Haploinsufficiency of the Mouse Forkhead Box f1 Gene Causes Defects in Gall Bladder Development

Abstract

The forkhead box f1 (Foxf1) transcription factor is expressed in the visceral (splanchnic) mesoderm, which is involved in mesenchymal-epithelial signaling required for development of organs derived from foregut endoderm such as lung, liver, gall bladder, and pancreas. Our previous studies demonstrated that haploinsufficiency of the Foxf1 gene caused pulmonary abnormalities with perinatal lethality from lung hemorrhage in a subset of Foxf1؉/؊ newborn mice. During mouse embryonic development, the liver and biliary primordium emerges from the foregut endoderm, invades the septum transversum mesenchyme, and receives inductive signaling originating from both the septum transversum and cardiac mesenchyme. In this study, we show that Foxf1 is expressed in embryonic septum transversum and gall bladder mesenchyme. Foxf1؉/؊ gall bladders were significantly smaller and had severe structural abnormalities characterized by a deficient external smooth muscle cell layer, reduction in mesenchymal cell number, and in some cases, lack of a discernible biliary epithelial cell layer. This Foxf1؉/؊ phenotype correlates with decreased expression of vascular cell adhesion molecule-1 (VCAM-1), ␣ 5 integrin, plateletderived growth factor receptor ␣ (PDGFR␣) and hepatocyte growth factor (HGF) genes, all of which are critical for cell adhesion, migration, and mesenchymal cell differentiation.At 9-days postcoitum (dpc) 1 during mouse embryogenesis, the liver primordium emerges from the foregut endoderm, invades the septum transversum mesenchyme, and receives bone morphogenetic protein 4 (Bmp-4) and fibroblast growth factor 2 (Fgf2) signaling originating from the septum transversum and cardiac mesenchyme, respectively (1, 2). This hepatic specification is associated with expression of the Foxa2 (HNF-3␤) and Gata4 transcription factors (3). Liver morphogenesis involves a proliferative expansion period and development of the bipotential hepatoblasts that begin to differentiate into hepatocytes and bile duct epithelial cells of the intrahepatic bile ducts (IHBD) at 13.5 dpc (3,4). Interestingly, targeted disruption of either the homeodomain Hex gene or the Hgf gene allows normal development of the mouse hepatic diverticulum, but these cells fail to migrate into the septum transversum and undergo liver morphogenesis (5-7).In the adult liver, bile is synthesized in hepatocytes from cholesterol, secreted into the bile canaliculi and transported through the intrahepatic and extrahepatic biliary system to the gall bladder, where it is stored for secretion into the digestive tract to emulsify lipids (8). The gall bladder and extrahepatic bile ducts (EHBD) develop from the caudal portion of the liver primordium at 10 dpc of mouse embryogenesis (9). However, little is known regarding visceral (splanchnic) mesoderm transcription factors that regulate expression of genes involved in mesenchymal-epithelial induction of gall bladder development from the liver primordium. Visceral mesenchymal expression of the homeodomain Hlx gene is required for the proli...

A familial case of alveolar capillary dysplasia with misalignment of pulmonary veins supports paternal imprinting of FOXF1 in human

“…15 The mouse Foxf1 and the human FOXF1 proteins act in a haploinsufficient manner. 15,[21][22][23] FOXF1 has been predicted bioinformatically to be paternally imprinted in the humans; 24 however, a paternal uniparental disomy of chromosome 16 (UPD16pat) resulted in an otherwise normal child with only some fetal and postnatal growth retardation. 25 Interestingly, all the genic and upstream deletions in our cohort of patients reported previously 15 occurred on the maternal chromosome 16, which suggested that FOXF1 might be paternally imprinted.…”

Myocardium defects and ventricular hypoplasia in mice homozygous null for the Forkhead Box m1 transcription factor

“…We also used mouse monoclonal antibodies against p21 Cip1 (1:200; SXM30; BD Biosciences), goat polyclonal antibodies against GATA-4 (1:100; C-20; Santa Cruz), and rat monoclonal antibodies against Pecam-1 (1:50; MEC 13.3, Pharmingen). Antibody-antigen complexes were detected using biotinylated secondary antibody, avidin-horseradish peroxidase (HRP) complex, and DAB substrate (all from Vector Labs) as described (Kalinichenko et al, 2002;Kim et al, 2005b). For immunofluorescent detection of phosphohistone H3 (PH3), we used rabbit polyclonal PH3 antibody (1:200; MC463; Upstate) followed by anti-rabbit antibody conjugated with FITC (Vector Labs) as described previously (Kalinichenko et al, 2003).…”

“…Total mouse heart RNA was prepared from 14.5-dpc hearts of WT or Foxm1 Ϫ/Ϫ embryos using RNA-STAT-60 (Tel-Test "B" Inc. Friendswood, TX) and then used for reverse transcriptase (RT)-PCR analysis as described (Kalinichenko et al, 2002). The following sense and antisense primers were used for amplification: laminin ␣4 (Lama4), 5Ј-ggatcccgactggtcattgatggtctacgag and 5Ј-gtcgaccgccttctgtggaaaaataagttc; Foxm1, 5Ј-ggatcctgccaccccagaccttgttc and 5Ј-gtcgactccctgatgcttttcgctgtc; Cdc25B, 5Ј-attccagctctgcccaagctttggc and 5Ј-tccacaaatccgtcatcttctca; Aurora B, 5Ј-ttgacaactttgagattggg and 5Ј-gctggtcgtagaagtagttgt; Plk-1, 5Ј-ctcctggagctgcacaagaggaggaa and 5Ј-tctgtctgaagcatcttctggatgag; Cyclin B1, 5Ј-atcggggaacctctgatttt and 5Ј-tcacacacaggcaccttctc; Skp2, 5Ј-gtatgttagggaaccatttgcgag and 5Ј-ttagaagggcacttggaagagtt; Cks1, 5Ј-gacctcaaagccctcgtgt and 5Ј-tgaaacataaatccataagtcatca; laminin ␣2 (Lama2), 5Ј-tgtcgtggggattctgtatgtc and 5Ј-caagaaggtccaatccaacttt; integrin ␤1, 5Ј-attggctttggctcatttgtgg and 5Ј-ccagcagtcgtgttacattcc; NFATc3, 5Ј-tggatctcagtatcctttaa and 5Ј-cacacgaaatacaagtcgga; NFATc4, 5Ј-cattggcactgcagatgag and 5Ј-cgtagctcaatgtctgaat; H11 Kinase, 5Ј-cttgcttggttgcgttaggt and 5Ј-tggtggtgatggtttgagag; 5-HT 2B receptor, 5Ј-ctcttttcaactgcctccatc and 5Ј-ccagcattgccaccttttc; calmodulin, 5Ј-tccgttcttccttcttcgctcgcaccatggc and 5Ј-gtgtgtggacagaggggcttctgacatcag; FoxO3a, 5Ј-gtcacactacggcaaccaga and 5Ј-cctgagagagagtccgagagg; cyclophilin, 5Ј-agctctgagcactggagagaaa and 5Ј-tcctgagctacagaaggaatgg.…”

“…Two different cDNA concentrations were used for RT-PCR reactions to ensure that RT-PCR conditions were in the linear range. Quantitation of expression levels was determined with Tiff files of ethidium bromide-stained gels by using the BioMax 1D program (Kodak) as described (Kalinichenko et al, 2002).…”

Foxf1 +/− mice exhibit defective stellate cell activation and abnormal liver regeneration following CCl4 injury

“…10 Liver development initiates at 9 days postcoitum (dpc) of mouse embryogenesis, when the cardiac mesenchyme induces the hepatic primordium to emerge from the foregut endoderm that invades the septum transversum mesenchyme. 6 Previous expression studies have shown that Foxf1 is expressed in the splanchnic mesoderm 11,12 and septum transversum mesenchyme, 13 the latter of which provides inductive signaling for the liver primordium. 14 Consistent with this expression pattern, Foxf1 ϩ/Ϫ mice exhibited severe defects in gallbladder mesenchyme formation.…”