Vertebrate Lrig3-ErbB Interactions Occur In Vitro but Are Unlikely to Play a Role in Lrig3-Dependent Inner Ear Morphogenesis

Background

The Lrig genes encode a family of transmembrane proteins that have been implicated in tumorigenesis, psoriasis, neural crest development, and complex tissue morphogenesis. Whether these diverse phenotypes reflect a single underlying cellular mechanism is not known. However, Lrig proteins contain evolutionarily conserved ectodomains harboring both leucine-rich repeats and immunoglobulin domains, suggesting an ability to bind to common partners. Previous studies revealed that Lrig1 binds to and inhibits members of the ErbB family of receptor tyrosine kinases by inducing receptor internalization and degradation. In addition, other receptor tyrosine kinase binding partners have been identified for both Lrig1 and Lrig3, leaving open the question of whether defective ErbB signaling is responsible for the observed mouse phenotypes.

Methodology/Principal Findings

Here, we report that Lrig3, like Lrig1, is able to interact with ErbB receptors in vitro. We examined the in vivo significance of these interactions in the inner ear, where Lrig3 controls semicircular canal formation by determining the timing and extent of Netrin1 expression in the otic vesicle epithelium. We find that ErbB2 and ErbB3 are present in the early otic epithelium, and that Lrig3 acts cell-autonomously here, as would be predicted if Lrig3 regulates ErbB2/B3 activity. However, inhibition of ErbB activation in the chick otic vesicle has no detectable effect on Netrin gene expression or canal morphogenesis.

Conclusions/Significance

Our results suggest that although both Lrig1 and Lrig3 can interact with ErbB receptors in vitro, modulation of Neuregulin signaling is unlikely to contribute to Lrig3-dependent processes of inner ear morphogenesis. These results highlight the similar binding properties of Lrig1 and Lrig3 and underscore the need to determine how these two family members bind to and regulate different receptors to affect diverse aspects of cell behavior in vivo.     

Hmx homeobox gene function in inner ear and nervous system cell-type specification and development

The Hmx homeobox gene family is comprised of three members in mammals, Hmx1, Hmx2, and Hmx3, which are conserved across the animal kingdom and are part of the larger NKL clustered family of homeobox genes. Expression domains of Hmx genes in distantly related species such as Drosophila and mouse suggest an ancestral function in rostral central nervous system development. During vertebrate evolution, the Hmx genes appear to have been recruited into additional roles in inner ear morphogenesis and specification of vestibular inner ear sensory and supporting cell types. Being derived from a common ancestor, the vertebrate Hmx gene family is thus a strong candidate to investigate functional overlap versus the unique roles played by multiple genes belonging to the same family. The functions of Hmx2 and Hmx3 were investigated via directed gene mutagenesis and the primary regions where Hmx2 and Hmx3 exert their individual functions are consistent with their expression domains, such as the vestibule and uterus. Meanwhile, it is notable that some tissues where both Hmx2 and Hmx3 are extensively expressed were not severely affected in either of the Hmx2 or Hmx3 single mutant mice, suggesting a possible functional overlap existing between these two genes. Compound Hmx2 and Hmx3 double mutant mice showed more severe defects in the inner ear than those displayed by either single knockout. Furthermore, novel abnormalities in the hypothalamic–neuroendocrine system, which were never observed in either of the single mutant mice, confirmed a hypothesis that Hmx2 and Hmx3 also function redundantly to control embryonic development of the central nervous system.     

A Late Role for bmp2b in the Morphogenesis of Semicircular Canal Ducts in the Zebrafish Inner Ear

Background

The Bone Morphogenetic Protein (BMP) genes bmp2 and bmp4 are expressed in highly conserved patterns in the developing vertebrate inner ear. It has, however, proved difficult to elucidate the function of BMPs during ear development as mutations in these genes cause early embryonic lethality. Previous studies using conditional approaches in mouse and chicken have shown that Bmp4 has a role in semicircular canal and crista development, but there is currently no direct evidence for the role of Bmp2 in the developing inner ear.

Methodology/Principal Findings

We have used an RNA rescue strategy to test the role of bmp2b in the zebrafish inner ear directly. Injection of bmp2b or smad5 mRNA into homozygous mutant swirl (bmp2b^−/−) embryos rescues the early patterning defects in these mutants and the fish survive to adulthood. As injected RNA will only last, at most, for the first few days of embryogenesis, all later development occurs in the absence of bmp2b function. Although rescued swirl adult fish are viable, they have balance defects suggestive of vestibular dysfunction. Analysis of the inner ears of these fish reveals a total absence of semicircular canal ducts, structures involved in the detection of angular motion. All other regions of the ear, including the ampullae and cristae, are present and appear normal. Early stages of otic development in rescued swirl embryos are also normal.

Conclusions/Significance

Our findings demonstrate a critical late role for bmp2b in the morphogenesis of semicircular canals in the zebrafish inner ear. This is the first demonstration of a developmental role for any gene during post-embryonic stages of otic morphogenesis in the zebrafish. Despite differences in the early stages of semicircular canal formation between zebrafish and amniotes, the role of Bmp2 in semicircular canal duct outgrowth is likely to be conserved between different vertebrate species.     

Hmx homeobox gene function in inner ear and nervous system cell-type specification and development

The Hmx homeobox gene family is comprised of three members in mammals, Hmx1, Hmx2, and Hmx3, which are conserved across the animal kingdom and are part of the larger NKL clustered family of homeobox genes. Expression domains of Hmx genes in distantly related species such as Drosophila and mouse suggest an ancestral function in rostral central nervous system development. During vertebrate evolution, the Hmx genes appear to have been recruited into additional roles in inner ear morphogenesis and specification of vestibular inner ear sensory and supporting cell types. Being derived from a common ancestor, the vertebrate Hmx gene family is thus a strong candidate to investigate functional overlap versus the unique roles played by multiple genes belonging to the same family. The functions of Hmx2 and Hmx3 were investigated via directed gene mutagenesis and the primary regions where Hmx2 and Hmx3 exert their individual functions are consistent with their expression domains, such as the vestibule and uterus. Meanwhile, it is notable that some tissues where both Hmx2 and Hmx3 are extensively expressed were not severely affected in either of the Hmx2 or Hmx3 single mutant mice, suggesting a possible functional overlap existing between these two genes. Compound Hmx2 and Hmx3 double mutant mice showed more severe defects in the inner ear than those displayed by either single knockout. Furthermore, novel abnormalities in the hypothalamic-neuroendocrine system, which were never observed in either of the single mutant mice, confirmed a hypothesis that Hmx2 and Hmx3 also function redundantly to control embryonic development of the central nervous system.     

BH3-Only Protein BIM Mediates Heat Shock-Induced Apoptosis

Acute heat shock can induce apoptosis through a canonical pathway involving the upstream activation of caspase-2, followed by BID cleavage and stimulation of the intrinsic pathway. Herein, we report that the BH3-only protein BIM, rather than BID, is essential to heat shock-induced cell death. We observed that BIM-deficient cells were highly resistant to heat shock, exhibiting short and long-term survival equivalent to Bax^−/−Bak^−/− cells and better than either Bid^−/− or dominant-negative caspase-9-expressing cells. Only Bim^−/− and Bax^−/−Bak^−/− cells exhibited resistance to mitochondrial outer membrane permeabilization and loss of mitochondrial inner membrane potential. Moreover, while dimerized caspase-2 failed to induce apoptosis in Bid^−/− cells, it readily did so in Bim^−/− cells, implying that caspase-2 kills exclusively through BID, not BIM. Finally, BIM reportedly associates with MCL-1 following heat shock, and Mcl-1^−/− cells were indeed sensitized to heat shock-induced apoptosis. However, pharmacological inhibition of BCL-2 and BCL-X_L with ABT-737 also sensitized cells to heat shock, most likely through liberation of BIM. Thus, BIM mediates heat shock-induced apoptosis through a BAX/BAK-dependent pathway that is antagonized by antiapoptotic BCL-2 family members.     

Failure of Fluid Absorption in the Endolymphatic Sac Initiates Cochlear Enlargement that Leads to Deafness in Mice Lacking Pendrin Expression

Mutations of SLC26A4 are among the most prevalent causes of hereditary deafness. Deafness in the corresponding mouse model, Slc26a4^−/−, results from an abnormally enlarged cochlear lumen. The goal of this study was to determine whether the cochlear enlargement originates with defective cochlear fluid transport or with a malfunction of fluid transport in the connected compartments, which are the vestibular labyrinth and the endolymphatic sac. Embryonic inner ears from Slc26a4^+/− and Slc26a4^−/− mice were examined by confocal microscopy ex vivo or after 2 days of organ culture. Culture allowed observations of intact, ligated or partially resected inner ears. Cochlear lumen formation was found to begin at the base of the cochlea between embryonic day (E) 13.5 and 14.5. Enlargement was immediately evident in Slc26a4^−/− compared to Slc26a4^+/− mice. In Slc26a4^+/− and Slc26a4^−/− mice, separation of the cochlea from the vestibular labyrinth by ligation at E14.5 resulted in a reduced cochlear lumen. Resection of the endolymphatic sacs at E14.5 led to an enlarged cochlear lumen in Slc26a4^+/− mice but caused no further enlargement of the already enlarged cochlear lumen in Slc26a4^−/− mice. Ligation or resection performed later, at E17.5, did not alter the cochlea lumen. In conclusion, the data suggest that cochlear lumen formation is initiated by fluid secretion in the vestibular labyrinth and temporarily controlled by fluid absorption in the endolymphatic sac. Failure of fluid absorption in the endolymphatic sac due to lack of Slc26a4 expression appears to initiate cochlear enlargement in mice, and possibly humans, lacking functional Slc26a4 expression.     

Heat Shock Factor Hsf1 Cooperates with ErbB2 (Her2/Neu) Protein to Promote Mammary Tumorigenesis and Metastasis

ErbB2/Neu oncogene is overexpressed in 25% of invasive/metastatic breast cancers. We have found that deletion of heat shock factor Hsf1 in mice overexpressing ErbB2/Neu significantly reduces mammary tumorigenesis and metastasis. Hsf1^+/−ErbB2/Neu⁺ tumors exhibit reduced cellular proliferative and invasive properties associated with reduced activated ERK1/2 and reduced epithelial-mesenchymal transition (EMT). Hsf1^+/+Neu⁺ mammary epithelial cells exposed to TGFβ show high levels of ERK1/2 activity and EMT; this is associated with reduced expression of E-cadherin and increased expression of Slug and vimentin, a mesenchymal marker. In contrast, Hsf1^−/−Neu⁺ or Hsf1^+/+Neu⁺ cells do not exhibit activated ERK1/2 and show reduced EMT in the presence of TGFβ. The ineffective activation of the RAS/RAF/MEK/ERK1/2 signaling pathway in cells with reduced levels of HSF1 is due to the low levels of HSP90 in complex with RAF1 that are required for RAF1 stability and maturation. These results indicate a powerful inhibitory effect conferred by HSF1 downstream target genes in the inhibition of ErbB2-induced breast cancers in the absence of the Hsf1 gene.     

Slc26a7 Chloride Channel Activity and Localization in Mouse Reissner’s Membrane Epithelium

Several members of the SLC26 gene family have highly-restricted expression patterns in the auditory and vestibular periphery and mutations in mice of at least two of these (SLC26A4 and SLC26A5) lead to deficits in hearing and/or balance. A previous report pointed to SLC26A7 as a candidate gene important for cochlear function. In the present study, inner ears were assayed by immunostaining for Slc26a7 in neonatal and adult mice. Slc26a7 was detected in the basolateral membrane of Reissner’s membrane epithelial cells but not neighboring cells, with an onset of expression at P5; gene knockout resulted in the absence of protein expression in Reissner’s membrane. Whole-cell patch clamp recordings revealed anion currents and conductances that were elevated for NO₃ ⁻ over Cl⁻ and inhibited by I⁻ and NPPB. Elevated NO₃ ⁻ currents were absent in Slc26a7 knockout mice. There were, however, no major changes to hearing (auditory brainstem response) of knockout mice during early adult life under constitutive and noise exposure conditions. The lack of Slc26a7 protein expression found in the wild-type vestibular labyrinth was consistent with the observation of normal balance. We conclude that SLC26A7 participates in Cl⁻ transport in Reissner’s membrane epithelial cells, but that either other anion pathways, such as ClC-2, possibly substitute satisfactorily under the conditions tested or that Cl⁻ conductance in these cells is not critical to cochlear function. The involvement of SLC26A7 in cellular pH regulation in other epithelial cells leaves open the possibility that SLC26A7 is needed in Reissner’s membrane cells during local perturbations of pH.     

Lack of Developmental Redundancy between Unc45 Proteins in Zebrafish Muscle Development

Since the majority of protein-coding genes in vertebrates have intra-genomic homologues, it has been difficult to eliminate the potential of functional redundancy from analyses of mutant phenotypes, whether produced by genetic lesion or transient knockdown. Further complicating these analyses, not all gene products have activities that can be assayed in vitro, where the efficiency of the various family members can be compared against constant substrates. Two vertebrate UNC-45 homologues, unc45a and unc45b, affect distinct stages of muscle differentiation when knocked down in cell culture and are functionally redundant in vitro. UNC-45 proteins are members of the UCS (UNC-45/CRO1/She4p) protein family that has been shown to regulate myosin-dependent functions from fungi to vertebrates through direct interaction with the myosin motor domain. To test whether the same functional relationship exists between these unc45 paralogs in vivo, we examined the developmental phenotypes of doubly homozygous unc45b^−/−; unc45a^−/− mutant zebrafish embryos. We focused specifically on the combined effects on morphology and gene expression resulting from the zygotic lack of both paralogs. We found that unc45b^−/− and unc45b^−/−; unc45a^−/− embryos were phenotypically indistinguishable with both mutants displaying identical cardiac, skeletal muscle, and jaw defects. We also found no evidence to support a role for zygotic Unc45a function in myoblast differentiation. In contrast to previous in vitro work, this rules out a model of functional redundancy between Unc45a and Unc45b in vivo. Instead, our phylogenetic and phenotypic analyses provide evidence for the role of functional divergence in the evolution of the UCS protein family.     

Vestibular Role of KCNQ4 and KCNQ5 K+ Channels Revealed by Mouse Models

The function of sensory hair cells of the cochlea and vestibular organs depends on an influx of K⁺ through apical mechanosensitive ion channels and its subsequent removal over their basolateral membrane. The KCNQ4 (K_v7.4) K⁺ channel, which is mutated in DFNA2 human hearing loss, is expressed in the basal membrane of cochlear outer hair cells where it may mediate K⁺ efflux. Like the related K⁺ channel KCNQ5 (K_v7.5), KCNQ4 is also found at calyx terminals ensheathing type I vestibular hair cells where it may be localized pre- or postsynaptically. Making use of Kcnq4^−/− mice lacking KCNQ4, as well as Kcnq4^dn/dn and Kcnq5^dn/dn mice expressing dominant negative channel mutants, we now show unambiguously that in adult mice both channels reside in postsynaptic calyx-forming neurons, but cannot be detected in the innervated hair cells. Accordingly, whole cell currents of vestibular hair cells did not differ between genotypes. Neither Kcnq4^−/−, Kcnq5^dn/dn nor Kcnq4^−/−/Kcnq5^dn/dn double mutant mice displayed circling behavior found with severe vestibular impairment. However, a milder form of vestibular dysfunction was apparent from altered vestibulo-ocular reflexes in Kcnq4^−/−/Kcnq5^dn/dn and Kcnq4^−/− mice. The larger impact of KCNQ4 may result from its preferential expression in central zones of maculae and cristae, which are innervated by phasic neurons that are more sensitive than the tonic neurons present predominantly in the surrounding peripheral zones where KCNQ5 is found. The impact of postsynaptic KCNQ4 on vestibular function may be related to K⁺ removal and modulation of synaptic transmission.     

Protein-Trap Insertional Mutagenesis Uncovers New Genes Involved in Zebrafish Skin Development,Including a Neuregulin 2a-Based ErbB Signaling Pathway Required during Median Fin Fold Morphogenesis

Skin disorders are widespread, but available treatments are limited. A more comprehensive understanding of skin development mechanisms will drive identification of new treatment targets and modalities. Here we report the Zebrafish Integument Project (ZIP), an expression-driven platform for identifying new skin genes and phenotypes in the vertebrate model Danio rerio (zebrafish). In vivo selection for skin-specific expression of gene-break transposon (GBT) mutant lines identified eleven new, revertible GBT alleles of genes involved in skin development. Eight genes—fras1, grip1, hmcn1, msxc, col4a4, ahnak, capn12, and nrg2a—had been described in an integumentary context to varying degrees, while arhgef25b, fkbp10b, and megf6a emerged as novel skin genes. Embryos homozygous for a GBT insertion within neuregulin 2a (nrg2a) revealed a novel requirement for a Neuregulin 2a (Nrg2a) – ErbB2/3 – AKT signaling pathway governing the apicobasal organization of a subset of epidermal cells during median fin fold (MFF) morphogenesis. In nrg2a mutant larvae, the basal keratinocytes within the apical MFF, known as ridge cells, displayed reduced pAKT levels as well as reduced apical domains and exaggerated basolateral domains. Those defects compromised proper ridge cell elongation into a flattened epithelial morphology, resulting in thickened MFF edges. Pharmacological inhibition verified that Nrg2a signals through the ErbB receptor tyrosine kinase network. Moreover, knockdown of the epithelial polarity regulator and tumor suppressor lgl2 ameliorated the nrg2a mutant phenotype. Identifying Lgl2 as an antagonist of Nrg2a – ErbB signaling revealed a significantly earlier role for Lgl2 during epidermal morphogenesis than has been described to date. Furthermore, our findings demonstrated that successive, coordinated ridge cell shape changes drive apical MFF development, making MFF ridge cells a valuable model for investigating how the coordinated regulation of cell polarity and cell shape changes serves as a crucial mechanism of epithelial morphogenesis.     

Developmental Changes of ENaC Expression and Function in the Inner Ear of Pendrin Knock-Out Mice as a Perspective on the Development of Endolymphatic Hydrops

Pendrin mutations cause enlarged vestibular aqueducts and various degrees of sensorineural hearing loss. The selective abolition of pendrin causes dilation of the membranous labyrinth known as endolymphatic hydrops, loss of the endocochlear potential, and consequently loss of hearing function. Because Na⁺ transport is one of the most important driving forces for fluid transport, the epithelial Na⁺ channel (ENaC) is believed to play an important role in fluid volume regulation in the inner ear. Therefore, the dysfunction of Na⁺ transport through ENaC by the acidification of endolymph in Pendred syndrome is one of the potential causes of endolymphatic hydrops. We investigated the changes of ENaC expression and function during the development of the pendrin knock-out mouse. In the cochlea, the expression of β and γENaC was significantly increased at P56 in Pds^−/− mice compared with Pds^+/+ mice. In the vestibule, the expression of βENaC was significantly increased at P56, and γENaC expression significantly increased from P6 to P56 in Pds^−/− mice. The ENaC-dependent trans-epithelial current was not significantly different between Pds^+/+ and Pds^−/− mice in Reissner’s membrane or the saccular extramacular roof epithelium at P0, but the current was significantly increased in Pds^−/− mice at P56 compared with Pds^+/+ mice. These findings indicate that the expression and function of ENaC were enhanced in Pds^−/− mice after the development of endolymphatic hydrops as a compensatory mechanism. This result provides insight into the role of Na⁺ transport in the development and regulation of endolymphatic hydrops due to pendrin mutations.     

Developmental changes in the cochlear hair cell mechanotransducer channel and their regulation by transmembrane channel–like proteins

Vibration of the stereociliary bundles activates calcium-permeable mechanotransducer (MT) channels to initiate sound detection in cochlear hair cells. Different regions of the cochlea respond preferentially to different acoustic frequencies, with variation in the unitary conductance of the MT channels contributing to this tonotopic organization. Although the molecular identity of the MT channel remains uncertain, two members of the transmembrane channel–like family, Tmc1 and Tmc2, are crucial to hair cell mechanotransduction. We measured MT channel current amplitude and Ca²⁺ permeability along the cochlea’s longitudinal (tonotopic) axis during postnatal development of wild-type mice and mice lacking Tmc1 (Tmc1−/−) or Tmc2 (Tmc2−/−). In wild-type mice older than postnatal day (P) 4, MT current amplitude increased ∼1.5-fold from cochlear apex to base in outer hair cells (OHCs) but showed little change in inner hair cells (IHCs), a pattern apparent in mutant mice during the first postnatal week. After P7, the OHC MT current in Tmc1−/− (dn) mice declined to zero, consistent with their deafness phenotype. In wild-type mice before P6, the relative Ca²⁺ permeability, P_Ca, of the OHC MT channel decreased from cochlear apex to base. This gradient in P_Ca was not apparent in IHCs and disappeared after P7 in OHCs. In Tmc1−/− mice, P_Ca in basal OHCs was larger than that in wild-type mice (to equal that of apical OHCs), whereas in Tmc2−/−, P_Ca in apical and basal OHCs and IHCs was decreased compared with that in wild-type mice. We postulate that differences in Ca²⁺ permeability reflect different subunit compositions of the MT channel determined by expression of Tmc1 and Tmc2, with the latter conferring higher P_Ca in IHCs and immature apical OHCs. Changes in P_Ca with maturation are consistent with a developmental decrease in abundance of Tmc2 in OHCs but not in IHCs.     

Transient Receptor Potential Melastatin 1: A Hair Cell Transduction Channel Candidate

Sound and head movements are perceived through sensory hair cells in the inner ear. Mounting evidence indicates that this process is initiated by the opening of mechanically sensitive calcium-permeable channels, also referred to as the mechanoelectrical transducer (MET) channels, reported to be around the tips of all but the tallest stereocilia. However, the identity of MET channel remains elusive. Literature suggests that the MET channel is a non-selective cation channel with a high Ca²⁺ permeability and ∼100 picosiemens conductance. These characteristics make members of the transient receptor potential (TRP) superfamily likely candidates for this role. One of these candidates is the transient receptor potential melastatin 1 protein (TRPM1), which is expressed in various cells types within the cochlea of the mouse including the hair cells. Recent studies demonstrate that mutations in the TRPM1 gene underlie the inherited retinal disease complete congenital stationary night blindness in humans and depolarizing bipolar cell dysfunction in the mouse retina, but auditory function was not assessed. Here we investigate the role of Trpm1 in hearing and as a possible hair cell MET channel using mice homozygous for the null allele of Trpm1 (Trpm1^−/−) or a missense mutation in the pore domain of TRPM1 (Trpm1^{tvrm27/tvrm27}). Hearing thresholds were evaluated in adult (4–5 months old) mice with auditory-evoked brain stem responses. Our data shows no statistically significant difference in hearing thresholds in Trpm1^−/− or Trpm1^{tvrm27/tvrm27} mutants compared to littermate controls. Further, none of the mutant mice showed any sign of balance disorder, such as head bobbing or circling. These data suggest that TRPM1 is not essential for development of hearing or balance and it is unlikely that TRPM1 is a component of the hair cell MET channel.     

Loss of lkb1 Expression Reduces the Latency of ErbB2-Mediated Mammary Gland Tumorigenesis,Promoting Changes in Metabolic Pathways

The tumor suppressor kinase LKB1 is mutated in a broad range of cancers however, the role of LKB1 mammary gland tumorigenesis is not fully understood. Evaluation of human breast cancer tissue microarrays, indicate that 31% of HER2 positive samples lacked LKB1 expression. To expand on these observations, we crossed STK11^fl/fl mice with mice genetically engineered to express activated Neu/HER2-MMTV-Cre (NIC) under the endogenous Erbb2 promoter, to generate STK11^−/−/NIC mice. In these mice, the loss of lkb1 expression reduced the latency of ErbB2-mediated tumorigenesis compared to the latency of tumorigenesis in NIC mice alone. Analysis of STK11^−/−/NIC mammary tumors revealed hyperactivation of mammalian target of rapamycin (mTOR) through both mTORC1 and mTORC2 pathways as determined by the phosphorylation status of ribosomal protein S6 and AKT. Furthermore, STK11^−/−/NIC mammary tumors had elevated ATP levels along with changes in metabolic enzymes and metabolites. The treatment of primary mammary tumor cells with specific mTOR inhibitors AZD8055 and Torin1, that target both mTOR complexes, attenuated mTOR activity and decreased expression of glycolytic enzymes. Our findings underscore the existence of a molecular interplay between LKB1-AMPK-mTORC1 and ErbB2-AKT-mTORC2 pathways with mTOR at its epicenter, suggestive that loss of LKB1 expression may serve as a marker for hyperactivated mTOR in HER2 positive breast cancer and warranting further investigation into therapeutics that target LKB1-AMPK-mTOR and glycolytic pathways.     

Members of the Family Comamonadaceae as Primary Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate)-Degrading Denitrifiers in Activated Sludge as Revealed by a Polyphasic Approach

The distribution and phylogenetic affiliations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-degrading denitrifying bacteria in activated sludge were studied by a polyphasic approach including culture-independent biomarker and molecular analyses as well as cultivation methods. A total of 23 strains of PHBV-degrading denitrifiers were isolated from activated sludges from different sewage treatment plants. 16S ribosomal DNA (rDNA) sequence comparisons showed that 20 of the isolates were identified as members of the family Comamonadaceae, a major group of β-Proteobacteria. When the sludges from different plants were acclimated with PHBV under denitrifying conditions in laboratory scale reactors, the nitrate removal rate increased linearly during the first 4 weeks and reached 20 mg NO₃⁻-N h⁻¹ g of dry sludge⁻¹ at the steady state. The bacterial-community change in the laboratory scale sludges during the acclimation was monitored by rRNA-targeted fluorescence in situ hybridization and quinone profiling. Both approaches showed that the population of β-Proteobacteria in the laboratory sludges increased sharply during acclimation regardless of their origins. 16S rDNA clone libraries were constructed from two different acclimated sludges, and a total of 37 clones from the libraries were phylogenetically analyzed. Most of the 16S rDNA clones were grouped with members of the family Comamonadaceae. The results of our polyphasic approach indicate that β-Proteobacteria, especially members of the family Comamonadaceae, are primary PHBV-degrading denitrifiers in activated sludge. Our data provide useful information for the development of a new nitrogen removal system with solid biopolymer as an electron donor.     

Combined effect of cyclin D3 expression and abrogation of cyclin D1 prevent mouse skin tumor development

We have previously demonstrated that ras-mediated skin tumorigenesis depends on signaling pathways that act preferentially through cyclin D1 and D2. Interestingly, the expression of cyclin D3 inhibits skin tumor development, an observation that conflicts with the oncogenic role of D-type cyclins in the mouse epidermis. Here, we show that simultaneous up and downregulation of particular members of the D-type cyclin family is a valuable approach to reduce skin tumorigenesis. We developed the K5D3/cyclin D1^−/− compound mouse, which overexpresses cyclin D3 but lacks expression of cyclin D1 in the skin. Similar to K5D3 transgenic mice, keratinocytes from K5D3/cyclin D1^−/− compound mice show a significant reduction of cyclin D2 levels. Therefore, this model allows us to determine the effect of cyclin D3 expression when combined with reduced or absent expression of the remaining two members of the D-type cyclin family in mouse epidermis. Our data show that induced expression of cyclin D3 compensates for the reduced level of cyclin D1 and D2, resulting in normal keratinocyte proliferation. However, simultaneous ablation of cyclin D1 and downregulation of cyclin D2 via cyclin D3 expression resulted in a robust reduction in ras-mediated skin tumorigenesis. We conclude that modulation of the levels of particular members of the D-type cyclin family could be useful to inhibit tumor development and, in particular, ras-mediated tumorigenesis.Key words: cell cycle, D-type cyclins, skin, carcinogenesis, epidermis