Rbp4 is a plasma protein acting as a transporter of retinol in blood circulation. During early development rbp4 is expressed in the YSL of zebrafish [[18, 50], this study]. Because of peculiarities of embryonic development in fish and mammals, direct comparison of the YSL to the extraembryonic structures in mammals is difficult, but comparison of expression of developmental genes may help to solve this puzzle. Rbp4 is expressed in the extra-embryonic endoderm of the yolk sac during embryonic development of the rat, mouse and chick [15–17]. Interestingly, zebrafish rbp4 is expressed in the YSL on the surface of the yolk cell [[18, 50], this study], which, like the yolk sac in mammals, acts as a depot of maternal retinoids in zebrafish [4, 5]. These observations suggest functional similarity of these extraembryonic structures in mammals and fish. Further, as suggested by Thomas et al. , the YSL plays an important role in early embryonic patterning similar to that of the anterior visceral endoderm (AVE) in mouse embryos . Consistent with this, some murine genes homologous to Xenopus genes important in the organizer (e.g. hex, hesx1, lim1, otx2, cer-1, etc) are also expressed in the AVE ; interestingly, the zebrafish hhex is also expressed in the dorsal YSL in zebrafish during gastrula stage [45, 54].
Several early developmental genes, including sqt, cas and gata5, are expressed both in the YSL and adjacent vegetal blastomeres [55–58], but the specific roles of these genes in the YSL remain unknown since it is difficult to uncouple these functions from those in the marginal blastoderm. Now it is possible to target the YSL by injecting materials into the YSL only. It has been demonstrated that injections of RNAse into the YSL effectively eliminates YSL transcripts without affecting ubiquitously expressed genes in the blastoderm . By this approach, important information about early function of YSL in the formation of ventro-lateral mesoderm and induction of Nodal-related genes in the ventro-lateral marginal blastomeres has been obtained .
However, the specific function of genes expressed in the late YSL remains largely unknown. While a number of genes are expressed in the YSL ubiquitously (e.g. [38, 59]), zangptl2 is probably the only one with restricted posterior expression pattern in the YSL [29, 60]. Our current work on zebrafish rbp4 expression in the YSL provides another example illustrating patterning of the YSL. This non-uniform expression pattern suggests that at this stage the YSL could be patterned along the A-P and D-V axes. Consistent with this idea, the distinct morphogenetic domains in the YSL have been reported previously based on migration of its nuclei, though the underlying molecular mechanism remains unknown . The restricted expression of rbp4 in the YSL opens a question about mechanisms of such restriction, which we answered to some extent by demonstrating that the Nodal and Hh signalling pathways [62, 63] negatively control expression of rbp4 similar to that of ceruloplasmin . At the same time, RA seems to positively regulate expression of rbp4 in the YSL.
The effects of these signalling pathways on liver development require further experimental efforts. In our preliminary experiments, we noticed that both RA and DEAB led to no liver when the treatment was performed before but not after 16 hpf (data not shown). Although RA signalling could regulate rbp4 expression, it is difficult to conclude the involvement of Rbp4 in the RA signalling in regulation of liver development because of the pleitropic effect of RA. Nodal and Hh signalling pathways have been reported to play important roles in zebrafish endoderm development [64–71]. In our preliminary analyses using the Nodal and Hh mutants, most of them (cyc-/-, smu-/-, syu-/-, oep-/- and cas-/-) showed either smaller or no liver (data not shown). Only sqt-/- showed duplicated liver formation, which is probably due to its midline defect  as reported for another midline defect mutant flh-/- . However, liver bud duplication in Rbp4 morphant is unlikely due to the midline defect because the midline structure was remained normal in the Rbp4 morphant (Fig. 4G, H).
While it is widely accepted that the YSL plays a leading role during epiboly [73, 74], little information is available about the function of YSL after epiboly. It is not known whether the YSL at this stage influences the overlying cells just like that during early gastrulation or, alternatively, the embryo proper influences the patterning of the extraembryonic structures including the YSL. We discovered at least two different functions of Rbp4 within the YSL. First, Rbp4 deficiency results in abnormality of the YCE. Interestingly, rbp4 starts to be expressed in the YSL a few hours before the formation of YCE. While the exact molecular mechanism behind the YCE formation is not known, it has been suggested that the YCE formation is influenced by the posterior to anterior migration of cells between the yolk and EVL. These cells accumulate at the level of YCE and could be responsible for YCE formation .
Second, Rbp4 is involved in the formation of the liver bud. As Rbp4 is not expressed in the endoderm during this process, its contribution is probably indirect through its role in the YSL. During organogenesis different cell lineages migrate to establish anlage of various organs and differentiate thereafter. Recently it has been reported that the YSL-specific factor Mtx1 plays a role in migration of myocardial precursor cells and posterior endoderm, as knockdown of Mtx1 in the YSL results in cardia bifida due to a failure of myocardial cells to migrate to the midline. In parallel, 30% of Mtx1 morphants developed duplicated hepatic and pancreatic buds . Moreover, duplication of liver bud has been also observed in other studies. Ober et al  have reported that Vegfc is required for coalescence of anterior endoderm to the midline and knockdown of Vegfc results in formation of a forked gut tube and duplicated buds of liver and pancreas. Similarly, Matsui et al  have also reported a new role of non-canonical Wnt signalling during migration of precursor cells toward the midline. The down-regulation of Wnt/Dvl/RhoA signalling leads to the failure of fusion of the anterior gut tube and formation of duplicated livers and pancreas; in addition, migration of myocardial precursors toward the midline is also affected. In contrast to these observations, knockdown of Rbp4 has no effects on migration of heart precursors and the formation of the gut and pancreatic bud. Instead, the deficiency of Rbp4 causes a more limited effect resulting in formation of duplicated liver buds only. Consistent with this, we observed in the Rbp4 morphants that the cell migration molecule Fn1 is specifically reduced in the ventro-lateral region of the yolk, where the rbp4 is normally expressed, but not in the myocardia progenitors; Meanwhile, the ectopic expression of foxa3 appears specifically in the ventro-lateral region of the yolk. The ventro-lateral increase of foxa3 and decrease of fn1 suggested that the cells above the rbp4-expressing YSL are probable hepatocyte progenitors which will migrate from the ventro-lateral yolk toward the midline depending on a Rbp4-Fn1 signalling pathway. Thus, the effect of Rbp4 is limited probably only to hepatocyte progenitors.
Recently, two conflicting hypotheses of organogenesis of zebrafish visceral organs have been proposed. One emphasizes the formation of the endodermal rod by migration of endodermal cells towards the midline and budding of all major endodermal organs from the rod (, reviewed in ) while the other puts more weight on the establishment of independent primordia of these organs and their later assembly . Based on the data available, we suggest a unified theory of formation of endodermal organs; i.e. following the formation of the endodermal rod through convergence of the endodermal cells at the midline and the budding of organ primordia, there is continued cell migration from posterior to anterior and from lateral to medial, adding more cells to the buds of organs. Previously, based on analysis of expression pattern of ceruloplasmin in the wild type and mutant zebrafish, migration of liver progenitors from both sides of the yolk cell to the midline has been postulated during formation of the liver bud and a role of midline signaling in this process has been illustrated . Migration of liver progenitors is probably a part of a more general process of migration of endodermal cells that contributes also to organogenesis of the pancreas, interrenal gland and heart [14, 29, 41]. Thus cell migration during the late phase of formation of visceral organs seems to be rather common in zebrafish. Similarly, it has been shown in mice by cell fate mapping that there are different groups of liver precursor cells which migrate to form the liver bud . Our analysis of Rbp4 morphants demonstrated that on the surface of the yolk there are two spatially separated populations of liver precursor cells found on both sides of the midline as proposed earlier . It seems that these cells require an input from the YSL for proper migration. In the present study, we demonstrated that the YSL-expressed Rbp4 is necessary for migration of liver progenitors towards the midline and formation of a single liver bud.
Rbp4 is the extracellular transporter of retinol, a precursor of RA that has been implicated in regulation of cell migration as a stimulator and as an inhibitor of this process depending on the cellular context [77–79]. It stimulates neuronal migration in the zebrafish hindbrain . In view of these earlier observations, our current data suggest that retinoids could play a role in regulating migration of early hepatic cells during the process of liver formation. Given the fact that during the course of our analysis we only evaluated molecular markers and morphology of the heart, pancreas and liver, retinoids could be involved in regulation of cell migration during formation of some other visceral organs that were not analysed here. The developmental roles of Rbp4 in zebrafish revealed in this study should also be considered within a much more general context of metabolism of retinoids in extraembryonic structures that seems to be evolutionarily conserved in all vertebrates studied so far([4, 5], reviewed in ).