Apparent normal lung architecture in protein tyrosine phosphatase-sigma -deficient mice

Jane Batt1, Ernest Cutz2, Chris Fladd1, and Daniela Rotin1

Programs in 1 Cell Biology and 2 Pathology, The Hospital for Sick Children, and Biochemistry Department, University of Toronto, Toronto, Ontario, Canada M5G 1X8


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Protein tyrosine phosphatase-sigma (PTP-sigma ) is a member of the mammalian LAR family of phosphatases, which is characterized by a cell adhesion-like ectodomain, a single transmembrane segment, and two tandemly repeated intracellular catalytic domains. The expression of PTP-sigma is developmentally regulated in epithelial, neuronal, and neuroendocrine tissues. We previously showed that PTP-sigma is strongly expressed within the fetal, but not adult, rat lung and is localized to the Clara cells and type II pneumocytes. In view of the developmentally regulated pulmonary expression of PTP-sigma , we performed a detailed histological and ultrastructural study of the lungs of PTP-sigma knockout mice we have generated. Our findings indicate no apparent structural abnormalities in the lungs of PTP-sigma -/- mice, including airway and alveolar epithelium. In addition, pulmonary neuroendocrine cells also appear normal, in contrast to pituitary, pancreatic, and gastrointestinal endocrine cells, in the knockout mice, suggesting different developmental regulation of these neuroendocrine cells. These observations suggest compensation for the absence of PTP-sigma during development by related family member phosphatases, such as LAR.

pulmonary; knockout mice; neuroepithelial body


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PROTEIN TYROSINE PHOSPHATASE (PTP)-sigma is a transmembrane receptor PTP and a member of the mammalian LAR family, which consists of the three closely related enzymes: PTP-sigma , PTP-delta , and LAR. PTP-sigma contains a cell adhesion molecule (CAM)-like ectodomain, a single transmembrane domain, and two tandemly repeated intracellular phosphatase domains (13, 19, 22, 25, 27). The proximal phosphatase domain is catalytically active, whereas the second domain is inactive and serves a regulatory role (21). The CAM-like ectodomain of PTP-sigma bears a strong resemblance to the ectodomain of neural CAMs of the Ig superfamily, such as L1 and N-CAM (3, 6). These molecules regulate the development of specific axonal projections in the nervous system. The substrate and ligand for PTP-sigma and the signaling networks in which it functions remain unknown.

PTP-sigma expression is tightly controlled and developmentally regulated within epithelial, neuronal, and neuroendocrine tissues and organs (13, 15, 23, 25). It plays a critical role in the development of the pituitary, pancreas, enteroendocrine gut, and peripheral nerve. PTP-sigma knockout mice demonstrate pituitary and pancreatic islet hypoplasia associated with deficiencies of growth hormone (GH), prolactin, and insulin (1, 4, 20). Severe GH deficiency contributes to neonatal hypoglycemia in the PTP-sigma -/- mice and a high neonatal mortality rate (60%) (1). The peripheral nerve in the PTP-sigma -deficient mice shows developmental delay (20) and significant abnormalities of axon guidance and regenerative capacity after injury (12).

Within the rat lung, PTP-sigma is predominantly and strongly expressed in proliferating and differentiating epithelial cells (fetal Clara cells and type II pneumocytes) of the embryonic, fetal, and neonatal airway and alveolar sacs (9). PTP-sigma is not found in quiescent adolescent and adult rat pulmonary epithelium. On the basis of this cell-specific and developmentally regulated pulmonary expression and the absolute requirement for PTP-sigma in the developing nervous and neuroendocrine systems, we hypothesized that PTP-sigma would also play a role in the development of the mammalian respiratory system. We thus performed detailed histological and ultrastructural assessment of the lungs in PTP-sigma -deficient mice.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The full-length PTP-sigma gene was inactivated, and mice were characterized as previously described (20). The PTP-sigma +/- mice are phenotypically indistinguishable from PTP-sigma +/+ mice. The PTP-sigma -/- animals consisted of three cohorts: most (60%) died as neonates within hours of birth, 37.5% demonstrated growth retardation and succumbed to a wasting syndrome by 2-3 wk of age, and 2.5% survived to adulthood, appeared healthy, and were fertile.

All animal experimentation was conducted in accordance with accepted standards of humane animal care.

Lac Z staining. The knockout cassette used to generate the PTP-sigma knockout mice contains the beta -galactosidase gene, allowing Lac Z staining to determine the expression pattern of PTP-sigma . Animals were killed, and the lungs were harvested and fixed in 0.1 M sodium phosphate buffer (pH 7.9) containing 1% formaldehyde, 0.1% glutaraldehyde, 2 mM MgCl2, and 5 mM EGTA for 6 h. Organs were washed for 2 h with four exchanges of a wash buffer (2 mM MgCl2, 0.01% deoxycholate, and 0.02% NP-40 in 0.1 M sodium phosphate buffer, pH 7.9) at room temperature. The tissue was then incubated in PBS containing 5 mM ferricyanide, 5 mM ferrocyanide, 2 mM MgCl2, and X-gal (Roche; 0.1 mg/ml) overnight at 37°C. Tissues were subsequently rinsed in 70% ethanol, embedded in paraffin, and sectioned.

Lung wet-to-dry weight ratios. Newborn litters were killed via decapitation, and the right lung was immediately excised, gently blotted on a cotton towel, and weighed to obtain the lung wet weight. The lungs were then placed in a 50°C oven and weighed daily until a stable dry weight was obtained (>= 72 h). Wet-to-dry lung weight ratios were determined for PTP-sigma -/-, PTP-sigma +/-, and PTP-sigma +/+ newborns. An increased ratio is indicative of an increase in intravascular and/or interstitial lung water content.

Histopathology. Representative mice from each of the three PTP-sigma -/- cohorts and sibling controls were euthanized. Newborn mice were decapitated, and the lungs were dissected and fixed overnight in 4% paraformaldehyde at 4°C. Adult and 2- to 3-wk-old mice were sedated with an intraperitoneal injection of chloral hydrate and subsequently perfusion fixed with 4% paraformaldehyde. Alternatively, these two cohorts of mice were euthanized by lethal intraperitoneal injection of pentobarbital sodium. The lungs were then dissected and fixed overnight in 10% buffered formalin at 4°C. All lung tissue was subsequently embedded in paraffin, sectioned (5 µm), and stained with hematoxylin and eosin (H & E) or periodic acid-Schiff (PAS).

Electron microscopy. Representative newborn, 2- to 3-wk-old, and adult PTP-sigma -/- mice and appropriate controls were euthanized; the lungs were dissected and fixed overnight in 1% glutaraldehyde-4% formaldehyde in 0.1 M phosphate buffer. Lungs were washed once in 0.1 M phosphate buffer for 5 min and then postfixed in 2% OsO4. Reduced osmium fixation (2% OsO4 and ferrocyanide) was used to demonstrate cytoplasmic glycogen. Sections (1 µm) were dehydrated in graded acetone, embedded in Epon, and stained with toluidine blue; ultrathin sections were stained with uranyl acetate and lead citrate. Electron-microscopic examination was performed with a transmission electron microscope (model 201, Phillips).

Immunohistochemistry. Immunohistochemical localization of calcitonin gene-related peptide (CGRP), a marker of neuroepithelial bodies (NEB) in rodent lungs, was carried out as previously reported (5). Briefly, representative mice from each of the three PTP-sigma -/- cohorts were euthanized, and the lungs were dissected and fixed for 30 min (newborn) or 2 h (2-3 wk old and adult) at room temperature in Bouin's fixative. Lungs were subsequently washed four times with PBS overnight at 4°C, embedded in paraffin, and sectioned (5 µm). Sections were blocked and incubated with a rabbit polyclonal CGRP antiserum (Chemicon) at a dilution of 1:400 overnight at 4°C, followed by biotinylated secondary antibody, streptavidin-HRP (Vectastain Elite ABC kit; Vector Labs) and developed in diaminobenzidine (0.5 mg/ml) and H2O2 (0.03%). Appropriate positive and negative (omission of primary antibody) controls were performed.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Developmental expression of PTP-sigma in mouse lungs. The PTP-sigma knockout cassette contains the beta -galatosidase gene (20). Lac Z staining was therefore undertaken to localize the expression of PTP-sigma within the lungs of the three cohorts of PTP-sigma -/-, PTP-sigma +/-, and PTP-sigma +/+ mice. PTP-sigma is expressed within the walls of the alveolar sacs, airway epithelium, and pulmonary vessels of newborn mice (Fig. 1, A and B). At 2-3 wk of age, LacZ staining is evident in the alveolar walls and pulmonary vessels but is no longer expressed in the airway epithelium (Fig. 1, D and E). As in other tissues, PTP-sigma expression decreases dramatically as the animal matures and is absent from the lungs of the adult mice (Fig. 1, G and H). These results are in agreement with our previously observed decline of PTP-sigma expression with maturation of rat lung epithelia (9).


View larger version (119K):
[in this window]
[in a new window]
 
Fig. 1.   Lac Z staining of protein tyrosine phosphatase (PTP)-sigma -/-, PTP-sigma +/-, and PTP-sigma +/+ newborn (NB), 2- to 3-wk-old, and adult lungs. As the beta -galactosidase gene replaces PTP-sigma in the knockout cassette, blue Lac Z staining represents the PTP-sigma expression pattern. The pattern of Lac Z-positive staining in PTP-sigma -/- mice (A, D, and G) parallels that in PTP-sigma +/- mice (B, E, and H). Lac Z staining is evident in alveolar sacs, airway epithelium, and pulmonary vasculature of newborn mice (A and B). Lac Z staining is still evident in alveoli but absent from airway epithelium by 2-3 wk of age (D and E). Lac Z is no longer expressed in adult lung (G and H). PTP-sigma +/+ mice act as negative controls for Lac Z staining (C, F, and I). Scale bar, 40 µm. Insets depict higher magnification of segments of the tissue.

Analysis of pulmonary edema. During the original phenotypic characterization of the PTP-sigma -/- mice, pulmonary edema was noted in some of the newborn animals (20). To further investigate this observation and analyze its prevalence, the lung wet-to-dry weight ratio was determined for a large series of newborn mice. The presence of fluid within the lung manifests as an increase in the lung wet-to-dry weight ratio. Figure 2 shows no significant difference in the lung wet-to-dry weight ratio between PTP-sigma -/-, PTP-sigma +/-, and PTP-sigma +/+ newborn mice. This suggests that, overall, there is no significant pulmonary edema in the PTP-sigma -/- newborn.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 2.   Lung wet-to-dry weight ratios of newborn mice. An increase in lung wet-to-dry weight ratio indicates alveolar fluid or an increase in intravascular volume. There is no significant difference between lung wet-to-dry weight ratios of PTP-sigma -/- (n = 20) and sibling PTP-sigma +/- (n = 43) and PTP-sigma +/+ (n = 21) newborn mice.

Analysis of lung architecture. Analysis of H & E-stained sections of lungs from newborn and adult PTP-sigma -/- mice revealed no apparent abnormalities (Fig. 3, A and E). Specifically, the alveoli and epithelial lining of the bronchioles appeared normal, and alveolar pulmonary edema was not evident in the newborn PTP-sigma -/- mice (Fig. 3A). One 18-day-old PTP-sigma -/- mouse (2- to 3-wk-old cohort) demonstrated significant bronchial epithelial hyperplasia and possibly metaplasia (data not shown), but similar findings were not evident, and the lungs appeared normal in the large majority of this cohort (Fig. 3C).


View larger version (148K):
[in this window]
[in a new window]
 
Fig. 3.   Hematoxylin-and-eosin-stained sections of PTP-sigma -/- newborn (A), 2- to 3-wk-old (C), and adult (E) mice and PTP-sigma +/+ newborn (B), 2- to 3-wk-old (D), and adult control mice (F). Scale bar, 100 µm. All 3 cohorts of PTP-sigma -/- mice demonstrate normal histology; i.e., there are no differences in appearance of alveolar sac (AS), alveoli (AL), or bronchioles (BR) between PTP-sigma -/- mice and age-matched PTP-sigma +/+ controls.

The Clara cells of the airways of newborn mice contain a significant amount of cytoplasmic glycogen that is rapidly metabolized in the first 24-48 h after birth (11). Because PTP-sigma is expressed in the Clara cell, we used PAS staining with diastase digestion to visualize the glycogen stores of newborn PTP-sigma -/- animals. Our results show no difference in the distribution of glycogen or the duration of positive glycogen staining in the PTP-sigma -/- newborn mice (Fig. 4, A and B) compared with PTP-sigma +/- and PTP-sigma +/+ sibling controls (Fig. 4, C and D). Thus, although the PTP-sigma knockout mice exhibit a delay in several aspects of development, glycogen metabolism in Clara cells at birth is not altered.


View larger version (157K):
[in this window]
[in a new window]
 
Fig. 4.   Periodic acid-Schiff staining of newborn PTP-sigma -/- mice (A) and sibling PTP-sigma +/+ mice (C) reveals magenta-colored neutral mucopolysaccharides in epithelial cells of the airway representing Clara cells (arrows). Pretreatment with diastase (B and D) reveals loss of magenta staining (arrowheads), confirming presence of glycogen. No differences are noted between PTP-sigma -/- and PTP-sigma +/+ newborns. Scale bar, 40 µm.

Electron microscopy of lungs from all three cohorts of the PTP-sigma -/- mice was undertaken to assess subcellular components of the cell types expressing PTP-sigma , such as the lamellar bodies of the type II pneumocytes and the secretory granules of the Clara cells. The Clara cells of newborn, 2- to 3-wk-old, and adult PTP-sigma -/- mice (Fig. 5 A, C, and E) appear normal and indistinguishable from Clara cells of the PTP-sigma +/- or PTP-sigma +/+ control mice (Fig. 5, B, D, and F). The Clara cells of the newborn mouse contain significant cytoplasmic glycogen with few or no secretory granules. Postnatally there is an expected significant decrease in cytoplasmic glycogen content, with a corresponding increase in the cell organelles, including secretory granules, and a modest increase in the mitochondrial content (11). The cell organelles of type II pneumocytes, including their lamellar bodies, also appeared normal in all three cohorts of PTP-sigma -/- mice and indistinguishable from the type II cells of the PTP-sigma +/- and PTP-sigma +/+ sibling control mice (Fig. 6).


View larger version (130K):
[in this window]
[in a new window]
 
Fig. 5.   Electron micrographs of airway epithelium in PTP-sigma -/- newborn (A), 2- to 3-wk-old (C), and adult (E) mice and PTP-sigma +/+ newborn (B), 2- to 3-wk-old (D), and adult (F) mice. Magnification ×3,572. Cytoplasm of Clara cells in lungs of newborn mice contain large amounts of cytoplasmic glycogen (A and B) appearing as electron-dense material (GLY) with reduced osmium fixation (inset). Clara cell glycogen content decreases and secretory granules (arrowhead) and mitochondria (arrow) increase as the cell matures (C-F). Clara cells and ciliated cells of PTP-sigma -/- mice are indistinguishable from PTP-sigma +/+ mice.



View larger version (130K):
[in this window]
[in a new window]
 
Fig. 6.   Electron micrograph of a type II pneumocyte from a 2- to 3-wk-old PTP-sigma -/- mouse (A) and a sibling PTP-sigma +/+ mouse (B). Magnification ×11,324. Well-formed lamellar bodies (L) are evident in both micrographs. Type II cells from all 3 cohorts of PTP- sigma -/- mice appeared normal.

Pulmonary neuroendocrine cells. We previously described abnormalities of the neuroendocrine system (pituitary, pancreas, and enteroendocrine cells of the gut) of the PTP-sigma -/- mice (1, 20). To determine whether the pulmonary neuroendocrine cell (PNEC) system may be similarly affected by the loss of PTP-sigma , we immunostained the lungs of the PTP-sigma -/- mice for CGRP, a marker of PNEC and NEB (5). Our findings show that PNEC and NEB in the lungs of PTP-sigma -/- mice (Fig. 7, A and B) were comparable to those in the lungs of PTP-sigma +/- and PTP-sigma +/+ sibling control mice (Fig. 7, C and D).


View larger version (139K):
[in this window]
[in a new window]
 
Fig. 7.   Positive immunostaining of neuroepithelial bodies (NEB) for calcitonin gene-related peptide (arrows) in a 2- to 3-wk-old PTP-sigma -/- mouse (A and B) and a PTP-sigma +/+ sibling mouse (C and D). Calcitonin gene-related peptide-positive staining appears brown. NEB are normally sparse and appear as small clusters of intraepithelial cells preferentially located at the airway bifurcation. NEB in PTP-sigma -/- mouse are similar to those in control PTP-sigma +/+ mouse. Scale bars, 100 µm (A and C) and 20 µm (B and D).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously demonstrated by in situ hybridization and Northern blot analysis that PTP-sigma expression is developmentally regulated in the rat lung (9, 14). The highest levels of expression of PTP-sigma in lungs were evident in fetal and newborn epithelium of the bronchi, terminal bronchioles, budding air sacs, and alveoli, with significant downregulation of expression as the rat matures. Parallel immunostaining for surfactant proteins A and B, proliferating cell nuclear antigen, and [3H]thymidine uptake studies revealed that PTP-sigma was predominantly expressed in the undifferentiated and proliferating fetal alveolar type II cells and fetal Clara cells of the airways (9). Fetal type II cells are the precursors for adult type II cells (10). Fetal Clara cells are the stem cells of the small conducting airway epithelium and give rise to adult Clara cells and ciliated cells (10). Once differentiation was complete and proliferation was reduced to the low level of epithelial cell turnover normally seen in the quiescent healthy postnatal lung, PTP-sigma expression became undetectable (9).

In the present study, the pulmonary PTP-sigma expression pattern was determined by Lac Z staining of the PTP-sigma -/- and PTP-sigma +/- mice. In agreement with our previous in situ hybridization results in the rat lung (9), Lac Z staining demonstrates expression of PTP-sigma in the airways and respiratory epithelia of newborn mice; moreover, this expression is developmentally downregulated. Although we previously failed to detect PTP-sigma expression in the rat pulmonary vasculature using in situ hybridization (9), Lac Z staining (Fig. 1) clearly demonstrates significant expression of PTP-sigma in the blood vessels (smooth muscle and, possibly, endothelium). The reason for the lack of previous detection of PTP-sigma in the vasculature is not known but may stem from the different methodology employed, which may be less sensitive.

During the initial phenotypic analytic screen of the PTP-sigma -/- mice, the occasional neonate was found to have pulmonary edema (20). Subsequent systematic collection and assessment of a large number of newborn PTP-sigma -/- mice, however, demonstrated that this was not a general phenomenon. H & E analysis of PTP-sigma -/- newborn lungs did not reveal evidence of alveolar fluid (Fig. 3A). Wet-to-dry lung weight ratios were similar in PTP-sigma -/-, PTP-sigma +/-, and PTP-sigma +/+ newborns, arguing against the presence of alveolar fluid/pulmonary edema in the knockout neonate (Fig. 2). On the basis of our recent and present work, we conclude that the high neonatal mortality rate of the PTP-sigma -/- mice is the result of hypoglycemia, which at least in part results from GH deficiency, and is not due to pulmonary edema (1).

Airways of the PTP-sigma -/- mice appeared normal on H & E-stained sections and indistinguishable from the airways of PTP-sigma +/- and PTP-sigma +/+ mice (Fig. 3). As expected, PAS staining and diastase digestion revealed glycogen in cells of the airways of PTP-sigma -/-, PTP-sigma +/-, and PTP-sigma +/+ newborn mice (Fig. 4). Many cell types of the neonatal lung, including Clara cells of the airway and alveolar type II cells, have significant glycogen stores (11). In the rat Clara cell, glycogen is immediately and rapidly metabolized postpartum, so that within 48 h of birth there is a massive decrease in the volume density (fraction of cell volume) of glycogen. This is followed by a continual, but very gradual, decrease of glycogen into adulthood. Glycogen mobilization at birth is presumably important for the maturation of the secretory apparatus of the Clara cell. In addition, because Clara cells are the progenitors for the cells of the terminal bronchioles, glycogenolysis in Clara cells may be critical for the proliferative burst they undergo during the first 48 h after birth. Glycogen deposition and mobilization, as assessed by PAS staining and diastase digestion, were similar between PTP-sigma -/- and PTP-sigma +/+ mice.

Ultrastructural analysis of different pulmonary epithelial cells, including alveolar type II cells, Clara cells, and ciliated cells, did not reveal any significant differences between the PTP-sigma -/- and PTP-sigma +/- or the PTP-sigma +/+ mice (Figs. 5 and 6). The Clara cell population demonstrated typical postnatal maturation changes (11) when assessed in the three cohorts of animals. The type II cells and ciliated cells of the airway in all three cohorts of the PTP-sigma -/- mice also appeared ultrastructurally normal.

We recently showed that the PTP-sigma -/- mouse demonstrates significant abnormalities of the endocrine pancreas, pituitary, and enteroendocrine cells of the gut (1). Here we assessed the pulmonary neuroendocrine system by immunostaining for CGRP, a predominant neuropeptide of mouse NEB. The NEB appeared normal in the PTP-sigma -/- mice and indistinguishable from the immunostaining pattern in the PTP-sigma +/- and PTP-sigma +/+ sibling control mice. In knockout mice lacking mammalian achaete-scute homolog-1, a mammalian homolog of the Drosophila achaete-scute genes, pulmonary NEB are completely absent, but the pancreatic islets and enteroendocrine cells in the gut develop and appear normal (2). These contrasting phenotypes of the mammalian achaete-scute homolog-1 and PTP-sigma knockout mice suggest that the development and differentiation of the neuroendocrine cell component of different endodermal derivatives (i.e., gut, pancreas, and lung) are under tissue-specific regulatory control.

Although pulmonary mechanics were not formally assessed in this study, no obvious abnormalities of ventilation were evident during routine activity in the PTP-sigma -/- animals. Specifically, they were not found to be tachypneic, nor was there any evidence of increased respiratory effort.

In view of the tightly controlled and developmentally regulated expression of PTP-sigma within the lung, it was a surprise to see normal lung architectural development in the PTP-sigma -deficient mouse. This is particularly so when one considers that PTP-sigma is essential to the development of other organs (e.g., central nervous system, peripheral nervous system, pituitary), where a similar temporal expression pattern is seen, and that mice deficient in receptors bearing a marked resemblance to PTP-sigma demonstrate a dramatic lung phenotype. For example, Dutt-1/Robo-1 is a mammalian transmembrane receptor possessing an ectodomain very similar to PTP-sigma and the Ig family of neural CAMs (24). The ectodomains of these proteins consist of repeats of Ig-like domains and fibronectin type III repeats. Dutt-1 is probably a tumor suppressor gene in mammals (24). It has also been identified as the human homolog of the Drosophila roundabout (Robo) (8) and has been proposed to play an essential role in the guidance and migration of axons, myoblasts, and leukocytes in vertebrates. In mice homozygous for a targeted mutation in the Dutt-1/Robo-1 gene (which eliminates the first Ig domain of the ectodomain), the lungs demonstrate mesenchyme expansion and extensive bronchial epithelial hyperplasia (24). A 60% homozygote neonatal mortality rate results from respiratory failure. We also noted significant hyperplasia in the bronchial epithelium of one 2- to 3-wk-old PTP-sigma -/- mouse. However, this finding was not evident in the large majority of animals assessed.

In view of the normal lung architecture in the PTP-sigma -deficient mice, one may speculate that, despite strong developmental expression of PTP-sigma in the lung, either this protein does not play a significant role in pulmonary development or, more likely, lung development in the PTP-sigma knockout animals is "rescued" by the compensatory actions of a closely related family member, such as LAR. Katsura and colleagues (7) assessed the developmental expression profile of LAR in the rat lung via immunohistochemistry and in situ hybridization. They demonstrated that, in the lung, LAR is exclusively expressed in the epithelium of the fetal airways and alveoli with persistent Clara cell and alveolar type II cell expression throughout life. Thus LAR and PTP-sigma are expressed in the same cell types within the rat lung. In view of the similar expression patterns and extensive sequence homology between the two proteins, it is not unreasonable to speculate that LAR might rescue lung development in the PTP-sigma -deficient mouse. The LAR knockout mouse also lacks a lung phenotype (16-18, 26), and significant upregulation of PTP-sigma mRNA was observed in the lungs of these knockout mice (W. Skarnes, personal communication). Again, in this scenario, one might speculate that rescue of the LAR-deficient mice by PTP-sigma would permit normal pulmonary development.

PTP-delta , the third member of the mammalian LAR family, is also expressed in the lung. Katsura et al. (7) demonstrated by in situ hybridization that PTP-delta is expressed exclusively in the mesenchyme of the lung and that expression falls off significantly after birth. It is possible that PTP-delta also plays a role in rescue of the PTP-sigma knockout mouse, because mesenchymal-epithelial interactions are critical during lung morphogenesis.

In conclusion, we have generated a PTP-sigma -deficient mouse and found the lung architecture to be normal by detailed histological assessment. We speculate that the presence and activity of the closely related family members LAR and, possibly, PTP-delta rescue and normalize lung development in the absence of PTP-sigma in these mice. Although lung architecture is not affected by the loss of PTP-sigma , pulmonary mechanics, epithelial cell biology, and other lung functions of the PTP-sigma -/- mice have not been investigated and may be affected by the loss of this phosphatase.


    ACKNOWLEDGEMENTS

We thank A. Giffin for assistance with mouse colony maintenance, J. Hwong for assistance with electron microscopy, and V. Wong for performing the CGRP immunostaining.


    FOOTNOTES

This work was supported by grants from the Canadian Institutes of Health Research (CIHR) to D. Rotin. J. Batt is supported by a CIHR fellowship. D. Rotin is the recipient of a CIHR Investigator Award.

Address for reprint requests and other correspondence: D. Rotin, Programs in Cell Biology, The Hospital for Sick Children, 555 University Ave., Toronto, ON, Canada M5G 1X8 (E-mail: drotin{at}sickkids.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajplung.00069.2002

Received 25 February 2002; accepted in final form 19 August 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Batt, J, Asa S, Fladd C, and Rotin D. Pituitary, pancreatic and gut neuroendocrine defects in protein tyrosine phosphatase-sigma -deficient mice. Mol Endocrinol 16: 155-169, 2002[Abstract/Free Full Text].

2.   Borges, M, Linnoila RI, van de Velde HJ, Chen H, Nelkin BD, Mabry M, Baylin SB, and Ball DW. An achaete-scute homologue essential for neuroendocrine differentiation in the lung. Nature 386: 852-855, 1997[ISI][Medline].

3.   Crossin, KL, and Krushel LA. Cellular signaling by neural cell adhesion molecules of the immunoglobulin superfamily. Dev Dyn 218: 260-279, 2000[ISI][Medline].

4.   Elchebly, M, Wagner J, Kennedy TE, Lanctot C, Michaliszyn E, Itie A, Drouin J, and Tremblay ML. Neuroendocrine dysplasia in mice lacking protein tyrosine phosphatase-sigma . Nat Genet 21: 330-333, 1999[ISI][Medline].

5.   IJsselstijn, H, Hung N, de Jongste JC, Tibboel D, and Cutz E. Calcitonin gene-related peptide expression is altered in pulmonary neuroendocrine cells in developing lungs of rats with congenital diaphragmatic hernia. Am J Respir Cell Mol Biol 19: 278-285, 1998[Abstract/Free Full Text].

6.   Kamiguchi, H, and Lemmon V. IgCAMs: bidirectional signals underlying neurite growth. Curr Opin Cell Biol 12: 598-605, 2000[ISI][Medline].

7.   Katsura, H, Williams MC, Brody JS, and Yu Q. Two closely related receptor-type tyrosine phosphatases are differentially expressed during rat lung development. Dev Dyn 204: 89-97, 1995[ISI][Medline].

8.   Kidd, T, Brose K, Mitchell KJ, Fetter RD, Tessier-Lavigne M, Goodman CS, and Tear G. Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily conserved guidance receptors. Cell 92: 205-215, 1998[ISI][Medline].

9.   Kim, H, Yeger H, Han R, Wallace M, Goldstein B, and Rotin D. Expression of LAR-PTP2 in rat lung is confined to proliferating epithelia lining the airways and air sacs. Am J Physiol Lung Cell Mol Physiol 270: L566-L576, 1996[Abstract/Free Full Text].

10.   MacDonald, JA. Lung Growth and Development. New York: Dekker, 1997.

11.   Massaro, D. Lung Cell Biology. New York: Dekker, 1989.

12.   McLean, J, Batt J, Doering L, Rotin D, and Bain J. Enhanced rate of nerve regeneration and directional errors following sciatic nerve injury in PTP-sigma -knockout mice. J Neurosci 22: 5481-5491, 2002[Abstract/Free Full Text].

13.   Pulido, R, Serra-Pages C, Tang M, and Streuli M. The LAR/PTP-delta /PTP-sigma , a subfamily of transmembrane protein-tyrosine-phosphatases: multiple human LAR, PTP-delta , and PTP-sigma isoforms are expressed in a tissue-specific manner and associate with the LAR-interacting protein LIP.1. Proc Natl Acad Sci USA 92: 11686-11690, 1995[Abstract].

14.   Rotin, D, Goldstein BJ, and Fladd CA. Expression of the tyrosine phosphatase LAR-PTP2 is developmentally regulated in lung epithelia. Am J Physiol Lung Cell Mol Physiol 267: L263-L270, 1994[Abstract/Free Full Text].

15.   Schaapveld, RQ, Schepens JT, Bachner D, Attema J, Wieringa B, Jap PH, and Hendriks WJ. Developmental expression of the cell adhesion molecule-like protein tyrosine phosphatases LAR, RPTP-delta and RPTP-sigma in the mouse. Mech Dev 77: 59-62, 1998[ISI][Medline].

16.   Schaapveld, RQ, Schepens JT, Robinson GW, Attema J, Oerlemans FT, Fransen JA, Streuli M, Wieringa B, Hennighausen L, and Hendriks WJ. Impaired mammary gland development and function in mice lacking LAR receptor-like tyrosine phosphatase activity. Dev Biol 188: 134-146, 1997[ISI][Medline].

17.   Skarnes, WC, Moss JE, Hurtley SM, and Beddington RS. Capturing genes encoding membrane and secreted proteins important for mouse development. Proc Natl Acad Sci USA 92: 6592-6596, 1995[Abstract].

18.   Van Lieshout, EM, Van der Heijden I, Hendriks WJ, and Van der Zee CE. A decrease in size and number of basal forebrain cholinergic neurons is paralleled by diminished hippocampal cholinergic innervation in mice lacking leukocyte common antigen-related protein tyrosine phosphatase activity. Neuroscience 102: 833-841, 2001[ISI][Medline].

19.   Wagner, J, Boerboom D, and Tremblay ML. Molecular cloning and tissue-specific RNA processing of a murine receptor-type protein tyrosine phosphatase. Eur J Biochem 226: 773-782, 1994[Abstract].

20.   Wallace, MJ, Batt J, Fladd CA, Henderson JT, Skarnes W, and Rotin D. Neuronal defects and posterior pituitary hypoplasia in mice lacking the receptor tyrosine phosphatase PTP-sigma . Nat Genet 21: 334-338, 1999[ISI][Medline].

21.   Wallace, MJ, Fladd C, Batt J, and Rotin D. The second catalytic domain of protein tyrosine phosphatase-delta (PTP-delta ) binds to and inhibits the first catalytic domain of PTP-sigma . Mol Cell Biol 18: 2608-2616, 1998[Abstract/Free Full Text].

22.   Walton, KM, Martell KJ, Kwak SP, Dixon JE, and Largent BL. A novel receptor-type protein tyrosine phosphatase is expressed during neurogenesis in the olfactory neuroepithelium. Neuron 11: 387-400, 1993[ISI][Medline].

23.   Wang, H, Yan H, Canoll PD, Silvennoinen O, Schlessinger J, and Musacchio JM. Expression of receptor protein tyrosine phosphatase-sigma (RPTP-sigma ) in the nervous system of the developing and adult rat. J Neurosci Res 41: 297-310, 1995[ISI][Medline].

24.   Xian, J, Clark KJ, Fordham R, Pannell R, Rabbitts TH, and Rabbitts PH. Inadequate lung development and bronchial hyperplasia in mice with a targeted deletion in the Dutt1/Robo1 gene. Proc Natl Acad Sci USA 98: 15062-15066, 2001[Abstract/Free Full Text].

25.   Yan, H, Grossman A, Wang H, D'Eustachio P, Mossie K, Musacchio JM, Silvennoinen O, and Schlessinger J. A novel receptor tyrosine phosphatase-sigma that is highly expressed in the nervous system. J Biol Chem 268: 24880-24886, 1993[Abstract/Free Full Text].

26.   Yeo, TT, Yang T, Massa SM, Zhang JS, Honkaniemi J, Butcher LL, and Longo FM. Deficient LAR expression decreases basal forebrain cholinergic neuronal size and hippocampal cholinergic innervation. J Neurosci Res 47: 348-360, 1997[ISI][Medline].

27.   Zhang, WR, Hashimoto N, Ahmad F, Ding W, and Goldstein BJ. Molecular cloning and expression of a unique receptor-like protein-tyrosine-phosphatase in the leucocyte-common-antigen-related phosphate family. Biochem J 302: 39-47, 1994[ISI][Medline].


Am J Physiol Lung Cell Mol Physiol 284(1):L214-L223
1040-0605/03 $5.00 Copyright © 2003 the American Physiological Society




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Batt, J.
Articles by Rotin, D.
Articles citing this Article
PubMed
PubMed Citation
Articles by Batt, J.
Articles by Rotin, D.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online