Identification of 2 novel genes developmentally regulated in the mouse aorta-gonad-mesonephros region

Hematopoietic stem cells (HSCs) form the basis of the adult hematopoietic hierarchy.1 Adult-repopulating HSCs first appear at embryonic day 10.5 (E10.5) within the mouse embryo proper in the aorta-gonad-mesonephros (AGM) region.2,3More detailed studies show that HSCs are localized to the dorsal aorta.4-6 Because de novo production of HSCs is thought to take place during a limited time within the embryo, the AGM region is of interest to study the genes involved in the generation and regulation of the earliest HSCs.

To date, research on HSC development has focused on known transcription factors and signaling molecules. Transcription factors AML-1 and GATA-2 were shown to be important regulators of HSCs in the embryo.7-9 Also, members of the Hedgehog and Wnt (signaling) families have been implicated in playing instructive and/or regulatory roles on HSCs.10-12 To further increase our understanding of AGM HSC development, we set out to identify differentially expressed genes by using differential display reverse transcriptase–polymerase chain reaction (DD RT-PCR). We found several differentially expressed genes in the AGM region at the time of HSC induction, including β-catenin and novel mouse homologs of human TM9SF2 and TAB2. Characterization of mTM9SF2 and mTAB2 suggests a role for these genes in hematopoiesis. Interestingly, expression of mTAB2 in the E11 aortic endothelium suggests a role for mouse TAB2 (mTAB2) in HSC emergence and/or regulation.

AGM (sub)dissections of C57BL/6 or (CBA × C57BL/10)F1 embryos and cell cultures were performed as described.3,4,13 14 

RNA was isolated with Ultraspec (Biotecx, Houston, TX) or Trizol (Invitrogen/Life Technologies, Carlsbad, CA). For cDNA synthesis, 1 to 5 μg DNAse-treated RNA was reverse transcribed with Superscript II RT (Invitrogen/Life Technologies).

DD RT-PCR was essentially performed as described15 with 8 arbitrary primers. RT-PCRs were performed with Amplitaq (Roche, Branchburg, NJ) and 40 ng RNA equivalent of cDNA at optimized conditions for each primer set. Primer sequences are available on request.

For Northern blots 10 to 25 μg RNA, and for cDNA dot blots the equivalent of 1 μg RNA, was blotted to Genescreen membrane (NEN Technologies, Boston, MA) and probed with³²P-labeled nick-translated DNA fragments (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom).

For Western blotting and immunohistochemistry (6 to 8 μM cryosections) anti-TAB2 (sc-11851; Santa Cruz Biotechnology, CA) antibody was used according to the manufacturer's guidelines. Immunohistochemistry staining was visualized with ABC staining kit (Santa Cruz Biotechnology).

Three independent DD RT-PCR screenings were performed on early E10 (30 to 35 somite pairs) and E11 (40 to 50 somite pairs) AGM material to identify genes expressed prior to and after the induction of HSCs. For the first screening, cDNA populations from E10 and E11 AGM were compared. In the second and third screenings, E10 and E11 aorta and complete E10 AGM and subdissected E11 AGM cDNA populations were compared, respectively. In total, 39 differential bands were detected, and 62 cloned sequences were compared against National Center for Biotechnology Information (NCBI) nucleotide databases.

RT-PCR analyses (Figure 1) confirm that clones corresponding to fetal globin ε, enolase, TRAP150(not shown), β-catenin, mTM9SF2, andmTAB2 are differentially expressed between E10 and E11 in the AGM region. As expected, some clones are not differentially expressed, such as Dvl2, BUB1B, and Cu/Zn SOD.16 Hence, in 3 DD RT-PCR screenings, we identified a small number of differentially expressed genes. Most importantly, we were able to identify 2 novel genes not previously implicated in development or hematopoiesis. A complete overview of the DD RT-PCR clones is available on the Blood website; see the Supplemental Table link at the top of the online article.

Wnt family members are known regulators of developmental processes and hematopoiesis.10,12,17 Several Wnts and Wnt receptors are expressed in the yolk sac and fetal liver, both known hematopoietic sites during development. Identification of β-cateninand Dvl2 in our DD RT-PCR prompted us to examine whether other Wnt signaling members are expressed in the AGM and fetal liver (Figure 1B). In addition to β-catenin, Dvl3, Wnt5, andDvl1 (not shown) are up-regulated between E10 and E11 in the AGM. Additionally, TCF1 and LEF1 expression could be detected in the AGM and fetal liver. The expression ofTCF1 and LEF1 corresponds to previous findings of Oosterwegel et al.18 The simultaneous expression of several Wnt signaling components and the up-regulation of the key component β-catenin in the E11 AGM and liver suggests that the Wnt signaling pathway is functional. Further studies are required to address the role of Wnt signaling in the AGM, in particular in HSCs.

Protein sequence alignment of TM9SF2 homologs from several species, including mouse, human, Drosophila, andCaenorhabditis elegans, reveals high-level homology, indicative of a conserved function for this protein (data not shown). Human TM9SF2 is localized in endosomes and is proposed to function as channel or small molecule transporter.19 Alignment of themTM9SF2 mRNA sequence with genomic sequences reveals 17 exons (Figure 2A). Northern and cDNA dot blot analyses show mTM9SF2 expression in several adult tissues and hematopoietic cells lines. The highest levels of expression are observed in kidney and liver tissue (Figure 2B) and myeloid and B-lymphoid cell lines (Figure 2C). The expression pattern ofhTM9SF219 is partially consistent with our data in the mouse. Thus, the expression of mTM9SF2 in the AGM region and in hematopoietic cell lines makes this conserved gene an interesting candidate for future studies concerning its function and role in development and regulation of the hematopoietic system.

Human TAB2 is a binding partner of TAK1 and is involved in nuclear factor–κB (NFκB) and Jun N-terminal kinase (JNK) activation, in the interleukin-1 (IL-1) and receptor activator of NFκB (RANK) signaling pathways.20,21 Both IL-1 and RANK function within the hematopoietic system, and NFκB is an important hematopoietic transcription factor.22-24 Interestingly, studies in Xenopus reveal that TAK1 negatively regulates the Wnt pathway,25 making mTAB2 an interesting candidate for further study.

Alignment of the mTAB2 mRNA sequence with genomic sequences reveals 6 exons in the mTAB2 gene (Figure 2D). Northern blotting verifies the differential expression of mTAB2 in the AGM (Figure 2E) and demonstrates mTAB2 expression in several adult mouse tissues, including spleen, thymus, and bone marrow (BM) (Figure 2F). Additionally, mTAB2 is expressed at high levels in myeloid cell lines and at lower levels in lymphoid and erythroid cell lines (Figure 2C). Thus, mTAB2is expressed in several hematopoietic tissues and cell lines.

To investigate the expression pattern of mTAB2 in the AGM, we performed immunohistochemistry. First, a commercial antibody against hTAB2 was tested for its ability to recognize mTAB2 by Western blotting. In human and mouse cell lines, a single TAB2 protein band was detected at the predicted size of 77 kDa (Figure3A). Immunohistochemistry on cryosections of E10-E12 embryos reveals an increasing level of mTAB2 protein in the endothelium of the E11 (not shown) and E12 dorsal aorta and some underlying mesenchymal cells (Figure 3B). No mTAB2 protein expression was found in the E10 dorsal aorta, consistent with undetectable mTAB2 mRNA in the E10 AGM (not shown).

Taken together, the up-regulation of mTAB2 in the AGM coincides with the emergence of HSCs in this region, particularly in the dorsal aorta.4-6 Based on this spatial and temporal expression pattern of mTAB2, we suggest that mTAB2 is either directly or indirectly involved in the induction or regulation of HSCs in the AGM. Future studies should reveal whether mTAB2 is involved in IL-1 or RANK signaling and whether cross-talk between TAK1-TAB2 and Wnt signaling takes place in the AGM region.

Note added in proof. Recently, another group independently identified mouse TAB2 in neuronal cells.26 

The authors thank Drs Marella de Bruijn and Marian Peeters for help with embryo dissections, Karin van der Horn for embryo sectioning, and Sjozef van Baal and Ton Verkerk for help with the Celera database/bioinformatics. Drs R. Delwel, S. Tsai, and T. Langerak generously provided hematopoietic cell lines, and Drs M. de Bruijn, M. Peeters, and D. Meijer provided critical comments on the manuscript.