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The American Journal of Pathology logoLink to The American Journal of Pathology
. 2009 Jun;174(6):2015–2022. doi: 10.2353/ajpath.2009.090053

Loss of Matriptase Suppression Underlies Spint1 Mutation-Associated Ichthyosis and Postnatal Lethality

Roman Szabo *, Peter Kosa *, Karin List †, Thomas H Bugge *
PMCID: PMC2684167  PMID: 19389929

Abstract

Hepatocyte growth factor activator inhibitor-1 (HAI)-1 is an epithelial Kunitz-type transmembrane serine protease inhibitor that is encoded by the SPINT1 gene. HAI-1 displays potent inhibitory activity toward a large number of trypsin-like serine proteases. HAI-1 was recently shown to play an essential role in postnatal epithelial homeostasis. Thus, Spint1-deficient mice were found to display severe growth retardation and are unable to survive beyond postnatal day 16. The mice present histologically with overt hyperkeratosis of the forestomach, hyperkeratosis and acanthosis of the epidermis, and hypotrichosis associated with abnormal cuticle development. In this study, we show that loss of inhibition of a proteolytic pathway that is dependent on the type II transmembrane serine protease, matriptase, underlies the detrimental effects of postnatal Spint1 deficiency. Matriptase and HAI-1 precisely co-localize in all tissues that are affected by the Spint1 disruption. Spint1-deficient mice that have low matriptase levels, caused by a hypomorphic mutation in the St14 gene that encodes matriptase, not only survived the neonatal period but were healthy and displayed normal long-term survival. Furthermore, a detailed histological analysis of neonatal, young adult, as well as aged mice did not reveal any abnormalities in Spint1-deficent mice that have low matriptase levels. This study identifies matriptase suppression as an essential function of HAI-1 in postnatal tissue homeostasis.

Trypsin-like serine proteases constitute a large family of secreted and membrane-bound proteolytic enzymes that operate in the pericellular environment to facilitate embryonic development tissue homeostasis, tissue remodeling, and tissue repair. These enzymes are synthesized as inactive precursors, or zymogens, that are converted to their catalytically active form by endoproteolytic cleavage within a highly conserved activation site. This activation step is irreversible, and proteolysis is terminated by specific macromolecular protease inhibitors that bind directly to the active site of their cognate proteases.1,2,3 Inhibition of trypsin-like serine proteases is mediated by a large number of serine protease inhibitors belonging to one of three structurally and functionally distinct families: serpin-type inhibitors, Kazal-type inhibitors, and Kunitz-type inhibitors.1,2,3 Hepatocyte growth factor activator-1 (HAI-1) is a recently discovered membrane-associated Kunitz-type serine protease inhibitor. This peculiar serine protease inhibitor, which is structurally distinct from other previously described Kunitz-type inhibitors, is a type I transmembrane glycoprotein that consists of an N-terminal MANEC-domain followed by two extracellular Kunitz-type inhibitory domains that are separated by a single low-density lipoprotein receptor type a repeat, and a C-terminal transmembrane/cytoplasmic tail domain.4,5,6,7,8 HAI-1 is widely expressed in embryonic and extra-embryonic epithelia of the developing embryo, as well as in simple, stratified and pseudo-stratified epithelia of the adult organs of humans and mice.9,10,11,12,13 HAI-1 was originally identified as the cognate inhibitor of the trypsin-like serine protease, hepatocyte growth factor activator. However, the isolated Kunitz-type inhibitor domains of HAI-1 were subsequently shown to display potent inhibitory activity toward a large number of other trypsin-like serine proteases when analyzed in vitro, suggesting that HAI-1 could have multiple inhibitory targets and be a key player in many physiological processes.14,15,16,17,18,19,20

The widespread epithelial expression of HAI-1 in embryonic and adult epithelial tissues would also predict an essential function of the transmembrane serine protease inhibitor in mammalian development and adult tissue homeostasis. Indeed, inactivation of the Spint1 gene encoding HAI-1 in mice resulted in embryonic lethality at mid-gestation due to placental failure caused by agenesis of the placental labyrinth.13,21,22 This uniform lethality of Spint1 null embryos at mid-gestation has complicated the analysis of the function of HAI-1 in adult tissue homeostasis. However, a recent elegant study by Nagaike and co-workers bypassed the embryonic lethality imposed by the placental requirement for HAI-1 by injection of Spint1 null embryonic stem cells into wild-type blastocysts, leading to the generation of chimeric mice with high-grade deletion of Spint1 in the embryo proper, but sufficient placental Spint1 to support development to term.23 These high-grade Spint1−/− chimeric mice were unremarkable at birth. However, they uniformly suffered from severe growth retardation and were incapable of surviving beyond postnatal day 16. Histopathological examination revealed that Spint1−/− mice displayed a range of abnormalities of keratinized epithelium, including hyperplasia of the forestomach, severe ichthyosis of the skin with inflammatory cell infiltration, and abnormal hair development. However, the molecular basis for these dramatic manifestations of HAI-1 ablation was not determined, and several possible explanations were proposed by the authors of the study.23

Matriptase (also known as MT-SP1, epithin, and TADG15) is a modular trypsin-like serine protease encoded by the ST14 gene that belongs to the recently established type II transmembrane serine protease family.24,25,26,27,28,29,30 Like HAI-1, matriptase is widely expressed in both embryonic and adult epithelia of mice and humans.9,10,11,13,22,31 Homozygosity for mutations in the ST14 gene was recently shown to be the etiological origin of two human syndromes termed congenital ichthyosis, follicular atrophoderma, hypotrichosis, and hypohidrosis and autosomal recessive ichthyosis with hypotrichosis.32,33,34,35,36 Mice with wholesale deletion of the St14 gene complete embryonic development, but they die shortly after birth as a consequence of compromised epidermal barrier function.37,38 However, an St14 hypomorphic mouse strain, generated by insertion of a retroviral targeting vector into intron 1 of the St14 gene, which displays 1% residual matriptase mRNA levels in the epidermis and up to 18% residual mRNA levels in other tissues, has normal postnatal and long-term survival, revealing that low matriptase suffices to maintain epithelial homeostasis.39

In the present study, we hypothesized that the detrimental effects of postnatal loss of HAI-1 were caused by excess matriptase activity. Indeed, using a genetic approach, we demonstrate in this study that the requirement of HAI-1 for postnatal mouse development and epithelial homeostasis is mechanistically linked to the inhibition of matriptase. Thus, whereas Spint1−/− mice with one or two wild-type St14 alleles died in utero, Spint1−/− mice on the St14 hypomorphic mouse background not only completed embryonic development, but were viable, healthy, and displayed normal long-term survival. This study identifies matriptase as a critical inhibitory target for HAI-1 in keratinized squamous epithelium, shows that HAI-1 inhibition of protease targets besides matriptase is not essential for mouse health and long-term survival, and reveals that a delicate balance between matriptase and HAI-1 maintains homeostasis of adult mammalian tissues.

Materials and Methods

Mice and Tissue Acquisition

All procedures involving live animals were performed in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited vivarium, following Institutional Guidelines and standard operating procedures. The generation and genotyping of the Spint1 null mice and β-galactosidase-tagged St14 hypomorphic mice have been described.13,39,40 Histological analysis, immunohistochemistry and whole mount X-gal staining was performed exactly as described.10,13,31,38,40

Blood Chemistry Analysis

To isolate serum, blood was collected from the periportal vein of freshly euthanized animals, clotted for 2 hours at room temperature, and centrifuged at 800 × g for 10 minutes at room temperature. The clear upper phase was transferred into a new tube and immediately used for the analysis or stored at −80°C. The biochemical analysis of mouse sera from at least three animals of each genotype and sex was performed by the Department of Laboratory Medicine at the National Institutes of Health Clinical Center (Bethesda, MD).

Intestinal Epithelial Permeability

Ten μl per gram of body weight of 22 mg/ml fluorescein isothiocyanate (FITC)-labeled dextran (average 4,000 g/mol, Sigma, St. Louis, MO) in PBS was injected directly into the stomach of 6 month-old animals using a 1.5-inch long, bulb-tipped gastric gavage needle (Roboz, Gaithersburg, MD) attached to a 1-ml syringe. After four h, the animals were euthanized by CO2 inhalation, blood was extracted, and serum was prepared as described above. Fifty μl of serum was then diluted 1:3 in 1× PBS (Gibco-Invitrogen, Carlsbad, CA) and the concentration of FITC-dextran was determined by reading the fluorescence at 535 nm after excitation at 485 nm using a Victor3V spectrophotometer (PerkinElmer, Waltham, MA).

Detection of Matriptase Protein in Mouse Skin

Whole skins from newborn mice were homogenized in glass homogenizers in ice-cold RIPA buffer (50 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, 1% NP-40, 0.1% SDS) with a protease inhibitor cocktail (Sigma). The homogenate was cleared by centrifugation for 10 minutes at 16,000 × g at 4°C. The protein content was determined with the BCA protein assay kit (Thermo Scientific, Waltham, MA) according to the manufacturer’s instructions. Seventy-five μg of total protein and 15 ng of recombinant mouse matriptase serine protease domain (R&D Systems, Minneapolis, MN) was separated on 4% to 12% NuPAGE Bis/Tris gel (Invitrogen, Carlsbad, CA) under reducing conditions and transferred onto polyvinylidene difluoride membrane (Invitrogen). Matriptase was detected using 1 μg/ml sheep anti-human matriptase primary antibody (R&D Systems) and donkey anti-sheep secondary antibody conjugated to alkaline phosphatase (Sigma), diluted 1:10,000.

Body Fat Content Determination

Mice were anesthetized by isoflurane inhalation and the body area excluding the head and the tail was subjected to a dual energy X-ray absorptiometry whole body scan using a Lunar PIXImus small animal scanner (GE Lunar Corp., Madison, WI) according to the manufacturer’s instructions. The amount of body fat (g) and body fat content (%) were then determined using PIXImus software. At least four animals of each genotype and sex were analyzed.

Results

Low Matriptase Negates HAI-1 Requirement during Early Postnatal Development

Matriptase co-localizes with HAI-1 in the epidermis (Figure 1A, 1B) and can be detected in its proteolytically activated form (Figure 1C). Mice with a combined wholesale deletion of the Spint1 and St14 genes complete embryonic development, but die shortly after birth as a consequence of matriptase deficiency, thus precluding the analysis of the importance of matriptase regulation by HAI-1 in adult tissue homeostasis.13 On the other hand, greatly lowering matriptase levels in mice has no major adverse effect on postnatal health and survival.39 Therefore, to query the potential role of excess matriptase proteolytic activity in the postnatal lethality of mice devoid of HAI-1,23 we reduced the level of matriptase in Spint1−/− null mice, rather than eliminate the protease altogether. Spint1+/− mice were first bred with mice carrying one St14 hypomorphic allele and one St14 null allele (St14−/hypo) mice. Breeding of the ensuing St14+/hypo;Spint1+/− offspring to St14+/−;Spint1+/− offspring subsequently produced the following strains: Spint1+/+ and Spint1+/− mice on an St14+/+, St14+/hypo, or St14+/− background (hereafter termed St14+;Spint1+ mice), Spint1−/− mice on an St14+/+, St14+/hypo or St14+/− background (hereafter termed St14+;Spint1−/− mice), Spint1+/+ and Spint1+/− mice on an St14−/hypo background (hereafter termed St14−/hypo;Spint1+ mice), and Spint1−/− mice on an St14−/hypo background (hereafter termed St14−/hypo;Spint1−/− mice). In agreement with previous studies,13,21,22 no St14+;Spint1−/− mice were identified among 119 offspring genotyped at weaning (Figure 1D). Interestingly, however, St14−/hypo;Spint1−/− mice were found with the expected frequency in weaned offspring, revealing that reduced levels of matriptase negate not only the requirement for placental HAI-1 expression during development, but also the requirement of HAI-1 for survival within the postnatal period. The reported demise of high-grade Spint1−/− chimeric mice is preceded by severe growth retardation (30% at postnatal day 7 and 60% at day 13) when compared with their low-grade chimeric littermates.23 To determine the possible contribution of excess matriptase activity to the failure of HAI-1-deficient pups to gain weight during the pre-weaning period, we determined the weight of St14−/hypo;Spint1−/− pups and their St14−/hypo;Spint1+; and St14+;Spint1+ littermates every 24 hours between postnatal day 1 and 19 (Figure 1E). Interestingly, St14−/hypo;Spint1−/− pups displayed only a minimal residual growth retardation, when compared with either St14−/hypo;Spint1+ or St14+;Spint1+ littermates. Like high-grade Spint1−/− chimeric pups,23 St14−/hypo;Spint1−/− pups were unremarkable at birth (data not shown). Unlike high-grade Spint1−/− chimeric pups, however, St14−/hypo;Spint1−/− pups remained outwardly normal and did not develop pronounced hyperkeratosis, or grossly abnormal pelage hair and whiskers, displaying only the minimal phenotypes observed in their St14−/hypo;Spint1+ littermates39 (Figure 1F and 2A). In agreement with the outward healthy appearance, a careful histological examination of keratinized tissues (Figure 1G, H, I, and J, and data not shown) as well as non-keratinized tissues (data not shown) of pre-weaning St14−/hypo;Spint1−/− pups revealed no apparent abnormalities. Notably, the pervasive epidermal acanthosis with neutrophilic infiltration reported in high-grade Spint1−/− chimeric pups23 was entirely absent. The hair follicle morphology was also unremarkable, as was the appearance of the forestomach. Taken together, these data show that lowering the level of matriptase in mice negates the strict requirement of HAI-1 for growth, survival, and tissue homeostasis and restores normal early postnatal development.

Figure 1.

Figure 1

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Decrease in matriptase activity rescues postnatal lethality, ichthyosis and hair follicle defects in HAI-1-deficient mice. A–B: Matriptase (A, blue staining) and HAI-1 (B, brown staining) co-localize in hair follicle matrix (A, B arrowheads) and in suprabasal layers of interfolicullar epidermis (A, B arrows). C: Reduced SDS-polyacrylamide gel electrophoresis/Western blot of skin extracts from two pairs of newborn wild-type St14+/+ mice (lanes 1 and 3), littermate St14−/− mice (lane 2 and 4), and 15 ng purified mouse recombinant active matriptase serine protease domain (rMatSP, lane 5). The presence of a ∼30 kDa immunoreactive protein species in St14+/+ (indicated by arrow at right), but not in St14−/− extracts, which co-migrates with purified recombinant matriptase serine protease domain shows the presence of activated matriptase in the epidermis. Bands observed in both St14+/+ and St14−/− extracts represent cross reactivity. The positions of molecular weight markers in kDa are indicated at left. D: Distribution of genotypes of newborn mice from St14+/−;Spint1+/− × St14+/hypo;Spint1+/− breeding pairs. No Spint1−/− pups were detected that carried one or two wild-type St14 alleles (St14+), while Spint1−/− pups on an St14−/hypo background were present with the expected Mendelian frequency. E: Average pre-weaning weight gain of St14+;Spint1+ (N = 54), St14−/hypo;Spint1+ (N = 7), and St14−/hypo;Spint1−/− (N = 14) pups. F: Representative example of the outward appearance of St14−/hypo;Spint1−/−, St14−/hypo;Spint1+, and St14+;Spint1+ mice at day 17. G–J: H&E staining of histological sections of skin (G, H) and forestomach (I, J) of 17 day-old St14−/hypo;Spint1−/− mice (G, I) and their St14+;Spint1+ littermates (H, J). No hyperkeratosis, acanthosis, or neutrophil infiltration has been observed in HAI-1-deficient tissues with low matriptase. Scale bars = 25 μm (G, H); 50 μm (I, J). Scale bars for A and B = 50 μm.

Figure 2.

Figure 2

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HAI-1 is dispensable for postnatal development and long-term survival in the presence of low matriptase activity. A: Macroscopic image of the head region of a 17 day-old St14−/hypo;Spint1−/− mouse showing normal development of whiskers and lack of scaling on ears and around eyes. B: Long term survival of female St14+;Spint1+ (N = 29), St14−/hypo;Spint1+ (N = 25), and St14−/hypo;Spint1−/− (11), and male St14+;Spint1+ (N = 26), St14−/hypo;Spint1+ (N = 20), and St14−/hypo;Spint1−/− (N = 8) mice. No effect of the loss of HAI-1 on overall survival of mice with low matriptase activity was observed. C: Representative example of the appearance of 9 months-old control St14+;Spint1+, St14−/hypo;Spint1−/−, and St14−/hypo;Spint1+ mice. HAI-1-deficient mice with low matriptase are outwardly normal (D–O). H&E staining (D–N) and HAI-1 immunostaining (E–O) of histological sections of skin (D–G), esophagus (H–K), and forestomach (H–O) of 12 month-old St14+;Spint1+ (D, E; H, I; L, M) or St14−/hypo;Spint1−/− (F, G; J, K; N, O) mice. No apparent abnormalities were detected in any of the keratinized tissues of HAI-1-deficient mice with low matriptase. Arrows indicate the expression of HAI-1 protein. Scale bars = 25 μm (D–O).

Normal Long-Term Survival, but Reduced Body Fat Accumulation in HAI-1-Deficient Mice with Low Matriptase

Prospective cohorts of male and female St14−/hypo;Spint1−/− mice and their St14−/hypo;Spint1+ or St14+;Spint1+ littermates were next established to determine the long-term consequences of complete HAI-1 deficiency in mice with low matriptase. The mice were monitored by bi-weekly weight determination and visual inspection for 1 year. At the termination of the experiment, the mice were subjected to body-fat mass and blood chemistry analysis, as well as detailed histopathological examination. No increase in postweaning mortality was observed in neither male nor female St14−/hypo;Spint1−/− mice (Figure 2B). Likewise, the appearance of skin, fur, whiskers, teeth, eyes, and external genitalia of these mice was indistinguishable from St14−/hypo;Spint1+ or St14+;Spint1+ littermates (Figure 2C and data not shown). The microscopic appearance of the interfollicular and follicular epidermis (Figure 2, D–G), esophagus (Figure 2, H–K), and forestomach (Figure 2, L–O), as well as all other organs examined (data not shown) was unremarkable. These findings demonstrate that the complete loss of HAI-1 has no major adverse effect on the long-term health of mice with low matriptase in the absence of intrinsic or external challenges. However, both female and male mice were markedly leaner than St14−/hypo;Spint1+ or St14+;Spint1+ littermates (Figure 3A and B). Peripheral instantaneous X-ray imager analysis confirmed that body fat accumulation in St14−/hypo;Spint1−/− female mice was significantly reduced (71% and 65% relative to St14−/hypo;Spint1+ or St14+;Spint1+ mice, respectively), whereas the reduction in body fat content in St14−/hypo;Spint1−/− male mice did not reach significance. However, measurement of body length revealed that both male and female St14−/hypo;Spint1−/− mice were slightly shorter in stature (females: 90% and 89% relative to St14−/hypo;Spint1+ or St14+;Spint1+ mice, respectively, P < 0.01; males: 96% and 94% relative to St14−/hypo;Spint1+ or St14+;Spint1+ mice, respectively, did not reach significance). Both HAI-1 and matriptase are highly expressed throughout the mouse gastrointestinal tract.10,31,40 However, the intestinal barrier permeability in St14−/hypo;Spint1−/− mice, as measured by FITC-dextran gavage, was similar to that of control St14+;Spint1+ mice (Figure 3C). Likewise, blood chemistry analysis was uninformative as to the cause of the reduced body fat accumulation and shorter stature (Figure 3, D and E, and data not shown).

Figure 3.

Figure 3

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Decreased body weight but normal intestinal barrier function and biochemical parameters in HAI-1-deficient mice with low matriptase. A, B: Body weight of 6 to 52 week-old female (A) St14+;Spint1+ (N = 29), St14−/hypo;Spint1+ (N = 25), and St14−/hypo;Spint1−/− (N = 11), and male (B) St14+;Spint1+ (N = 26), St14−/hypo;Spint1+ (N = 20), and St14−/hypo;Spint1−/− (N = 8) mice. C: Concentration of FITC-labeled dextran in the blood of 12 month-old St14+;Spint1+ (females N = 6, males N = 12) and St14−/hypo;Spint1−/− (females N = 4, males N = 5) mice 4 hours after instillation of FITC-labeled dextran into the stomach by oral gavage. No significant increase in intestinal permeability was observed in HAI-1-deficient animals with low matriptase. Error bars indicate SD (D, E). Biochemical analysis of the blood of 12 month St14+;Spint1+ (females N = 10 (D), males N = 15 (E), St14−/hypo;Spint1+ (females N = 10, males N = 14), and St14−/hypo;Spint1−/− (females N = 3, males N = 5) mice. No significant differences between HAI-1-expressing and HAI-1-deficient animals were identified. Error bars indicate SD. Abbreviations: ALT/GPT, alanine aminotransferase/glutamate-pyruvate transaminase; AST/GOT, aspartate aminotransferase/glutamate-oxaloacetate transaminase; BUN, blood urea nitrogen; CK, creatine kinase; LD, lactate dehydrogenase.

Discussion

The current study provides the mechanistic explanation for the requirement of HAI-1 for maintenance of epithelial integrity within the postnatal period.23 Indeed, we identified the suppression of proteolysis dependent on the type II transmembrane serine protease matriptase as a critical in vivo function for HAI-1. Thus, whereas HAI-1-deficient mice displayed severe growth retardation and uniform lethality within the early postnatal period and presented with profound defects in keratinized epithelium, HAI-1-deficient mice with a low level of matriptase gained weight at near-normal rates in the postnatal period, had unaltered long-term survival, were outwardly healthy, and histologically unremarkable. Matriptase is an autoactivating protease that is capable of activating other serine protease zymogens, including the glycosylphosphatidylinositol-anchored membrane serine protease, prostasin, and receptor-bound urokinase plasminogen activator.41,42,43,44 Therefore, it is formally possible that HAI-1 does not exert its essential inhibitory functions through a direct interaction with matriptase, but rather acts on target proteases whose activation is matriptase-dependent. However, HAI-1 and matriptase co-localize in tissues affected by the absence of HAI-1 and a proteolytically shed form of HAI-1 has been found in a complex with shed matriptase in conditioned medium from immortalized epithelial cells and even in human milk.45 Taken together, this suggests that HAI-1 most likely maintains tissue homeostasis through direct inhibition of matriptase.

This study identifies HAI-1 as one critical postnatal inhibitor of matriptase. However, it does not exclude that other potential matriptase inhibitors, such as HAI-2,10 antithrombin III, α1-antitrypsin, and α2-antiplasmin46 have equally important roles in the regulation of matriptase activity in the epidermis or in other adult tissues.

Despite the widespread expression of HAI-1 and its potent inhibitory activity against multiple trypsin-like proteases, we nevertheless found that reduction of matriptase-dependent proteolysis in mice devoid of HAI-1 sufficed to ensure normal postnatal development, health, and long-term survival. Thus, the direct inhibition of hepatocyte growth factor activator, trypsin, hepsin, prostasin, or other trypsin-like serine proteases was not critical to maintain tissue homeostasis. Functional redundancy in the inhibition of these other potential targets is one plausible explanation. In this regard it should be noted that HAI-1 and the closely related inhibitor HAI-2 (placental bikunin) display highly overlapping protease specificity in vitro and co-localize in most adult epithelia.10,14,15,16,17,18,19,20 Alternatively, HAI-1 inhibition of these other candidate protease targets may not be physiologically relevant or may only become critical in the context of external environmental or intrinsic challenges, such as tissue injury, infection or metabolic restrictions.

Reduced accumulation of body fat was the only notable feature that distinguished HAI-1-deficient mice with low matriptase levels from mice with low matriptase. No obvious reason for the leanness of these mice was pinpointed by histological, intestinal barrier function, or blood chemistry analysis. Two molecular explanations for this are likely: inadequate inhibition of a target protease different from matriptase, or excessive or spatially undesired activity of the residual matriptase. In support of the latter, we have found that supraphysiological levels of matriptase can cause the rapid demise in multiple epithelia (Karin List, Peter Kosa, Roman Szabo, and Thomas H. Bugge, unpublished data). In summary, the current study has revealed that inhibition of the type II transmembrane serine protease, matriptase, is an essential function of HAI-1 that maintains adult tissue homeostasis.

Acknowledgments

We thank Dr. Alfredo Molinolo for expert pathology support, and Dr. Silvio Gutkind and Dr. Mary Jo Danton for critically reviewing this manuscript.

Footnotes

Address reprint requests to Thomas H. Bugge, Ph.D., Proteases and Tissue Remodeling Section, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, Room 211, Bethesda, MD 20892. E-mail: thomas.bugge@nih.gov.

Supported by the NIDCR Intramural Research Program (T.H.B.) and by startup funds from Wayne State University (K.L.).

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