Byeong-Moo Kim

Hedgehog signaling controls mesenchymal growth in the developing mammalian digestive tract.

Homeostasis of the vertebrate digestive tract requires interactions between an endodermal epithelium and mesenchymal cells derived from the splanchnic mesoderm. Signaling between these two tissue layers is also crucial for patterning and growth of the developing gut. From early developmental stages, sonic hedgehog (Shh) and indian hedgehog (Ihh) are secreted by the endoderm of the mammalian gut, indicative of a developmental role. Further, misregulated hedgehog (Hh) signaling is implicated in both congenital defects and cancers arising from the gastrointestinal tract. In the mouse, only limited gastrointestinal anomalies arise following removal of either Shh or Ihh. However, given the considerable overlap in their endodermal expression domains, a functional redundancy between these signals might mask a more extensive role for Hh signaling in development of the mammalian gut. To address this possibility, we adopted a conditional approach to remove both Shh and Ihh functions from early mouse gut endoderm. Analysis of compound mutants indicates that continuous Hh signaling is dispensable for regional patterning of the gut tube, but is essential for growth of the underlying mesenchyme. Additional in vitro analysis, together with genetic gain-of-function studies, further demonstrate that Hh proteins act as paracrine mitogens to promote the expansion of adjacent mesenchymal progenitors, including those of the smooth muscle compartment. Together, these studies provide new insights into tissue interactions underlying mammalian gastrointestinal organogenesis and disease.

Independent functions and mechanisms for homeobox gene Barx1 in patterning mouse stomach and spleen.

Homeobox genes convey positional information in embryos and their role in patterning the mammalian gut is a topic of considerable interest. Barx1 is expressed selectively in fetal stomach mesenchyme and directs differentiation of overlying endoderm. Recombinant tissue cultures and study of young mouse embryos previously suggested that Barx1 controls expression of secreted Wnt antagonists, which suppress endodermal Wnt signaling, to enable stomach epithelial differentiation. We overcame mid-gestational lethality of Barx1-/- mouse embryos and report here the spectrum of anomalies in a distinctive and unprecedented model of gastrointestinal homeotic transformation. Using various mouse models, we confirm the importance of attenuated Wnt signaling in stomach development and the role of Barx1 in suppressing endodermal Wnt activity. Absence of Barx1 also results in fully penetrant defects in positioning and expansion of the spleen, an organ that originates within the mesothelial lining of the stomach. Barx1 is absent from the spleen primordium but highly expressed in the mesogastrium, indicating an indirect effect on spleen development. However, our results argue against a role for Wnt antagonism in genesis of the spleen. Mouse spleen development relies on several homeodomain transcriptional regulators that are expressed in the spleen primordium. Loss of Barx1 does not affect expression of any of these genes but notably reduces expression of Wt1, a transcription factor implicated in spleen morphogenesis and expressed in the mesothelium. These observations place Barx1 proximally within a Wt1 pathway of spleen development and reveal how a homeotic regulator employs different molecular mechanisms to mold neighboring organs.

Activation of nestin-positive duct stem (NPDS) cells in pancreas upon neogenic motivation and possible cytodifferentiation into insulin-secreting cells from NPDS cells.

Stem cells in adult pancreas and their specific marker are poorly characterized. We hypothesized that pancreatic stem cells could evolve from the duct system in response to neogenic stimulation and may transiently express nestin during tissue regeneration. After partial pancreatectomy (Px), we found extensive formation of ductules consisting of nestin‐positive epithelial cells with higher replicating ability in the neogenic foci, particularly at day 3 after Px. Nestin was highly expressed in the earlier stages of ductule morphogenesis and then regressed as the cells evolved toward differentiated pancreatic cell types. The neogenic ductules were isolated for the culture of nestin‐positive duct stem cells. These nestin‐positive duct cells were numerous and displayed extensive self‐replication in the duct cell explants after 2–3 days of culture, thus depicted as nestin‐positive duct stem (NPDS) cells. As seen in the tissue of neogenic foci, NPDS cells were negative for cytokeratin‐20 and vimentin, the marker for duct epithelial and mesenchymal cells, respectively. Endocrine cells, mostly insulin cells, were present in the explants at day 2 as single cells or as small clusters adjacent to the NPDS cells, and formed islet‐like masses at day 3 of culture, suggesting islet cell differentiation from NPDS cells. In addition, insulin secretion from these beta cells responded to glucose stimulation. We found transient up‐regulation of PDX‐1 expression by reverse transcriptase‐polymerase chain reaction at day 3 after Px in pancreatic tissue. Higher expression of PDX‐1 was seen in the culture of neogenic ductules than that of ducts isolated from the sham‐operated pancreas. In particular, a subpopulation of nestin‐positive cells in the duct cell explants formed from the neogenic ductules expressed PDX‐1 in their nuclei. Taken together, this information suggests that NPDS cells could be generated from adult pancreas by neogenic motivations and they may differentiate into insulin‐secreting cells. Developmental Dynamics 230:1–11, 2004.

Role of the Homeodomain Transcription Factor Bapx1 in Mouse Distal Stomach Development

BACKGROUND & AIMS Expansion and patterning of the endoderm generate a highly ordered, multiorgan digestive system in vertebrate animals. Among distal foregut derivatives, the gastric corpus, antrum, pylorus, and duodenum are distinct structures with sharp boundaries. Some homeodomain transcription factors expressed in gut mesenchyme convey positional information required for anterior-posterior patterning of the digestive tract. Barx1, in particular, controls stomach differentiation and morphogenesis. The Nirenberg and Kim homeobox gene Bapx1 (Nkx3-2) has an established role in skeletal development, but its function in the mammalian gut is less clear. METHODS We generated a Bapx1(Cre) knock-in allele to fate map Bapx1-expressing cells and evaluate its function in gastrointestinal development. RESULTS Bapx1-expressing cells populate the gut mesenchyme with a rostral boundary in the hindstomach near the junction of the gastric corpus and antrum. Smooth muscle differentiation and distribution of early regional markers are ostensibly normal in Bapx1(Cre/Cre) gut, but there are distinctive morphologic abnormalities near this rostral Bapx1 domain: the antral segment of the stomach is markedly shortened, and the pyloric constriction is lost. Comparison of expression domains and examination of stomach phenotypes in single and compound Barx1 and Bapx1 mutant mice suggests a hierarchy between these 2 factors; Bapx1 expression is lost in the absence of Barx1. CONCLUSIONS This study reveals the nonredundant requirement for Bapx1 in distal stomach development, places it within a Barx1-dependent pathway, and illustrates the pervasive influence of gut mesenchyme homeobox genes on endoderm differentiation and digestive organogenesis.

Clusterin expression during regeneration of pancreatic islet cells in streptozotocin-induced diabetic rats

Abstract.Aims/hypothesis: Beta-cell regeneration has been reported after islet injury in an animal model for diabetes. Recently, we showed up-regulation of clusterin after islet injury and suggested that clusterin might be involved in cytoprotection and in the regeneration of islet cells. The aim of this study was to investigate the correlation of clusterin expression with islet regeneration and its effect on islet cell replication. Methods: Streptozotocin was administrated to rats to induce various types of diabetes. Islet regeneration and clusterin expression were examined after islet injuries. Clusterin cDNA was transfected to MIN6 cells and their proliferation activity was measured by a [3H]thymidine-incorporation assay. Results: A diabetogenic dose of streptozotocin injected in rats provoked an immediate degeneration of beta cells. In this model, islets showed increased clusterin expression with extensive proliferation of alpha cells but showed poor beta-cell replication. A subdiabetogenic dose of streptozotocin, however, led to the proliferation of beta cells with clusterin up-regulation. In streptozotocin-treated neonatal rats, up-regulation of clusterin was noted during beta-cell proliferation. In all experimental models, clusterin was expressed in alpha cells in close correlation with islet cell proliferation, higher transcription of insulin mRNA and MAPKs activation. Cell replication was increased by 31 % in the MIN6 cells transfected by the clusterin cDNA. Conclusion/interpretation: Up-regulation of clusterin in alpha cells might induce beta-cell proliferation and thus restore their population after islet injury. We suggest that clusterin could be considered as a growth factor-like molecule stimulating islet-cell proliferation by paracrine action. [Diabetologia (2001) 44: 2192–2202]

Clusterin expression in the early process of pancreas regeneration in the pancreatectomized rat

We have previously reported upregulation of clusterin at the time of islet cell regeneration after β-cell injury. This led us to speculate that clusterin might be involved in the neogenic regeneration of the pancreas. Clusterin expression was examined throughout the process of pancreatic neogenesis in pancreatectomized rats. For in vitro analysis, duct cells were isolated from the rat pancreas and clusterin cDNA was transfected for its overexpression. Clusterin and its mRNA increased significantly in the early phase of regeneration, particularly at 1–3 days after pancreatectomy. Clusterin was transiently expressed in the differentiating acinar cells but faded afterwards. Interestingly, these clusterin cells were negative for PCNA (proliferating cell nuclear antigen), whereas most epithelial cells in ductules in the regenerating tissue showed extensive proliferative activity. Clusterin expression was also detected in some endocrine cells of the regenerating tissue. Transfection of clusterin cDNA into primary cultured duct cells resulted in a 2.5-fold increase in cell proliferation and induced transformation of non-differentiated duct cells into differentiated cells displaying cytokeratin immunoreactivity. Taken together, these results suggest that clusterin may play essential roles in the neogenic regeneration of pancreatic tissue by stimulating proliferation and differentiation of duct cells.

Clusterin induces differentiation of pancreatic duct cells into insulin-secreting cells

Aims/hypothesisWe recently reported that expression of the gene encoding clusterin (Clu) is upregulated in the regenerating pancreas, particularly in tissues undergoing differentiation. This led us to propose that clusterin participates in the cytodifferentiation of pancreatic tissue, particularly the endocrine islet cells. The aim of this study was to investigate whether clusterin induces the differentiation of duct-lining cells into insulin-secreting cells.MethodsWe isolated ductal tissue from rat pancreas and cultured it to develop epithelial cell explants for transfection of the Clu cDNA as well as for treatment of clusterin protein.ResultsThe number of newly differentiated insulin cells increased 6.9-fold upon Clu overexpression compared with controls. Ins1 mRNA and peptide levels were also increased. Furthermore, glucose-stimulated insulin secretion was observed in the differentiated insulin cells. These cells were immunoreactive for insulin and C-peptide, but negative for other islet hormones and for cytokeratin-20, which indicates a fully differentiated state. Insulin cell differentiation was also increased in a dose-dependent manner by treating duct cells in culture with clusterin, indicating a growth-factor-like action of clusterin in insulin cell differentiation.Conclusions/interpretationThese results suggest that clusterin can be considered as a potential morphogenic factor that promotes differentiation of pancreatic beta cells.

Barx1-Mediated Inhibition of Wnt Signaling in the Mouse Thoracic Foregut Controls Tracheo-Esophageal Septation and Epithelial Differentiation

Mesenchymal cells underlying the definitive endoderm in vertebrate animals play a vital role in digestive and respiratory organogenesis. Although several signaling pathways are implicated in foregut patterning and morphogenesis, and despite the clinical importance of congenital tracheal and esophageal malformations in humans, understanding of molecular mechanisms that allow a single tube to separate correctly into the trachea and esophagus is incomplete. The homoebox gene Barx1 is highly expressed in prospective stomach mesenchyme and required to specify this organ. We observed lower Barx1 expression extending contiguously from the proximal stomach domain, along the dorsal anterior foregut mesenchyme and in mesenchymal cells between the nascent esophagus and trachea. This expression pattern exactly mirrors the decline in Wnt signaling activity in late development of the adjacent dorsal foregut endoderm and medial mainstem bronchi. The hypopharynx in Barx1−/− mouse embryos is abnormally elongated and the point of esophago-tracheal separation shows marked caudal displacement, resulting in a common foregut tube that is similar to human congenital tracheo-esophageal fistula and explains neonatal lethality. Moreover, the Barx1−/− esophagus displays molecular and cytologic features of respiratory endoderm, phenocopying abnormalities observed in mouse embryos with activated ß-catenin. The zone of canonical Wnt signaling is abnormally prolonged and expanded in the proximal Barx1−/− foregut. Thus, as in the developing stomach, but distinct from the spleen, Barx1 control of thoracic foregut specification and tracheo-esophageal septation is tightly associated with down-regulation of adjacent Wnt pathway activity.

Clusterin enhances proliferation of primary astrocytes through extracellular signal-regulated kinase activation.

Clusterin, a secretory glycoprotein, has been shown to be up-regulated in the reactive astrocytes in response to brain injury and neurodegenerative diseases, but its function has not been clearly elucidated. In this study, we investigate whether clusterin has growth-stimulatory activity in astrocytes. Suppression of clusterin with antisense oligonucleotide induced growth arrest, whereas transient overexpression of clusterin by cDNA transfection or exogenous treatment with purified clusterin promoted proliferation of the primary astrocytes in culture. This clusterin-stimulated proliferation was abrogated by PD98059, an inhibitor of mitogen-activated protein kinase kinase. These results suggest that clusterin might play an important role in astrogliosis by stimulating the proliferation of astrocytes through activation of the extracellular signal-regulated kinase 1/2 signaling pathway.

Endodermal Hedgehog signals modulate Notch pathway activity in the developing digestive tract mesenchyme.

The digestive tract epithelium and its adjoining mesenchyme undergo coordinated patterning and growth during development. The signals they exchange in the process are not fully characterized but include ligands of the Hedgehog (Hh) family, which originate in the epithelium and are necessary for mesenchymal cells to expand in number and drive elongation of the developing gut tube. The Notch signaling pathway has known requirements in fetal and adult intestinal epithelial progenitors. We detected Notch pathway activity in the embryonic gut mesenchyme and used conditional knockout mice to study its function. Selective disruption of the Notch effector gene RBP-Jκ (Rbpj) in the mesenchyme caused progressive loss of subepithelial fibroblasts and abbreviated gut length, revealing an unexpected requirement in this compartment. Surprisingly, constitutive Notch activity also induced rapid mesenchymal cell loss and impaired organogenesis, probably resulting from increased cell death and suggesting the need for a delicate balance in Notch signaling. Because digestive tract anomalies in mouse embryos with excess Notch activity phenocopy the absence of Hh signaling, we postulated that endodermal Hh restrains mesenchymal Notch pathway activity. Indeed, Hh-deficient embryos showed Notch overactivity in their defective gut mesenchyme and exposure to recombinant sonic hedgehog could override Notch-induced death of cultured fetal gut mesenchymal cells. These results reveal unexpected interactions between prominent signals in gastrointestinal development and provide a coherent explanation for Hh requirements in mesenchymal cell survival and organ growth.