The molecular mechanisms that pattern endoderm along the A-P axis.
We have used a mouse to chick xenograft approach to show that endoderm is not yet specified along the A-P axis at e7.5. Mouse endoderm that would normally contribute to the large intestines is re-specified toward a pancreatic fate by grafting it into the pancreatic domain of a chick embryo. However, endoderm one-day later can no longer be re-specified. What are molecules that specify A-P endoderm pattern at this stage in development?
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Figure 1: Endoderm specification assay (chick/mouse embryo assay). |
Figure 1 schematically shows grafting of e7.5 mouse endoderm into the pancreatic domain of the same stage chick embryo. The left 2 panels shows anterior and posterior endoderm that was dissected from e7.5 mouse embryos. The mouse endoderm was grafted into a tear that was made in the endoderm of a primitive streak stage chick embryo. In this example, either anterior or posterior mouse endoderm is grafted into the presumptive pancreatic domain of endoderm, and embryos are cultured for 2-3 days until organ buds begin to form. Figure 2 shows an example of this experiment.
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Figure 2: E7.5 mouse endoderm is not specified along the A-P axis. |
The endoderm in Figure 2 was dissected from e7.5 transgenic mouse embryos that express lac Z under the control of the Pdx 1 gene, and grafted into a primitive streak stage chick embryos (summarized in Figure 1). Both anterior (pancreatic) and posterior (intestinal) mouse endoderm is induced to express Pdx1-lacZ when grafted into the pancreatic domain of the chick embryo. The top panel shows expression of the pancreatic marker Pdx1 in mouse. The middle panel is a control chick embryo, and the bottom panel shows expression of Pdx1-lacZ in mouse endoderm that was grafted in to chick embryos.
FGF's as posterior determinants of endoderm pattern.
Our preliminary specification studies have shown that soluble factors expressed by the primitive streak act to posteriorize endoderm. We used an in vitro assay to test growth factors expressed by the primitive streak, and identified that FGF4 induced posterior patterning markers in isolated endoderm. We observed a dose-responsive gene induction indicating that a gradient of FGF4 could establish a gene expression boundary between large and small intestine. Currently, we are using chick and mouse embryos to perform a thorough analysis of the role of FGF signaling in posterior endoderm patterning in vivo.
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Figure 3: Model for FGFs acting as posterior morphogens |
Several FGFs are expressed at high levels in the posterior of the e7.5 embyo (for example see Figure 3). Expression patterns of FGFs suggest that endoderm is exposed to a posterior gradient of FGF ligands. Our data shows that endoderm differentially responds to FGF concentration. Low FGF levels induce the pancreatic/duodenal marker neuroD where as high levels repress neuro D, but induce the posterior marker somatostatin. These data suggest that a posterior gradient of FGF could establish a gene expression boundary between duodenum and intestine.
Wells, J. M. and Melton, D. A. Early mouse endoderm is patterned by soluble factors from adjacent germ layers. Development, 127: 1563-1572. (2000).
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The function of the transcription factor Sox 17 in specification of anterior endoderm.
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Figure 4: The 2 major Sox 17 isoforms are differentially expressed in endoderm. |
Anterior endoderm is not specified at e7.5, and our analysis of genes that are expressed in anterior endoderm revealed that two mRNA isoforms of Sox 17 are expressed in different domains of anterior endoderm. One isoform, expressed in a small domain of anterior endoderm, encodes a truncated form of Sox 17 that inhibits the activity of full length Sox 17 in cell culture. Published reports in frog suggest that Sox 17 can impinge on TGFb and Wnt signaling pathways by binding to Smads and b-catenin. We are currently studying how Sox 17 and dominant negative Sox17 interact with co-factors to regulate expression of target genes, and how these interactions regulate anterior endoderm patterning.
RT-PCR analyses illustrated in Figure 4 show that at e7.5, full length Sox 17 is expressed predominantly in endoderm, but at low levels in mesoderm/ectoderm, and also at e10.5 in other tissues. Whole mount insitu analysis of e7-e8 embryos show that Sox 17 is expressed in a broad domain of anterior endoderm, where as truncated Sox 17 is expressed in a very restricted domain of anterior endoderm.
How to Reach Us
The Wells Laboratory is part of the Division of Developmental Biology at Cincinnati Children's Hospital Medical Center. The lab is located in Location R (Research Foundation Building), Room 2543.
For more information, contact Jim Wells, 513-636-8767 (james.wells@chmcc.org).
