Identification of 22 RA-responsive genes

Unlike other signaling molecules, RA enters the cell independent of cell-surface receptors and binds directly to nuclear receptors (Rochette-Egly and Germain, 2009;

Huang et al., 2014b). However, no studies are available dealing with the question in which time scale nuclear receptors are activated to induce target gene expression.

We could show that the known direct RA-target Cyp26a1 (Loudig et al., 2000) is induced within one hour after addition. Thus, for the identification of early RA-targets, the transcriptome of programed explants was analyzed one and two hours after RA-addition. 102 genes were found to be differentially expressed in the presence of RA-signaling. Almost half of these genes were affected by RA in the presence of CHX, indicating them as putative direct RA-targets. Twelve genes were found to be differentially expressed upon CHX-treatments but not affected by RA-signaling. This might be due to the translational inhibition of transcriptional repressors. Under these conditions, genes are possibly transcribed that are usually repressed. The list of 102 differentially expressed genes arise from the alignment of 50 nucleotide short sequence reads to the genome of two Xenopus species, tropicalis and laevis. The inclusion of the tropicalis genome was necessary as the laevis genome is not fully sequenced. Thus, six mismatches were allowed during the process of alignment to target also the tropicalis genome. Beside the frequently occurring errors using small sequence reads, this further increased the risk of false

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positive candidates (González and Joly, 2013). Thus, several approaches were used to validate the RA-responsiveness of the candidates.

By this, the list of RA-responsive genes was diminished to 22 candidates. This list contains nine homeobox transcription factors (Hnf1b, Lhx1, Gbx2.1, Meis3, Nkx6.2, Hoxd1, Hoxd4, Hoxa1, Hoxb1), three other transcription factors (Znf703, Foxh1, Cebpd), two enzymes (Dhrs3 and Cyp26a1), two signaling components (Fzd4, Igf3), an intermediate filament protein (Prph), an autocrine glycoprotein (Fst) and four candidates with unknown identity. A review by Balmer and Blomhoff in 2002 evaluated published data from in total 1,191 papers covering 532 described target genes and classified them into categories according to the probability of RA-regulation (Balmer and Blomhoff, 2002). However, most of these data were obtained in cell cultures without any developmental aspect. Nevertheless, 13 of the listed 22 RA-responsive genes in our study were already described as RA-regulated genes in the review of Balmer and Blomhoff or in following studies.

The homeobox-containing transcription factor Hnf1b was initially described as RA-inducible in a mouse stem cell line (De Simone et al., 1991). Furthermore, Hnf1b was found to be RA-induced in the mouse and zebrafish hindbrain (Hernandez et al., 2004; Pouilhe et al., 2007). It is described in several studies using zebrafish, mouse and Xenopus embryos that an RA-gradient during neural development contributes to the anterior-posterior expression pattern of homeobox-containing genes in the hindbrain (reviewed in Glover et al., 2006). Thus, the RA-inducibility of most of the Hox-genes in our list was previously described including Hoxd4 (Nolte et al., 2003), Hoxa1 (Boylan et al., 1993; Frasch et al., 1995), Hoxb1 (Huang et al., 2002; Ishioka et al., 2012), Hoxd1 (Kolm and Sive, 1994), Gbx2.1 (Bouillet et al., 1995) and Lhx1 (Strate et al., 2009). Furthermore, Fzd4 and Fst were previously shown to be up-regulated by RA in mouse embryonal tumor cells (Hashimoto et al., 1992; Katoh, 2002). For the RA-metabolic enzymes Cyp26a1 (Abu-Abed et al., 1998) and Dhrs3 (Kam et al., 2013), the zinc-finger transcription factor Znf-703 (Mitchell et al., 2015) and the intermediate filament Prph (De Genaro et al., 2013) the RA-inducibility was also shown previously. Beside these known RA-regulated genes detected in our screen, 9 additional candidates are identified as novel RA-target genes.

Based on the CHX-treatment data, we can distinguish between putative direct and indirect targets. However, the presence of retinoic acid response elements (RARE), as additional indicators for direct RA-regulation, remains to be proven. For Xenopus laevis genes this is a challenging venture as intronic and enhancer sequences are

99 not fully sequenced. RAREs are characterized by two direct repeats of the hexameric motif RGKTSA (R=A/G, K=G/T, S=C/G) separated by either one, two or five nucleotide spacers. RAREs were found to be located between 10 kb upstream and downstream from the gene locus and were bound by RXR/RAR heterodimers that display distinct motif affinities. Both classes of RA-receptors exist as α-, β-, γ-isoforms and further numerous γ-isoforms by differential promotor usage or alternative splicing exist, varying in RARE recognition (reviewed in Germain et al., 2006;

Lalevee et al., 2011). Several studies in mouse and zebrafish detect RAREs within the gene loci of RA-responsive homeodomain-containing transcription factors.

These include Hnf1b (Pouilhe et al., 2007), Hoxd4 (Nolte et al., 2003), Hoxa1 (Langston et al., 1997) and Hoxb1 (Huang et al., 2002).

The expression characteristics of the identified 22 RA-responsive genes during gastrulation gave us further indications about their possible function in pancreas development. Based on their RA-responsive endodermal expression domains, Hnf1b and Fzd4 were selected for functional analysis. Through loss and gain of function approaches, the requirement of Hnf1b for pancreas development could be confirmed and for Fzd4 a function in pancreatic fate determination is strongly suggested. These findings will be discussed in detail in the next sections. However, it remains to be urgently examined if the combined activity of Hnf1b and Fzd4 is sufficient to substitute for RA in pancreas specification.

If this is not the case, the list of RA-responsive genes from our screen contains some further interesting candidates. The results of a previous study, using different combinations of dorsal and ventral endodermal and mesodermal explants, suggests that RA-signaling acts simultaneously in both, dorsal endoderm and mesoderm, to promote pancreatic fate (Pan et al., 2007). In our list, four genes were found to be expressed in the dorsal internal involuting mesoderm which corresponds to the Raldh2 expression domain. Cebpd, a transcription factor of the C/EBP family, is linked to β-cell survival as it has an anti-apoptotic function in rat and human insulin-producing cells (Moore et al., 2012). The enzyme Dhrs3 exhibits a retinal reductase activity that converts retinal to retinol counteracting against RA-generation (Haeseleer et al., 1998). Fst (Follistatin), an autocrine glycoprotein, is an Activin inhibitor (Kogawa et al., 1991). In the context of pancreas development the inhibition of Activin was shown to expand pancreatic epithelium, but decrease the number of differentiated β-cells. Thus, it is suggested that Fst is required to regulate Activin activity to acquire the homeostasis of growth and differentiation in this context (Zhang et al., 2004). A further candidate, expressed in the dorsal involuting mesoderm, is the transcription factor Lhx1 for which no connection to pancreas

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development is described. Not expressed in the internal involuting mesoderm but linked to pancreas development is Nkx6.2. In mouse, this transcription factor was found to be expressed in pancreatic progenitors regulating pancreatic subtype specification (Henseleit et al., 2005). Therefore, functional studies for the listed candidates appear to be important.