HoxA and HoxB cluster genes subdivide the digestive tract into morphological domains during chick development

The digestive tract exhibits region-specific morphology and cytodifferentiation along the anteroposterior axis. We analyzed the spatial expression patterns of Hox genes belonging to the HoxA and HoxB cluster (Hoxa-4 approximately a-9, Hoxb-5 approximately b-9) in the developing chick digestive tract. The expression domains of these Hox genes correlated with morphological subdivision of the digestive tract along the anteroposterior axis.     

Hoxb-5 expression in the developing mouse lung suggests a role in branching morphogenesis and epithelial cell fate

 Hoxb-5 is one of the few homeobox genes strongly expressed in the developing mouse lung. To explore the hypothesis that Hoxb-5 acts to regulate epithelial cell fate and branching morphogenesis in the developing lung, we studied the temporal, spatial, and cell-specific expression of Hoxb-5 from gestational day (d) 13.5 to postnatal day (P) 2. Immunocytochemistry demonstrated regional localization of Hoxb-5 protein to developing conducting airways and surrounding mesenchyme. The cellular expression pattern changed from diffusely positive nuclei of mesenchymal cells on d13.5 to become more localized to nuclei of subepithelial fibroblasts and some adjacent columnar and cuboidal epithelial cells on d14.5. After d14.5, Hoxb-5 protein expression continued to decrease in mesenchymal cells distal from developing airways, but persisted in fibroblasts underlying conducting airways. Hoxb-5 protein expression persisted in nuclei of columnar and cuboidal epithelial cells on d16.5 and d17.5, with expression in low cuboidal epithelial cells as well from d17.5 to P2. Western blot analysis showed temporal and quantitative changes in Hoxb-5 protein expression with peak expression on d14.5–15.5. We conclude that Hoxb-5 protein is developmentally regulated in a temporal, spatial, and cell-specific manner throughout the pseudoglandular, canalicular, and terminal saccular periods of lung development in the mouse. This localization and expression pattern suggests that Hoxb-5 may influence branching morphogenesis, cell–cell communication, cell fate, and differentiation of conducting airway epithelia. Accepted: 5 May 1997     

Multiple roles and interactions of tbx4 and tbx5 in development of the respiratory system

Normal development of the respiratory system is essential for survival and is regulated by multiple genes and signaling pathways. Both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea; and, although multiple genes are known to be required in the epithelium, only Fgfs have been well studied in the mesenchyme. In this study, we investigated the roles of Tbx4 and Tbx5 in lung and trachea development using conditional mutant alleles and two different Cre recombinase transgenic lines. Loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Mutants deficient in Tbx4 and Tbx5 show severely reduced lung branching at mid-gestation. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. Lung branching undergoes arrest ex vivo when Tbx4 and Tbx5 are both completely lacking. Lung-specific Tbx4 heterozygous;Tbx5 conditional null mice die soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings. Sox9, a master regulator of cartilage formation, is expressed in the trachea; but mesenchymal cells fail to condense and consequently do not develop cartilage normally at birth. Tbx4;Tbx5 double heterozygous mutants show decreased lung branching and fewer tracheal cartilage rings, suggesting a genetic interaction. Finally, we show that Tbx4 and Tbx5 interact with Fgf10 during the process of lung growth and branching but not during tracheal/bronchial cartilage development.     

Hoxb-5 control of early airway formation during branching morphogenesis in the developing mouse lung

Hox proteins control structural morphogenesis, pattern formation and cell fate in the developing embryo. To determine if Hoxb-5 participates in patterning of early airway branching during lung morphogenesis, gestational day 11.5 embryonic lung cultures were treated with retinoic acid (RA) to up-regulate and antisense oligonucleotides to down-regulate Hoxb-5 protein expression. RA (10^?6 M) and Hoxb-5 antisense oligonucleotide (20 μM) treatment each significantly decreased branching morphogenesis (P<0.001), but the morphology of branching under these conditions was very different. RA-treated lungs had elongated primary branches but decreased further branching with increased Hoxb-5 immunostaining in subepithelial regions underlying these elongated airways. Western blots confirmed that Hoxb-5 protein was increased by 189±20% (mean±S.E.M., P<0.05) in RA-treated lungs compared to controls. In contrast, lungs treated with Hoxb-5 antisense oligos plus RA had foreshortened primary branches with rudimentary distal clefts resulting in decreased numbers of primary and subsequent branches. Immunohistochemistry confirmed that Hoxb-5 antisense oligos inhibited Hoxb-5 protein expression even in the presence of RA. We conclude that regional and quantitative changes in Hoxb-5 protein expression influence morphogenesis of the first airway divisions from the mainstem bronchi. RA-induced alterations in branching are mediated in part through regulated Hoxb-5 expression.     

Mesenchymal control of branching pattern in the fetal mouse lung

The effect of mesenchyme on specialization of respiratory epithelium in the fetal mouse was tested in organ cultures. Heterologous combinations were made between respiratory and non-respiratory lung epithelia and the corresponding mesenchymes. Isolated terminal respiratory buds of fetal mouse lungs were recombined with mesenchyme from chick lung parabronchi, mouse trachea or from the avascular, non-respiratory air sacs of chick lungs. Isolated non-branching chick air sacs were combined with mouse terminal bud mesenchyme or mesenchyme from the respiratory branches of chick lungs. Air sac epithelia branched in a pattern characteristic of the chick lung when combined with chick respiratory mesenchyme and in a pattern characteristic of mouse lung when combined with mouse terminal bud mesenchyme. Mouse terminal bud epithelia did not branch with either mouse tracheal mesenchyme or chick air sac mesenchyme but branched in a chick pattern with chick parabronchial mesenchyme. Electron microscopic examination of the cultures showed that all chick air sac epithelial cultures failed to produce surfactant (lamellar bodies) even when they branched. Control cultures of mouse terminal buds contained large numbers of lamellar bodies; mesenchyme which suppressed branching reduced the number of lamellar bodies to only a few in a small proportion of the cells. Culture medium supplemented with growth factors and hormones increased the number of lamellar bodies in heterologous mouse combinations but did not bring the number to control levels. Supplemented medium had no effect on lamellar body production by chick air sac epithelium. The results indicate that branching pattern is determined by the mesenchyme surrounding the epithelial primordium. However, the capacity to synthesize surfactant is determined by the source of the epithelium; mesenchyme may control the degree of expression but not the absolute presence or absence of the differentiated condition.     

Wnt signaling in lung organogenesis

Reporter transgene, knockout, and misexpression studies support the notion that Wnt/β-catenin signaling regulates aspects of branching morphogenesis, regional specialization of the epithelium and mesenchyme, and establishment of progenitor cell pools. As demonstrated for other foregut endoderm-derived organs, β-catenin and the Wnt/β-catenin signaling pathway contribute to control of cellular proliferation, differentiation and migration. However, the contribution of Wnt/β-catenin signaling to these processes is shaped by other signals impinging on target tissues. In this review, we will concentrate on roles for Wnt/β-catenin in respiratory system development, including segregation of the conducting airway and alveolar compartments, specialization of the mesenchyme, and establishment of tracheal asymmetries and tracheal glands.Key words: morphogenesis, respiratory, airway, alveolar, mesenchyme, endoderm     

Spatial gene expression in the T-stage mouse metanephros

The E11.5 mouse metanephros is comprised of a T-stage ureteric epithelial tubule sub-divided into tip and trunk cells surrounded by metanephric mesenchyme (MM). Tip cells are induced to undergo branching morphogenesis by the MM. In contrast, signals within the mesenchyme surrounding the trunk prevent ectopic branching of this region. In order to identify novel genes involved in the molecular regulation of branching morphogenesis we compared the gene expression profiles of isolated tip, trunk and MM cells using Compugen mouse long oligo microarrays. We identified genes enriched in the tip epithelium, sim-1, Arg2, Tacstd1, Crlf-1 and BMP7; genes enriched in the trunk epithelium, Innp1, Itm2b, Mkrn1, SPARC, Emu2 and Gsta3 and genes spatially restricted to the mesenchyme surrounding the trunk, CSPG2 and CV-2, with overlapping and complimentary expression to BMP4, respectively. This study has identified genes spatially expressed in regions of the developing kidney involved in branching morphogenesis, nephrogenesis and the development of the collecting duct system, calyces, renal pelvis and ureter.     

Suppression of embryonic lung branching morphogenesis by antisense oligonucleotides against HOM/C homeobox factors

The role of HOM/C homeobox genes on rat embryonic lung branching morphogenesis was investigated using the lung bud explant culture system in an air/liquid interface. Knock down of homeobox b3 and b4 expression by antisense oligonucleotide treatment repressed airway branch formation, while antisense oligonucleotide against homeobox a3 showed no effect. Addition of antisense Hoxb3 oligonucleotide resulted in upregulation of collagen type III mRNA and fibroblast growth factor 10 mRNA, while that of the T-box regulatory factor-4 was decreased. Consequently, expression of Clara cell-specific secretory protein was decreased. These results suggest a critical role for homeobox b3 and b4 genes in lung airway branching morphogenesis.     

Thyroid hormone affects embryonic mouse lung branching morphogenesis and cellular differentiation

Although thyroid hormone (T(3)) influences epithelial cell differentiation during late fetal lung development, its effects on early lung morphogenesis are unknown. We hypothesized that T(3) would alter embryonic lung airway branching and temporal-spatial differentiation of the lung epithelium and mesenchyme. Gestational day 11.5 embryonic mouse lungs were cultured for 72 h in BGJb serum-free medium without or with added T(3) (0.2, 2.0, 10.0, or 100 nM). Evaluation of terminal bud counts showed a dose- and time-dependent decrease in branching morphogenesis. Cell proliferation was also significantly decreased with higher doses of T(3). Morphometric analysis of lung histology showed that T(3) caused a dose-dependent decrease in mesenchyme and increase in cuboidal epithelia and airway space. Immunocytochemistry showed that with T(3) treatment, Nkx2.1 and surfactant protein SP-C proteins became progressively localized to cuboidal epithelial cells and mesenchymal expression of Hoxb5 was reduced, a pattern resembling late fetal lung development. We conclude that exogenous T(3) treatment during early lung development accelerated epithelial and mesenchymal cell differentiation at the expense of premature reduction in new branch formation and lung growth.     

Wnt-4 signaling is involved in the control of smooth muscle cell fate via Bmp-4 in the medullary stroma of the developing kidney

Wnt-4, a member of the Wnt family of secreted signaling molecules, is essential for nephrogenesis, but its expression in the presumptive medulla suggests additional developmental roles in kidney organogenesis. We demonstrate here that Wnt-4 signaling plays also a role in the determination of the fate of smooth muscle cells in the medullary stroma of the developing kidney, as a differentiation marker, smooth muscle alpha-actin (alpha-SMA), is markedly reduced in the absence of its signaling. Wnt-4 probably performs this function by activating the Bmp-4 gene encoding a known differentiation factor for smooth muscle cells, since Bmp-4 gene expression was lost in the absence of Wnt-4 while Wnt-4 signaling led to a rescue of Bmp-4 expression and induction of alpha-SMA-positive cells in vitro. Recombinant Bmp-4 similarly rescued the differentiation of alpha-SMA-expressing cells in cultured Wnt-4-deficient embryonic kidney. The lack of smooth muscle cell differentiation leads to an associated deficiency in the pericytes around the developing vessels of the Wnt-4-deficient kidney and apparently leads to a secondary defect in the maturation of the kidney vessels. Thus, besides being critical for regulating mesenchymal to epithelial transformation in the cortical region in nephrogenesis, Wnt-4 signaling regulates the fate of smooth muscle cells in the developing medullary region.     

Platelet-derived growth factor receptor regulates salivary gland morphogenesis via fibroblast growth factor expression

A coordinated reciprocal interaction between epithelium and mesenchyme is involved in salivary gland morphogenesis. The submandibular glands (SMGs) of Wnt1-Cre/R26R mice have been shown positive for mesenchyme, whereas the epithelium is beta-galactosidase-negative, indicating that most mesenchymal cells are derived from cranial neural crest cells. Platelet-derived growth factor (PDGF) receptor alpha is one of the markers of neural crest-derived cells. In this study, we analyzed the roles of PDGFs and their receptors in the morphogenesis of mouse SMGs. PDGF-A was shown to be expressed in SMG epithelium, whereas PDGF-B, PDGFRalpha, and PDGFRbeta were expressed in mesenchyme. Exogenous PDGF-AA and -BB in SMG organ cultures demonstrated increased levels of branching and epithelial proliferation, although their receptors were found to be expressed in mesenchyme. In contrast, short interfering RNA for Pdgfa and -b as well as neutralizing antibodies for PDGF-AB and -BB showed decreased branching. PDGF-AA induced the expression of the fibroblast growth factor genes Fgf3 and -7, and PDGF-BB induced the expression of Fgf1, -3, -7, and -10, whereas short interfering RNA for Pdgfa and Pdgfb inhibited the expression of Fgf3, -7, and -10, indicating that PDGFs regulate Fgf gene expression in SMG mesenchyme. The PDGF receptor inhibitor AG-17 inhibited PDGF-induced branching, whereas exogenous FGF7 and -10 fully recovered. Together, these results indicate that fibroblast growth factors function downstream of PDGF signaling, which regulates Fgf expression in neural crest-derived mesenchymal cells and SMG branching morphogenesis. Thus, PDGF signaling is a possible mechanism involved in the interaction between epithelial and neural crest-derived mesenchyme.     

Involvement of Wnt-5a in chondrogenic pattern formation in the chick limb bud

Members of the Wnt family are known to play diverse roles in the organogenesis of vertebrates. The full-coding sequences of chicken Wnt-5a were identified and the role it plays in limb development was examined by comparing its expression pattern with that of two other Wnt members, Wnt-4 and Wnt-11, and by misexpressing it with a retrovirus vector in the limb bud. Wnt-5a expression is detected in the limb-forming region at stage 14, and in the apical ectodermal ridge and distal mesenchyme of the limb bud. The signal was graded along the proximal-distal axis at stages 20-28 and also along the anterior-posterior axis during early stages. It disappeared in the cartilage-forming region after stage 26, and was restricted to the region surrounding the phalanges at stage 34. Wnt-4 and Wnt-11, other members of the Wnt-5a-subclass, were expressed with a distinct spatiotemporal pattern during the later phase. Wnt-4 was expressed in the articular structure and Wnt-11 was expressed in the dorsal and ventral mesenchyme adjacent to the ectoderm. Wnt-5a expression was partially reduced after apical ectodermal ridge removal, whereas Wnt-11 expression was down-regulated by dorsal ectoderm removal. Therefore, expression of these Wnt was differentially regulated by the ectodermal signal. Misexpression of Wnt-5a in the limb bud with the retrovirus resulted in truncation of long bones predominantly in the zeugopod because of retarded chondrogenic differentiation. Distal elements, such as the phalanges and metacarpals, were not significantly reduced in size. These results suggest that Wnt-5a is involved in pattern formation along the proximal-distal axis by regulation of chondrogenic differentiation.