HNF1β is a mediator for RA-signalling in the endoderm

The posterior expansion of XlHbox8 in the dorsal endoderm and the increase of insulin positive cells that was induced upon pan-endodermal activation of HNF1β resembled the result observed upon activation of RA- signaling (Chen et al., 2004). It was demonstratedIt was demonstrated that RA caused a caudal extension of the XlHbox8 positive expression domain and pro-moted insulin expression on the expense of exocrine tissue in the dorsal endoderm (Chen et al., 2004). In addition early RA promoted ventral exocrine tissue and also HNF1β pro-moted ventro-posterior expansion of the XlHbox8 domain.

Nevertheless, some discrepancies between ectopic HNF1β activity and increased RA si-some discrepancies between ectopic HNF1β activity and increased RA si-gnalling were observed. Head structures in RA treated embryos were nearly abolished,n RA treated embryos were nearly abolished,were nearly abolished, while they were only reduced in HNF1β depleted embryos. In contrast to posterior Xl-In contrast to posterior Xl-Hbox8 extension upon HNF1β overexpression, exogenous RA treatment also lead to an anterior reposition of insulin positive cells. This indicated that not only HNF1β is required but other signalling mechanism control correct A-P position of differentiating cells along the gut tube (table 4).

HNF1β expression was initiated after MBT in the vegetal hemisphere of the blastula stage embryo that gives rise to the endodermal germlayer (Vignali et al., 2000). By the onset of gastrulation the RA synthesizing enzyme RALDH2 is expressed in the involuting meso-derm, adjacent to the endogenous HNF1β expression domain (figure 1.5). Earlier studies in Xenopus laevis (Demartis et al., 1994) and zebrafish (Song et al., 2006) indicated that HNF1β was a RA responsive gene within the endoderm. Hence it was hypothesised thatHence it was hypothesised that HNF1β could link early RA induced prepatterning steps in the dorsal endoderm in order to promote pancreas developement, in particular endocrine cell differentiation (Stafford et al., 2004; Chen et al., 2004). However it remained unclear when HNF1β expression wasHowever it remained unclear when HNF1β expression was activated by RA and how it could mediate induction of pancreas development. So it was intriguing to investigate the role of RA to promote HNF1β gene activation. Therefore RA-Therefore RA- signalling was activated in blastula stage embryos upon RA treatment and inhibited using the RAR antagonist BMS453 (Chen et al., 2004).

Expression analysis of HNF1β in RA treated embryos revealed slight induction within the endoderm at early stages while BMS453 treatment revealed no effect compared to cont-rol. During subsequent development it was evident that endodermal expression level of HNF1β was elevated whereas BMS453 treatment rather repressed HNF1β transcription (fi-gure 3.1.11). In particular, expression by the stage of pancreatic budding (stage 34), HNF1β expression was strongly increased in the dorsal pancreatic region. It was an establishedIt was an established idea that RA is required for specification of the dorsal but not the ventral pancreas. This was conserved between vertebrates (Stafford and Prince 2002, Chen et al., 2004, Martin

et al., 2005). The increased HNF1β expression at stage 34 showed that the RA effect wasThe increased HNF1β expression at stage 34 showed that the RA effect was persistent through time and not a transient event. This result favored the idea of a putative role of HNF1β as RA mediator for dorsal pancreas specification.

In order to analyze direct activation of endodermal HNF1β expression, RA and BMS453 treatments were done on whole endodermal explants and analysed by semquantitative RT-PCR. HNF1β gene activation was altered upon exogenous application of RA and downre-gulated upon BMS453 treatment. This data provided evidence that endodermal HNF1β expression was responsive to direct RA- signalling.direct RA- signalling.RA- signalling.

A recent study reported that dorsal and ventral endoderm was differentially responsive to RA- signalling (Pan et al., 2007). As early HNF1β expression was in particular seen in the dorsal involuting endoderm, representing the prospective pancreatic tissue, it was of further interest to reveal whether HNF1β expression in the dorsal versus the ventral en-doderm differed in responsivity to RA (figure 3.1.12). Therefore whole enen-doderm explants were dissected and treated with RA or BMS453. HNF1β was slightly elevated in dorsal and ventral endoderm explants and its expression was remarkably reduced in dorsal and ventral explants upon BMS453 treatment. These findings indicate that HNF1β expressionHNF1β expression is regulated by RA within the endoderm. But HNF1β was not obviously differentially re-sponsive to RA signalling in the dorsal versus the ventral endoderm. However, this data remained to be confirm by quantitative RT-PCR.confirm by quantitative RT-PCR.RT-PCR.

The morphogen RA is synthesised by RALDH2 in the adjacent dorsal involuting mesoderm and secreted into the neighbouring endoderm. Assuming the formation of a dorso-ven-tral morphogen gradient, it can be speculated that under phyisological conditions, dorsal HNF1β expression is stronger influenced by RA than ventral HNF1β expression and that

Table 4 Comparison of RA and HNF1β induced effects on pancreas development. First ��olumn in�i��ate�

mananipulation of the embryos. The next ��olums in�i��ate expression levels of �lHbox� in the �orsal or ventral en�o�erm, level of en�o��rine ��ell �ifferentiation �en�o�, size of �orsal exo��rine ��ompartment �exo�, an� ��uut ��oilin�� effe��t. �+� elevate�, �-� re�u��e�, �++� e��topi��, �u��� un��han��e�.

-enous RA or BMS453 treatment lacked this differential gradient formation and therefore might activate or inhibit ventral HNF1β expression to the same extend than in the dorsal explant tissue. As consequence, this resulted in a comparable HNF1β gene activation or inhibition in dorsal and ventral explants.

Taken together, the results of this study support the idea that HNF1β is responsive to early RA signalling within the endoderm to direct dorsal pancreas development and endocrine cell differentiation, whereas it palys a distinct role in ventral pancreas specification. Hence, it would be of interest to investigate the effect on dorsal or ventral HNF1β expression upon region specific manipulation of RA- signalling.

A previous study in zebrafish was investigating the role of RA in directly acting on theprevious study in zebrafish was investigating the role of RA in directly acting on the endoderm to induce β-cell fate (Stafford et al., 2006) and it was shown that RA was not able to rescue insulin expression in the absence of vHNF1, the zebrafish homologue, whereas vHNF1 overexpression restored insulin expression when RA- signalling was inhibited (Song et al., 2006). A recent study suggested a role of RALDH1 within the pancreas to pro-mote endocrine cell differentiation (Öström et al., 2008). Data obtained in this study, as the RA-induced dorsal expression of HNF1β at stage 34, the HNF1β induced posterior expan-sion of XlHbox8 and the increase in insulin positive cells, together with previous findings, provided strong evidence that HNF1β acts as RA downstream target mediating instructive signals for dorsal pancreas formation and endocrine cell differentiation.

Apart from RA, many other signalling pathways were related to pancreas formation as the secreted factors SHH, BMPs and FGF (Hebrok et al., 1998; Kim et al., 2001; Hart et al., 2000). In the study by Chen et al., it was reported that RA induced expansion of XlHbox8 expression within the dorsal endoderm was associated with a decreased expression of SHH.

As shown in chick repression of SHH was crucial to permit Pdx1/ XlHbox8 expression in the pancreatic epithelium (Hebrok et al., 1998). Otherwise inhibition of RA caused an ex-pansion of the SHH expression domain in the endoderm. Antagonising action of SHH and RA was also demonstrated in chicken limb bud and zebrafish fin formation (Helm et al., 1994; Niswanderer et al.,1994). In zebrafish, endoderm formation and patterning relied on BMP signalling (Tiso et al., 2002). In the same study it was demonstrated that vHNF1 was dependent on early active BMP signaling and it was responsible to mediate BMP depend-ent endocrine cell differdepend-entiation in zebrafish.

Conversely, in Xenopus laevis it was shown that BMP- and also Wnt- signalling must bet was shown that BMP- and also Wnt- signalling must be repressed in the early dorsal endoderm in order to make the future anterior endoderm competent to acquire pancreatic cell fate (Pan et al., 2007; McLin et al., 2007; Spagnoli et al.,(Pan et al., 2007; McLin et al., 2007; Spagnoli et al.,

2008). In addition, it was shown that RA- and BMP- signalling interacted with each other. In addition, it was shown that RA- and BMP- signalling interacted with each other.

Therefore it would be intriguing to analyses changes in early HNF1β expression within the dorsal endoderm in response to modulated BMP- and Wnt- signalling.