FGF signaling facilitates postinjury recovery of mouse hematopoietic system.

Previous studies have shown that fibroblast growth factor (FGF) signaling promotes hematopoietic stem and progenitor cell (HSPC) expansion in vitro. However, it is unknown whether FGF promotes HSPC expansion in vivo. Here we examined FGF receptor 1 (FGFR1) expression and investigated its in vivo function in HSPCs. Conditional knockout (CKO) of Fgfr1 did not affect phenotypical number of HSPCs and homeostatic hematopoiesis, but led to a reduced engraftment only in the secondary transplantation. When treated with 5-fluorouracil (5FU), the Fgfr1 CKO mice showed defects in both proliferation and subsequent mobilization of HSPCs. We identified megakaryocytes (Mks) as a major resource for FGF production, and further discovered a novel mechanism by which Mks underwent FGF-FGFR signaling dependent expansion to accelerate rapid FGF production under stress. Within HSPCs, we observed an up-regulation of nuclear factor κB and CXCR4, a receptor for the chemoattractant SDF-1, in response to bone marrow damage only in control but not in Fgfr1 CKO model, accounting for the corresponding defects in proliferation and migration of HSPCs. This study provides the first in vivo evidence that FGF signaling facilitates postinjury recovery of the mouse hematopoietic system by promoting proliferation and facilitating mobilization of HSPCs.

Interplay of glucagon-like peptide-1 and transforming growth factor-beta signaling in insulin-positive differentiation of AR42J cells.

The differentiation of pancreatic exocrine AR42J cells into insulin-expressing endocrine cells has served as an important model for both endogenous in vivo beta-cell differentiation as well as potential application to beta-cell engineering of progenitor cells. Exogenous activin, possibly working through intracellular smad 2 and/or smad 3, as well as exogenous exendin-4 (a long-acting glucagon-like peptide-1 agonist) have both been shown to induce insulin-positive/endocrine differentiation in AR42J cells. In this study, we present evidence of significant interplay and interdependence of these two pathways as well as potential synergy between the pathways. In particular, insulin-positive differentiation seems to entail an exendin-4-induced drop in smad 2 and elevation in smad 3 in RNA levels. The latter appears to be dependent on endogenous transforming growth factor (TGF)-beta isoform release by the AR42J cells and may serve as a mechanism to promote beta-cell maturation. The drop in smad 2 may mediate early endocrine commitment. The coapplication of exogenous exendin-4 and, specifically, low-dose exogenous TGF-beta1 led to a dramatic 20-fold increase in insulin mRNA levels, supporting a novel synergistic and codependent relationship between exendin-4 signaling and TGF-beta isoform signaling.

Defective sonic hedgehog signaling in esophageal atresia with tracheoesophageal fistula.

BACKGROUND: The pathogenesis of esophageal atresia and tracheoesophageal fistula (EA/TEF) remains unknown. We have found previously that an initial esophageal atresia, followed by an abnormal (absent) branching pattern of the middle branch of a trifurcation of the lung/tracheal bud, leads to the neonatal finding of TEF. Mice null mutant for hedgehog signaling can experience the development of EA/TEF, but the mechanism for this development is also unknown. Given that EA/TEF in humans appears not to be due to genetic defects, a hedgehog mutation cause seems very unlikely. However, defective hedgehog signaling that is caused by environmental effects in the human embryo likely could be implicated. We studied a teratogen-induced model of EA/TEF to determine the mechanism by which defective hedgehog signaling may lead to EA/TEF. METHODS: We injected Adriamycin into pregnant rats to induce EA/TEF in rat embryos. We first quantified sonic hedgehog (Shh) signaling pathway molecule expression using real-time, semiquantitative reverse-transcriptase polymerase chain reaction for Shh, Shh receptors (patched and smoothened), and downstream intracellular targets of those receptors (Gli family members). On the basis of these findings, we then developed an in vitro culture system for the day-12 embryonic TEF and manipulated Shh signaling using either exogenous Shh or Shh inhibitors. RESULTS: By reverse transcriptase-polymerase chain reaction, a unique difference between the fistula tract and control tissues was that Gli-2 (downstream signaling molecule of Shh) messenger RNA levels were much lower in the fistula tract than in the adjacent esophagus (P =.002). Surprisingly, in the culture experiments, the fistula tract was induced to branch by exogenous Shh. Such branching of the fistula was unexpected and further supports the presumed respiratory origin of the fistula tract because the normal lung, but not normal esophagus, branched in response to Shh. The Shh inhibitor had no effect, which indicated that defective signaling, rather than hyperfunctioning Shh, is critical to the nonbranching phenotype of the fistula tract in TEF. CONCLUSIONS: The recapitulation of respiratory developmental morphogenesis by the fistula tract of TEF in the presence of exogenous Shh, together with the quantitative reduction in normal, endogenous levels of Gli-2, strongly suggests that 1 mechanism for the formation of the fistula tract is the lack of proper Shh signaling because of Gli-2 deficiency, with subsequent straight, nonbranching caudal growth of the fistula tract. This deficiency can be rescued by excess exogenous Shh, thus reestablishing respiratory morphogenesis.

N-cadherin expression level distinguishes reserved versus primed states of hematopoietic stem cells.

Osteoblasts expressing the homophilic adhesion molecule N-cadherin form a hematopoietic stem cell (HSC) niche. Therefore, we examined how N-cadherin expression in HSCs relates to their function. We found that bone marrow (BM) cells highly expressing N-cadherin (N-cadherin(hi)) are not stem cells, being largely devoid of a Lineage(-)Sca1(+)cKit(+) population and unable to reconstitute hematopoietic lineages in irradiated recipient mice. Instead, long-term HSCs form distinct populations expressing N-cadherin at intermediate (N-cadherin(int)) or low (N-cadherin(lo)) levels. The minority N-cadherin(lo) population can robustly reconstitute the hematopoietic system, express genes that may prime them to mobilize, and predominate among HSCs mobilized from BM to spleen. The larger N-cadherin(int) population performs poorly in reconstitution assays when freshly isolated but improves in response to overnight in vitro culture. Their expression profile and lower cell-cycle entry rate suggest N-cadherin(int) cells are being held in reserve. Thus, differential N-cadherin expression reflects functional distinctions between two HSC subpopulations.

Bone morphogenetic protein expression patterns in human esophageal atresia with tracheoesophageal fistula.

The organogenesis of esophageal atresia with tracheoesophageal fistula (EA/TEF) remains unknown. The fistula tract appears to develop from a non-branching trifurcation of the embryonic lung bud. The non-branching growth of the fistula differs from the other lung buds and suggests a deficiency in bone morphogenetic protein (BMP) signaling, since BMPs are critical to proper lung development and branching. With IRB approval, portions of newborn human proximal esophageal pouch and distal fistula samples were recovered at the time of surgical repair of EA/TEF. The tissues were processed for immunohistochemistry. Commercially available fetal tissues were used as controls. In control tissues, BMP ligands (BMP 2, 4, and 7) were all present in the esophagus but absent in the trachea. BMPRIA was absent in both tissues. BMPRIB was detected in trachea but not in esophagus and BMPRII was detected in esophagus but not in trachea. In the EA/TEF specimens, all BMP ligands were present in the proximal esophageal pouch but absent in the fistula tract. BMPRIA and BMPRIB were not detected in either tissue. However, BMPRII was found in both fistula tract and proximal pouch. The submucosa of the fistula appears to maintain a mixed (identical neither to lung, esophagus, or trachea) BMP signaling pattern, providing one mechanism which could potentially explain the esophageal dismotility and lack of lung branching seen in the fistula/distal esophagus.

Faulty bone morphogenetic protein signaling in esophageal atresia with tracheoesophageal fistula.

BACKGROUND: The organogenesis of esophageal atresia with tracheoesophageal fistula remains unclear. We have previously demonstrated that the fistula tract develops from a trifurcation of the embryonic lung bud and displays pulmonary lineage traits. Unlike the lung, the fistula grows without branching. Bone morphogenetic proteins (BMPs) are known to be important in lung branching. We studied possible BMP signaling defects as a potential cause for the absence of branching in the fistula tract. METHODS: Adriamycin was administered to pregnant rats on days 6-9 of gestation to induce tracheoesophageal fistula. Microdissection was performed at E13 and E17 isolating the foregut. Tissues were analyzed using immunohistochemistry for BMP ligand (BMP2, BMP4, BMP7) and receptor (BMPRIA, BMPRIB, BMPRII) expression. RESULTS: Immunohistochemistry revealed the presence of all 3 BMP ligands at E13, localized specifically to the esophageal mucosa but absent in the fistula and lung. At E17, the ligands were again present in the esophageal mucosa, and additionally in the fistula tract mucosa, but remained absent in the lung. At E17, all of the BMP receptors were also localized to the luminal surface of esophagus and fistula. However, in the lung epithelium, only BMPRII was found, whereas BMPRIA and BMPRIB remained absent. CONCLUSIONS: The normal expression pattern of BMP4 was increased at the branch tips and low between branches. Among other results, we show here a constant expression level of BMP ligands throughout the entire epithelium of the fistula tract. This diffuse expression suggests defective BMP signaling in the fistula tract and explains its nonbranching phenotype.

Retinoid signaling controls mouse pancreatic exocrine lineage selection through epithelial-mesenchymal interactions.

BACKGROUND & AIMS: The early embryonic pancreas gives rise to exocrine (ducts and acini) and endocrine lineages. Control of exocrine differentiation is poorly understood, but may be a critical avenue through which to manipulate pancreatic ductal carcinoma. Retinoids have been shown to change the character of pancreatic ductal cancer cells to a less malignant phenotype. We have shown that 9-cis retinoic acid (9cRA) inhibits acinar differentiation in the developing pancreas, in favor of ducts, and we wanted to determine the role of retinoids in duct versus acinar differentiation. METHODS: We used multiple culture systems for the 11-day embryonic mouse pancreas. RESULTS: Retinoic acid receptor (RAR)-selective agonists mimicked the acinar suppressive effect of 9cRA, suggesting that RAR-RXR heterodimers were critical to ductal differentiation. RARalpha was only expressed in mesenchyme, whereas RXRalpha was expressed in epithelium and mesenchyme. Retinaldehyde dehydrogenase 2, a critical enzyme in retinoid synthesis, was expressed only in pancreatic epithelium. 9cRA did not induce ductal differentiation in the absence of mesenchyme, implicating a requirement for mesenchyme in 9cRA effects. Mesenchymal laminin is necessary for duct differentiation, and retinoids are known to enhance laminin expression. In 9cRA-treated pancreas, immunohistochemistry for laminin showed a strong band of staining around ducts, and blockage of laminin signaling blocked all 9cRA effects. Western blot and RT-PCR of pancreatic mesenchyme showed laminin-beta1 protein and mRNA induction by 9cRA. CONCLUSIONS: Retinoids regulate exocrine lineage selection through epithelial-mesenchymal interactions, mediated through up-regulation of mesenchymal laminin-1.

Lectin as a marker for staining and purification of embryonic pancreatic epithelium.

The embryonic pancreatic epithelium, and later the ductal epithelium, is known to give rise to the endocrine and exocrine cells of the developing pancreas, but no specific surface marker for these cells has been identified. Here, we utilized Dolichos Biflorus Agglutinin (DBA) as a specific marker of these epithelial cells in developing mouse pancreas. From the results of an immunofluorescence study using fluorescein-DBA and pancreatic specific cell markers, we found that DBA detects specifically epithelial, but neither differentiating endocrine cells nor acinar cells. We further applied this marker in an immunomagnetic separation system (Dynabead system) to purify these putative multi-potential cells from a mixed developing pancreatic cell population. This procedure could be applied to study differentiation and cell lineage selections in the developing pancreas, and also may be applicable to selecting pancreatic precursor cells for potential cellular engineering.

Fibroblast growth factor signaling in the developing tracheoesophageal fistula.

BACKGROUND/PURPOSE: The Adriamycin-induced rat model of esophageal atresia and tracheoesophageal fistula (EA/TEF) provides a reliable system for the study of EA/TEF pathogenesis. The authors previously hypothesized that faulty branching lung morphogenesis pathways were a critical component of its pathogenesis. The authors have found evidence for faulty fibroblast growth factor (FGF) signaling related to epithelial-mesenchymal interactions in the fistula tract. To better define FGF signaling, the differential expression of FGF ligands and their receptors between lung, fistula tract, and esophagus are described. METHODS: Time-dated pregnant, Sprague-Dawley rats were injected with Adriamycin (2 mg/kg intraperitoneally) on days 6 through 9 of gestation. Tissues were processed for histology and reverse transcriptase polymerase chain reaction. FGF-1, -7 and -10 were measured from whole lung, fistula tract, and esophagus of TEF or normal embryos. Expression of FGF2RIIIb and FGF2RIIIc receptors was measured in isolated epithelium and mesenchyme of lung and fistula tract of TEF embryos as well as lung and esophagus from normal controls. RESULTS: FGF-1 mRNA was present in the fistula tract and normal and Adriamycin-exposed lung but absent from whole esophagus. Interestingly, FGF-7 mRNA was present only in normal lung. FGF-10 was present in all tissues examined. FGF2RIIIb mRNA was absent in fistula mesenchyme but present in all other tissues examined. However, the splice variant FGF2RIIIc mRNA was present in all tissues examined. CONCLUSIONS: These findings support defective FGF signaling in the rat model of EA/TEF. Absence of FGF-7 mRNA in Adriamycin-exposed tissues suggests the primary effect of Adriamycin may be to inhibit FGF-7 expression. Moreover, absence of FGF2RIIIb in fistula mesenchyme may be caused by loss of positive feedback from FGF-7, its normal obligate ligand. Understanding these specific defects in FGF signaling may provide insight into faulty mechanisms of EA/TEF.

Aberrant fibroblast growth factor receptor 2 signalling in esophageal atresia with tracheoesophageal fistula.

BACKGROUND: Although the pathogenesis of esophageal atresia with tracheoesophageal fistula (EA/TEF) remains unknown, it has been shown that despite its esophageal appearance, the fistula tract originates from respiratory epithelium. The authors now hypothesize that defects in fibroblast growth factor (FGF) signaling contribute to the esophaguslike phenotype of the fistula tract. FGF2R is critical to normal lung morphogenesis and occurs in 2 isoforms (FGF2RIIIb and FGF2RIIIc), each with different ligand-binding specificity. To characterize FGF signaling in the developing EA/TEF, the authors analyzed levels of FGF2R splice variants in experimental EA/TEF. METHODS: The standard Adriamycin-induced EA/TEF model in rats was used. Individual foregut components from Adriamycin-treated and control embryos were processed for real-time, fluorescence-activated semiquantitative reverse transcriptase polymerase chain reaction on gestational days 12.5 and 13.5. RESULTS: Both fistula tract and Adriamycin-treated or normal esophagus showed significantly lower levels of FGF2RIIIb than either Adriamycin-treated lung buds (E12.5, P =.02; E13.5, P <.005) or normal lung buds (E12.5, P <.005; E13.5, P <.01). At E13.5, the fistula tract had lower levels of FGF2RIIIc than either treated (P <.01) or normal lung (P <.05). CONCLUSIONS: Levels of FGF2R in the developing fistula tract resemble that of distal esophagus rather than developing lung. This defect in FGF2RIIIb signaling may account for the nonbranching, esophaguslike phenotype of the fistula, despite its respiratory origin.

Complete discontinuity of the distal fistula tract from the developing gut: direct histologic evidence for the mechanism of tracheoesophageal fistula formation.

The embryogenesis of tracheoesophageal anomalies remains controversial. The purpose of this study was to better define the embryogenesis of developing esophageal atresia with tracheoesophageal fistula (EA/TEF), with specific attention to the controversial issue of whether a discontinuity exists in the foregut during its development of EA/TEF. Pregnant outbred rats were injected with adriamycin (2 mg/kg i.p.) on days 6-9 of gestation (E6-E9). At E12.5 and 13.5, microdissection of the entire foregut was performed. Foreguts were examined by phase microscopy, and serial, precisely transverse sections were created for hematoxylin and eosin (H&E) staining. Gross microdissection of the developing foregut at E12.5 (n = 9) revealed a blind-ending, bulbous fistula tract arising from the middle branch of the tracheal trifurcation (as seen by direct and phase microscopy). No connection with the gut could be appreciated at E12.5, but by E13.5 (n = 10) there was an obvious connection between the fistula and the stomach. Serial H&E transverse sections also demonstrated a blind-ending fistula tract arising from the trachea at E12.5. This fistula tract was clearly discontinuous from the developing stomach, which appeared much further caudal to the end of the fistula tract. These results strongly support a model of experimental TEF wherein the fistula tract arises from a trifurcation of the trachea, and (only during a specific gestational window between days 12.5 and 13.5) there is discontinuity between the fistula tract and the stomach. By day 13.5, the fistula joins with the stomach anlage. These observations in the developing EA/TEF should help to resolve the controversy about the mechanism of EA/TEF formation.