Approaches:

• Mouse Models
• Transgenic Embryo Explant Culture Assays
• Genetically Manipulated Embryonic Stem (ES) Cells

Embryo explant culture studies implicate Indian hedgehog and BMP signaling in activation of embryonic hematopoiesis and vascular development. In these assays, we take apart the embryo and ask what must be added back in order for blood and endothelial cells to form.

Indian hedgehog (Ihh) is confined to the VE during gastrulation and is expressed in a graded manner. Red: anti-Hh staining; eee: extraembryonic ectoderm; epi: epiblast; ps: primitive streak. Dashed lines indicate approximate location of sections shown at right.

Explant culture assays demonstrating that recombinant IHH protein can substitute for VE to activate formation of primitive blood cells (globin-lacZ transgenic embryos) or endothelial cells (Flk1-lacZ embryos) in anterior epiblast pieces, which normally form neural tissue (thus, they are respecified or reprogrammed). Addition of HH-blocking antibody to epiblast + VE recombinants (right) ablates induction of hematopoiesis in these explants.

Translation to clinical practice: We are interested in exploring the possibility that activating or inhibiting the hedgehog signaling pathway may provide a means to treat a variety of human diseases in which these processes are abnormal. Components of the pathway may prove to be good targets for development of new drugs.

The explant culture assays we have developed can be used to identify other novel regulators of stem and progenitor cell development. Candidates can then be tested in mouse models in vivo.

Mouse Mix (aka Mml, Mixl, Mrx1) is a paired-class DNA-binding homeodomain protein.

Mix is expressed in VE and later in mesoderm. While it is not expressed in definitive endoderm (e.g., gut and associated organs), it appears to function in patterning the endoderm.

Immunostaining with anti-Mix serum: Mix is a nuclear protein, as expected for a transcription factor. (Tub: tubulin)

Approaches:

• Overexpression of Wild Type and Mutated Forms in Embryonic Stem (ES) Cells; Analysis of Effect on Differentiation in Vitro
• Analysis of Transcriptional Regulation Function and Interactions with Other Proteins in Vitro and in Vivo (cultured cells, ES cell- derived embryoid bodies)
• Mouse Models (transgenic and knockout)

Early and transient activation of mouse Mix in differentiating ES cells. Mix activation is slightly more restricted than that of another mesodermal marker, T, and precedes that of Flk1 (a hemangioblast marker) and Gata1 (a hematopoietic marker).

We have generated transgenic mouse lines expressing fluorescent reporters under the control of Mix regulatory elements. GFP+ cells can be isolated by FACS and studied in vitro and in vivo to assess their developmental potential.

Conditional targeting of the Mix locaus. This approach will allow us to inactivate Mix in specific tissues at specific stages of development.

Generation of inducible Mix ES cell lines (iMix). Mix is induced when differentiating ES cells are treated with doxycycline (DOX). We can now analyze the properties of cells formed in ES cell-derived embryoid bodies.

These studies are in the planning stages, but the possibilities are obvious from the various assays in use in the lab.

Confocal image of a cultured embryo in which GFP reporter is expessed specifically in primitive erythroblasts (red blood cells), around E8.5. The vascular network of the yolk sac and the heart tubes are easily seen.

Transgenic embryos expressing GFP reporter in primitive erythroid cells. The bright signal is easily detected. Note the ring of blood islands in the 8.5 day embryo and the outlined vasculature (including aorta and developing heart) in the 9.5 day embryos.

Laser scanning confocal images showing primitive vascular plexus (left) and high magnification of blood cells flowing through yolk sac microvasculature (right).