Liver Bud Morphogenesis
| StemBook |
| This research on liver development was published on StemBook by Dr. Zorn and his laboratory. |
| Table of Contents |
|
|
Figure 7. Liver bud morphogenesis. |
| The schematics depict transverse sections through the gut tube at the level of the liver diverticulum at different developmental stages in liver bud formation. After hepatic specification by FGF and BMP signals from the heart and septum transversum mesenchyme (STM) the hepatoblasts begin to express liver markers, the hepatic epithelium thickens then and transitions from a columnar to a pseudostratified epithelium (e8.75-e9.0). At this stage the hepatic epithelium is embedded in the STM and surrounded by a laminin rich basement membrane and endothelial cell precursors. By e9.5 the basal lamina breaks down and hepatoblasts delaminate and migrate into the STM to form the liver bud. Signals from the endothelial cells, the STM, as well as the activity of the indicated genes are required for this process. |
Figure 8. An example of hepatoblast delamination.
|
| (A) An X-gal stained e9.5 double transgenic Foxa3cre;Rosa26-reporter embryo expresses cre-recombinase in the endoderm (Lee et al., 2005), which activates the Rosa26-reporter allele resulting in permanent β-galactosidase expression in the gut epithelium and liver diverticulum (l.d.). (B) A transverse section through the liver diverticulum of the embryo in (A) stained with anti-HNF4α antibodies, shows the endoderm in blue and the HNF4α-expressing hepatoblasts invading the STM. |
Hhex and Gata | Prox1, Onecut and the ECM | Endothelial Signals
Shortly after hepatic specification (e8.5 to e9.0) the epithelium begins to express liver genes (Albumin, Afp, Hnf4α) and thickens as the cells transition from a simple cuboidal to a pseudostratified columnar epithelium, thus forming the liver diverticulum (Bort et al., 2006). Between e9.0 to e9.5 the laminin-rich basal layer surrounding the hepatic endoderm breaks down, and the hepatoblasts delaminate and migrate into the STM to form the nascent liver bud (see Fig. 7 and Fig. 8; Bort et al., 2006; Houssaint, 1980; Margagliotti et al., 2008; Medlock and Haar, 1983; Shiojiri and Sugiyama, 2004). Several transcription factors, as well as signals from endothelial cells are required for this process.
Return to top.
Hhex and Gata
Initially expressed throughout the ventral foregut endoderm, the homeodomain factor Hhex becomes enriched in the hepatic endoderm by e8.5 (see Fig. 6) and then persists in all the hepato-biliary cell lineages throughout development (Bogue et al., 2000; Hunter et al., 2007; Thomas et al., 1998). Hhex-/- embryos lack liver, gall bladder and ventral pancreas buds at e10.5 (Keng et al., 2000; Martinez Barbera et al., 2000) and studies indicate that Hhex function is required at multiple time points in the development of these lineages (Bort et al., 2004; Hunter et al., 2007; sections 4, 7 and 8). In addition to controlling foregut endoderm proliferation, Hhex is required for hepatoblast delamination. In e9.0 Hhex-/- embryos the hepatic endoderm transiently expresses liver genes, but the epithelium arrests in a simple columnar state, the hepatoblasts fail to invade the STM (Bort et al., 2006).
The zinc finger transcription factors Gata4 and Gata6 are expressed in a variety of tissues including the foregut endoderm, heart and STM. Mutations in either Gata4 or Gata6 result in early lethality due to defects in the extra embryonic tissue (Duncan, 2005). However in tetraploid chimeric embryos where only the epiblast was derived from Gata4-/- or Gata6-/- ES cells, hepatic development arrested at ∼e9.5 (Watt et al., 2007; Zhao et al., 2005). Similar to the Hhex mutants, hepatic gene expression was initiated but not maintained and the hepatoblasts failed to delaminate. Gata4-/- embryos also lack STM suggesting that part of the defect could be non-cell autonomous (Watt et al., 2007). Compound Gata4-/-; Gata6-/- mutants have early defects in foregut morphogenesis but the liver phenotype has not yet been reported in these embryos (Zhao et al., 2008). The proposed role for Gata factors in hepatic competence (section 3.2) predicts that liver development should not initiate in the compound mutants, which is consistent with the antisense depletion of Gata4, Gata5 and Gata6 in zebrafish and Xenopus embryos (Afouda et al., 2005; Holtzinger and Evans, 2005; Shin et al., 2007).
Return to top.
Prox1, Onecut and the ECM
The homeodomain transcription factors Prox1, Onecut-1 (OC-1, also known as Hnf6) and Onecut-2 (OC-2) also regulate hepatoblast delamination, but act slightly later than Hhex and Gata. Prox1 is expressed in the hepatic epithelium by e8.5 (Burke and Oliver, 2002). In Prox1-/- embryos hepatoblasts are specified and begin to proliferate, but the basal lamina fails to degrade and the cells remain trapped in the hepatic diverticulum (Sosa-Pineda et al., 2000). OC-1 and OC-2 are expressed in the foregut endoderm and hepatoblasts, and are redundantly required for the timely degradation of the basal lamina (Margagliotti et al., 2007). At e9.5, OC-1;OC-2 double mutants resemble the Prox1-/- phenotype, but later hepatoblast invasion recovers resulting in a hypoplastic fetal liver. These mutant livers also have bile duct and gall bladder defects due to later roles of OC-1 and OC-2 (sections 7.2 & 8).
Hepatoblasts normally down regulate E-cadherin as they migrate into the STM, however this does not occur properly in Prox1 and OC1;OC2 mutants. Studies suggest that Prox1 and Onecut factors control hepatoblast migration by regulating the expression of extra cellular matrix (ECM) proteins and ECM remodeling enzymes such as matrix metalloproteinases (MMPs; Margagliotti et al., 2007; Medico et al., 2001; Papoutsi et al., 2007). Hepatoblasts and the STM express several MMPs at e9.5 and pharmacological inhibition of MMP activity suppresses hepatoblast migration in culture (Margagliotti et al., 2008). The importance of cell-ECM interactions is also illustrated by the fact that hepatoblasts deficient for the laminin receptor β1-integrin are unable to colonize the liver bud (Fassler and Meyer, 1995). Small GTPases, well known for regulating cell migration, also appear to be involved because hepatoblasts fail to invade the STM in embryos with null mutations in the Pccmt gene, which encodes a GTPase modifying enzyme (Lin et al., 2002).
Return to top.
Endothelial Signals
At e9.0, prior to vascularization of the liver bud, endothelial precursor cells lay between the hepatic epithelium and the STM (see Fig. 7). This close contact with blood vessels persists as hepatoblasts migrate into the stroma. Null mutations in the vascular endothelial growth factor receptor gene Vegfr-2 (also known as Flk-1) results in embryos that lack endothelial cells and the hepatoblasts fail to delaminate in these embryos (Matsumoto et al., 2001). In addition angiogenesis inhibitors repress liver bud growth in culture, suggesting that endothelial cells provide unknown paracrine factors promoting hepatoblast migration and/or proliferation. One candidate that has emerged from chick embryo studies is the Glial-derived neurotrophic factor, Neurturin, which is secreted from blood vessels and acts as a hepatoblast chemoattractant via the GFRα2 receptor (Tatsumi et al., 2007).
