bypass the inhibitory effect of Noggin on hematopoieisis after d2.5 of EB development. In serum-free conditions, either BMP4 addition or Cdx4 expression was sufficient to specify hematopoietic progenitors at equal or greater levels than serum cultures. These data demonstrate that Cdx4 complements the BMP requirement for specification of primitive streak cells to blood, presumably by activating the Hox gene signature for blood. We are currently exploring whether direct biochemical interactions can be established between the BMP/Smad pathway and Cdx genes.

We investigated what alternative fates the posterior primitive streak cells might adopt, if not specified to blood by BMPs. Angioblasts were not affected, but cardiac differentiation displayed a striking inverse relationship to hematopoietic fate. Using quantitative assays of hematopoietic and cardiac potential, we determined that conditions promoting hematopoiesis (BMP4 addition or Cdx4 activation), simultaneously strongly suppressed cardiac development. We reproduced these observations with an ES cell line carrying a GFP reporter gene driven by the cardiac-specific NKX2.5 promoter (Wu et al., submitted). Hox gene analysis in purified cardiac (NKX2.5-GFP+) and hematopoietic progenitors (CD41+ckit+), showed posterior Hox genes to be suppressed in cardiac, and elevated in hematopoietic cells, supporting our hypothesis of positional patterning by differential Hox gene activation. We conclude that BMPs act on common progenitors via the Cdx-Hox pathway to pattern hematopoietic and cardiac fates within the primitive streak.