Biomedical subjects

Marko E Horb

Publications and source records attributed to Marko E Horb.

9 recordsLinked to original sources

PubMed2006

The Kruppel-like transcription factor KLF13 is a novel regulator of heart development.

In humans, congenital heart defects occur in 1-2% of live birth, but the molecular mechanisms and causative genes remain unidentified in the majority of cases. We have uncovered a novel transcription pathway important for heart morphogenesis. We report that KLF13, a member of the Krüppel-like family of zinc-finger proteins, is expressed predominantly in the heart, binds evolutionarily conserved regulatory elements on cardiac promoters and activates cardiac transcription. KLF13 is conserved across species and knockdown of KLF13 in Xenopus embryos leads to atrial septal defects and hypotrabeculation similar to those observed in humans or mice with hypomorphic GATA-4 alleles. Physical and functional interaction with GATA-4, a dosage-sensitive cardiac regulator, provides a mechanistic explanation for KLF13 action in the heart. The data demonstrate that KLF13 is an important component of the transcription network required for heart development and suggest that KLF13 is a GATA-4 modifier; by analogy to other GATA-4 collaborators, mutations in KLF13 may be causative for congenital human heart disease.

Alleles↗

PubMed2005

Germ layers to organs: using Xenopus to study "later" development.

The amphibian embryo is a highly successful model system with great promise for organogenesis research. Since the late 1800s, amphibians have been employed to understand vertebrate development and since the 1950s, the African clawed frog Xenopus laevis has been the amphibian of choice. In the past two decades, Xenopus has led the way forward in, among other things, identifying transcription factors, gene regulatory networks and inter- and intracellular signaling pathways that control early development (from fertilization through gastrulation and neurulation). Perhaps the best measure of how successful Xenopus has been as a model for early mammalian development is the observation that much of the knowledge gleaned from Xenopus studies has subsequently directly translated to discoveries of similar mechanisms operating in mouse development. Despite this great success in early development, research on organogenesis in Xenopus has lagged behind the mouse. However, recent technical advances now make Xenopus amenable for studies on later development, including organogenesis. Here, we discuss why Xenopus is well suited for such research and, we believe, permits addressing questions that have been difficult to approach using other model systems. We also highlight how Xenopus researchers have already begun studying a number of major organs, pancreas, liver, kidney and heart, and suggest how Xenopus might contribute more to these areas in the near future.

Animals↗

PubMed2005

In vitro transdifferentiation of hepatoma cells into functional pancreatic cells.

We have characterised the transdifferentiation of human HepG2 (hepatoma) cells to pancreatic cells following introduction of an activated version of the pancreatic transcription factor Pdx1 (XlHbox8-VP16). The following questions are addressed: (1) are all types of pancreatic cells produced? (2) is the requirement for expression of the transgene temporary or permanent? (3) are the transdifferentiated beta-cells responsive to physiological stimuli? The results showed that both pancreatic exocrine cells (by detection of amylase protein), and endocrine cells (by detecting insulin, glucagon and somatostatin proteins) are induced after XlHbox8VP16 transfection. Moreover, the hepatic phenotype becomes suppressed during transdifferentiation of hepatocytes to pancreatic cells. Requirement for the transgene is only temporary and it is no longer required once the pancreatic differentiation program is activated. Finally, we provided results to suggest that the transdifferentiated cells are functional by detecting: (1) functional markers for pancreatic beta-cells including prohormone convertase 1/3 (PC1/3), insulin C-peptide and glucagon-like peptide 1 receptor (GLP-1R), (2) increased insulin mRNA expression after treatment of cells with GLP-1 and betacellulin, physiological stimuli that regulate pancreatic function and (3) elevated insulin secretion after glucose challenge. The transdifferentiation of hepatic to pancreatic cells represents one possible source of beta-cells for human islet transplantation and this study shows that such a transdifferentiation can be achieved in vitro.

Active Transport, Cell Nucleus↗

PubMed2004

Differential requirement for ptf1a in endocrine and exocrine lineages of developing zebrafish pancreas.

Mammalian studies have implicated important roles for the basic helix-loop-helix transcription factor Ptf1a-p48 in the development of both exocrine and endocrine pancreas. We have cloned the Ptf1a-p48 ortholog in Danio rerio. Early zebrafish ptf1a expression is observed in developing hindbrain and in endodermal pancreatic precursors. Analysis of ptf1a and insulin expression reveals a population of exocrine precursors that, throughout early development, are temporally and spatially segregated from endocrine elements. Morpholino-mediated knockdown of ptf1a confirms early divergence of these endocrine and exocrine lineages. Ptf1a morphants lack differentiated exocrine pancreas, but maintain normal differentiation and organization of the principal islet. In addition to the exocrine phenotype, ptf1a knockdown also reduces the prevalence of a small population of anterior endocrine cells normally found outside the principal islet. Together, these findings suggest the presence of distinct ptf1a-dependent and ptf1a-independent precursor populations in developing zebrafish pancreas.

Amino Acid Sequence↗

PubMed2004

Differential requirement for ptf1a in endocrine and exocrine lineages of developing zebrafish pancreas.

Amino Acid Sequence↗

PubMed2004

High glucose is necessary for complete maturation of Pdx1-VP16-expressing hepatic cells into functional insulin-producing cells.

Pdx1 has been shown to convert hepatocytes into both exocrine and endocrine pancreatic cells in mice, but it fails to selectively convert hepatocytes into pure insulin-producing cells (IPCs). The molecular mechanisms underlying the transdifferentiation remain unclear. In this study, we generated a stably transfected rat hepatic cell line named WB-1 that expresses an active form of Pdx1 along with a reporter gene, RIP-eGFP. Our results demonstrate that Pdx1 induces the expression of multiple genes related to endocrine pancreas development and islet function in these liver cells. We do not however find any expression of the late-stage genes (Pax4, Pax6, Isl-1, and MafA) related to beta-cell development, and the cells do not secrete insulin upon the glucose challenge. Yet when WB-1 cells are transplanted into diabetic NOD-scid mice, these genes become activated and hyperglycemia is completely reversed. Detailed comparison of gene expression profiles between pre- and posttransplanted WB-1 cells demonstrates that the WB-1 cells have similar properties as that seen in pancreatic beta-cells. In addition, in vitro culture in high-glucose medium is sufficient to induce complete maturation of WB-1 cells into functional IPCs. In summary, we find that Pdx1-VP16 is able to selectively convert hepatic cells into pancreatic endocrine precursor cells. However, complete transdifferentiation into functional IPCs requires additional external factors, including high glucose or hyperglycemia. Thus, transdifferentiation of hepatocytes into functional IPCs may serve as a viable therapeutic option for patients with type 1 diabetes.

Animals↗

PubMed2003

Experimental conversion of liver to pancreas.

BACKGROUND: The liver and the pancreas arise from adjacent regions of endoderm in embryonic development. Pdx1 is a key transcription factor that is essential for the development of the pancreas and is not expressed in the liver. The aim of this study was to determine whether a gene overexpression protocol based on Pdx1 would be able to cause conversion of liver to pancreas. RESULTS: We show that a modified form of Pdx1, carrying the VP16 transcriptional activation domain, can cause conversion of liver to pancreas, both in vivo and in vitro. Transgenic Xenopus tadpoles carrying the construct TTR-Xlhbox8-VP16:Elas-GFP were prepared. Xlhbox8 is the Xenopus homolog of Pdx1, the TTR (transthyretin) promoter directs expression to the liver, and the GFP is under the control of an elastase promoter and provides a real-time visible marker of pancreatic differentiation. In the transgenic tadpoles, part or all of the liver is converted to pancreas, containing both exocrine and endocrine cells, while liver differentiation products are lost from the regions converted to pancreas. The timing of events is such that the liver is differentiating by the time Xlhbox8-VP16 is expressed, so we consider this a transdifferentiation event rather than a reprogramming of embryonic development. Furthermore, this same construct will bring about transdifferentiation of human hepatocytes in culture, with formation of both exocrine and endocrine cells. CONCLUSIONS: We consider that the conversion of liver to pancreas could be the basis of a new type of therapy for insulin-dependent diabetes. Although expression of the transgene is transient, once the ectopic pancreas is established, it persists thereafter.

Animals↗

PubMed2003

Transdifferentiation of pancreas to liver.

Transdifferentiation is the name used to describe the direct conversion of one differentiated cell type into another. Cells which have the potential to interconvert by transdifferentiation generally arise from adjacent regions in the developing embryo. For example, the liver and pancreas arise from the same region of the endoderm. The transdifferentiation of pancreas to liver (and vice versa) has been observed in animal experiments and in certain human pathologies. Understanding transdifferentiation is important to developmental biologists because it will help elucidate the cellular and molecular differences that distinguish neighbouring regions of the embryo. While the in vivo models for the transdifferentiation of liver to pancreas have been valuable, it is more difficult to extrapolate from these studies to individual changes at the cellular or molecular levels. The recent development of two in vitro systems (AR42J cells and embryonic pancreatic cultures) for the transdifferentiation of pancreas to liver has shown that an environmental change in the form of an exogenous glucocorticoid can cause the conversion of pancreatic exocrine cells into hepatocytes. The AR42J cell system has been used to elucidate the cell lineage and the molecular basis of transdifferentiation of pancreas to liver.

Animals↗

PubMed2002

Expression of amylase and other pancreatic genes in Xenopus.

To better understand the relationship between the endocrine and exocrine cell types in the Xenopus pancreas, we have cloned the Xenopus amylase cDNA and compared its expression profile with that of four other pancreatic markers: insulin, glucagon, elastase and trypsinogen. Our results demonstrate that the first pancreatic marker to be expressed is insulin, exclusively in the dorsal pancreas. These insulin-expressing cells form small groups which resemble islets, but no insulin is detected in the ventral pancreas until stage 47. In contrast, the exocrine markers, amylase, elastase and trypsinogen are first expressed only in the ventral pancreas beginning at stage 41; by stage 45 their expression extends into the dorsal pancreas. Glucagon, on the other hand, is not expressed in the pancreas until stage 45. In the endocrine cell clusters we do not find glucagon-expressing cells surrounding insulin-expressing cells, either in the tadpole or in the mature frog pancreas.

Amino Acid Sequence↗