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. 2008 Oct 30:9:511.
doi: 10.1186/1471-2164-9-511.

Microarray analysis of Foxa2 mutant mouse embryos reveals novel gene expression and inductive roles for the gastrula organizer and its derivatives

Owen J Tamplin  1 , Doris Kinzel, Brian J Cox, Christine E Bell, Janet Rossant, Heiko Lickert
Affiliations

Microarray analysis of Foxa2 mutant mouse embryos reveals novel gene expression and inductive roles for the gastrula organizer and its derivatives

Owen J Tamplin et al. BMC Genomics. .

Abstract

Background: The Spemann/Mangold organizer is a transient tissue critical for patterning the gastrula stage vertebrate embryo and formation of the three germ layers. Despite its important role during development, there are still relatively few genes with specific expression in the organizer and its derivatives. Foxa2 is a forkhead transcription factor that is absolutely required for formation of the mammalian equivalent of the organizer, the node, the axial mesoderm and the definitive endoderm (DE). However, the targets of Foxa2 during embryogenesis, and the molecular impact of organizer loss on the gastrula embryo, have not been well defined.

Results: To identify genes specific to the Spemann/Mangold organizer, we performed a microarray-based screen that compared wild-type and Foxa2 mutant embryos at late gastrulation stage (E7.5). We could detect genes that were consistently down-regulated in replicate pools of mutant embryos versus wild-type, and these included a number of known node and DE markers. We selected 314 genes without previously published data at E7.5 and screened for expression by whole mount in situ hybridization. We identified 10 novel expression patterns in the node and 5 in the definitive endoderm. We also found significant reduction of markers expressed in secondary tissues that require interaction with the organizer and its derivatives, such as cardiac mesoderm, vasculature, primitive streak, and anterior neuroectoderm.

Conclusion: The genes identified in this screen represent novel Spemann/Mangold organizer genes as well as potential Foxa2 targets. Further investigation will be needed to define these genes as novel developmental regulatory factors involved in organizer formation and function. We have placed these genes in a Foxa2-dependent genetic regulatory network and we hypothesize how Foxa2 may regulate a molecular program of Spemann/Mangold organizer development. We have also shown how early loss of the organizer and its inductive properties in an otherwise normal embryo, impacts on the molecular profile of surrounding tissues.

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Figures

Figure 1
Figure 1
Overview of the method used to derive and collect Foxa2 mutant epiblasts for analysis.
Figure 2
Figure 2
Validation of genes reduced in the Foxa2 mutant by whole mount in situ hybridization. (A-C) Wild-type and (D-F) Foxa2 mutant embryos at E7.75. (A, D) Foxd4 expression is absent from node and AME, and reduced in the ANE in mutant embryos compared to wild-type. (B, E) Gal is completely absent from node, AME and PS in the mutant embryos compared to wild-type. (Note: the Foxa2 mutant embryo in (E) has some background signal due to over-staining to detect any residual Gal expression). (C, F) Itga3 is severely reduced in the mutant embryo compared to wild-type and is only expressed in a small area around the APS. Black arrowheads indicate node; black lines indicate AME; red arrowheads indicate ANE; black arrows indicate DE. Scale bars are 200 μm.
Figure 3
Figure 3
Genes we identified that are expressed in the node at E7.5. (A, B) Gal and (C, D) Pim1 are expressed in the PS and node. (E) Prnp, (F) Cyb561, (G) Smoc1, (I) 1700027A23Rik, (J) Mlf1, (K) 1700009P17Rik, and (L) Josd2 are all specifically expressed in the node (Note: later at E7.75, Smoc1 is expressed in the node and more broadly in the mesoderm; Additional File 3). (H) Gstm5 has widespread expression throughout the embryo, but is expressed strongly in the periphery of the node. (A, C, I) lateral view, anterior left; (B, D) distal view, anterior top; (E-H, J-L) anterior view.
Figure 4
Figure 4
Genes we identified that are expressed in the DE. (A) Cldn4 is expressed in the anterior DE and in extra-embryonic regions at E7.25, (B) in the foregut pocket, lateral DE, and 37 extra-embryonic regions at E7.75, (C-D) and in the gut endoderm and otic vesicle from E8.5–9.0. (E) Itga3 is strongly expressed throughout the gut endoderm at E8.5. (F) Cpm is expressed in the anterior DE at E7.5, (G) strongly in the foregut pocket and throughout the DE at E7.75, (H) and in the ventral aspect of the gut at E8.5 (Note: at longer exposure, Cpm can be seen in more DE cells; Additional File 3). (I) Ppp1r14a is expressed in the ventral aspect of the gut at E8.5. (J) Efhd2 is highly expressed in the rostral foregut and caudal hindgut at E8.5. (A, F) lateral view, anterior left; (B, G) anterior view; (C-E) lateral view; (H-J) ventral view.
Figure 5
Figure 5
A network model for Foxa2-dependent gene regulation in organizer derivatives. (A) An example of binding motif prediction results visualized using the UCSC genome browser; [105]. Conserved Foxa2 and Brachyury/T binding motifs were found in the 10 kb region upstream of the endoderm-specific Cer1 gene. (B) A hypothetical network model based on existing genetic data (see Discussion for details and references), and binding motif predictions around putative Foxa2 target genes (Additional Files 1, 9, 10, 11, 12). Brachyury/T is active in node, notochord and axial mesendoderm (ND/NC/AME), but not in definitive endoderm (DE). The transcription factors Foxa2, Brachyury/T, Noto, Foxa1, and/or Foxd4 could regulate putative ND/NC/AME target genes; Foxa2, Foxa1, and/or Sox17 could regulate DE target genes. We propose a role for Brachyury/T in repression of DE targets in the ND/NC/AME lineages. Foxa2 likely regulates itself in both ND/NC/AME and DE. (Note: the network diagram was created using BioTapestry; [106]).
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