Hox expression pattern in embryos 94


Hox genes have been shown to play important roles in the establishment of the body axis during development (Krumlauf, 1994). Hox genes are a particular subgroup of homeobox genes, which code for a group of transcription factors. Special of Hox genes is not only their organization in chromosomal clusters, but the striking phenomenon of spatial colinearity (McGinnis and Krumlauf, 1992). In vertebrates, also a temporal colinearity has been detected (Duboule, 1994; Kmita and Duboule, 2003).

Hox proteins have been proved to be difficult to stain in whole mount immunohistochemistry approaches. Reviewing the current literature, the sensitivity of the different methodical approaches resulted in diverse Hoxc8 gene expression borders (Juan et al., 2006; Kwon et al., 2005b; Le Mouellic et al., 1992; van den

Akker et al., 2001). Recently, researchers replaced the Hoxc8 allele with either a GFP or a LacZ reporter. It could be shown that already the introduction of a reporter may lead to differences in endogenous Hox gene expression (Blackburn et al., 2009). The embryonic stage also seems to play an important role because the expression domains are labile within larger domains till embryonic day 12.5 dpc (McIntyre et al., 2007). In this study Hoxc8 expression in mouse embryos was analyzed using the Western blot method. The analysis of embryo parts revealed a specific protein expression in the posterior half of the embryo (Figure 28a).

Comparing mRNA and protein levels of Hoxc8 in wildtype embryos at 12.5 dpc, Hoxc8 mRNA could be detected in a broader area, while high expression of protein was restricted to a smaller region in the center of the mRNA expression area (Figure 28a,b). This difference in the mRNA and protein expression pattern do not exclude a regulation on the protein level, but strongly suggest a regulation on the post- transcriptional level causing an absence of Hoxc8 protein at the anterior and posterior expression borders. MiRNAs have been shown to repress the translation of target genes as well as to reduce the target mRNA expression levels and they have been proposed to act as fine-tuners of developmental gene expression programs (Stark et al., 2005). Consequently, it may be assumed that miRNAs inhibit Hoxc8 mRNA translation at sites of low expression.

Hoxc8 and Hoxb8 mRNAs could be identified to be targets of Eri1 during embryonic development. The Hoxc8 and Hoxb8 mRNA expression levels showed distinct phenotypes in Eri1-knockout and wildtype littermates (Figure 28b,c, Figure S-35). In the mutants the anterior expression border of Hoxc8 as well as Hoxb8 was shifted into posterior direction on the A-P axis of the embryo. In the critical expression area (embryo part 3) a 16-fold change for Hoxc8 and a 4-fold change for Hoxb8 was measured. However, no significant impact of Eri1 was seen in the posterior region of high Hoxc8 and Hoxb8 expression. The shift of Hox gene expression into the posterior direction in Eri1-knockout mice may account for both Eri1-knockout mouse phenotypes, the attachment of an 8th rib pair to the sternum as well as the development of an additional rib pair at the first lumbar vertebra. The posterior dominance model predicts that the more posterior expressed Hox genes have a greater effect on the pattering information than the more anterior expressed genes (Duboule, 1994). It can therefore be proposed that the Hoxc8 and Hoxb8 prevalence

starts only in the more posterior situated lower thoracic region in the Eri1-knockout situation.

Although the presented data cannot rule out a possible positive effect of Eri1 on the transcription rate of Hoxc8 and Hoxb8 mRNAs, there is compelling evidence for Eri1- mediated derepression of posttranscriptional silencing of Hoxc8. In support of this proposed mechanism, a very recently published paper showed an anterior expansion of Hoxb8 gene expression as the consequence of antagomir-mediated neutralization of the Hox miR-196 in chicken embryos (McGlinn et al., 2009).

The levels of mature miR-196a were measured in Eri1-knockout and wildtype animals. Although a tendency for a shift into the anterior direction was observed in Eri1-knockout mice, no significant difference could be detected comparing three Eri1- knockout and wildtype pairs by quantitative reverse transcription PCRs (Figure 29a, Figure S-36). This might be due to various reasons: First, the analyses were performed at 12.5 dpc, a time point when the identity of the segments has already been established. And second, the analysis is imprecise by measuring whole slices including the limbs, which express miR-196 as well. Therefore follow-up experiments using microdissection are planned in which exclusively the somites will be examined for their mature miR-196a expression. Further, whole mount in situ hybridizations on embryos at 11.5 and 12.5 dpc have been performed (Figure 29c), but the protocol still has to be established for earlier developmental stages closer to the time period of vertebrae anlage. A problem for the Eri1-knockout and wildtype comparison by in situ-hybridization is that the locked nucleic acids probe recognizes mature miR-196a as well as its precursor molecules, pre-miR-196a and pri-miR-196a. Regulation of precursor miRNAs by Eri1 has not been demonstrated yet.