Cracd Marks the First Wave of Meiosis during Spermatogenesis and Is Mis-Expressed in Azoospermia Mice.

Testicular development starts in utero and maturation continues postnatally, requiring a cascade of gene activation and differentiation into different cell types, with each cell type having its own specific function. As we had previously reported that the Capping protein inhibiting regulator of actin (Cracd) gene was expressed in the adult mouse testis, herein we examine when and where the ß-catenin associated Cracd is initially expressed during postnatal testis development. Significantly, Cracd mRNA is present in both the immature postnatal and adult testis in round spermatid cells, with highest level of expression occurring during the first wave of meiosis and spermatogenesis. In the juvenile testes, Cracd is initially expressed within the innermost region but as maturation occurs, Cracd mRNA switches to a more peripheral location. Thereafter, Cracd is downregulated to maintenance levels in the haploid male germ cell lineage. As Cracd mRNA was expressed within developing round spermatids, we tested its effectiveness as a biomarker of non-obstructive azoospermia using transgenic knockout mice models. Meaningfully, Cracd expression was absent in Deleted in azoospermia like (Dazl) null testis, which exhibit a dramatic germ cell loss. Moreover, Cracd was abnormally regulated and ectopically mis-expressed in Polypyrimidine tract binding protein-2 (Ptbp2) conditional germ cell restricted knockout testis, which exhibit a block during spermatid differentiation and a reduction in the number of late stage spermatocytes coincident with reduced ß-catenin expression. Combined, these data suggest that Cracd is a useful first wave of spermatogenesis biomarker of azoospermia phenotypes, even prior to an overt phenotype being evident.

Periostin and matrix stiffness combine to regulate myofibroblast differentiation and fibronectin synthesis during palatal healing.

Although the matricellular protein periostin is prominently upregulated in skin and gingival healing, it plays contrasting roles in myofibroblast differentiation and matrix synthesis respectively. Palatal healing is associated with scarring that can alter or restrict maxilla growth, but the expression pattern and contribution of periostin in palatal healing is unknown. Using periostin-knockout (Postn-/-) and wild-type (WT) mice, the contribution of periostin to palatal healing was investigated through 1.5 mm full-thickness excisional wounds in the hard palate. In WT mice, periostin was upregulated 6 days post-wounding, with mRNA levels peaking at day 12. Genetic deletion of periostin significantly reduced wound closure rates compared to WT mice. Absence of periostin reduced mRNA levels of pivotal genes in wound repair, including α-SMA/acta2, fibronectin and ßigh3. Recruitment of fibroblasts and inflammatory cells, as visualized by immunofluorescent staining for fibroblast specific factor-1, vimentin, and macrophages markers Arginase-1 and iNOS was also impaired in Postn-/-, but not WT mice. Palatal fibroblasts isolated from the hard palate of mice were cultured on collagen gels and prefabricated silicon substrates with varying stiffness. Postn-/- fibroblasts showed a significantly reduced ability to contract a collagen gel, which was rescued by the exogenous addition of recombinant periostin. As the stiffness increased, Postn-/- fibroblasts increasingly differentiated into myofibroblasts, but not to the same degree as the WT. Pharmacological inhibition of Rac rescued the deficient myofibroblastic phenotype of Postn-/- cells. Low stiffness substrates (0.2 kPa) resulted in upregulation of fibronectin in WT cells, an effect which was significantly reduced in Postn-/- cells. Quantification of immunostaining for vinculin and integrinß1 adhesions revealed that Periostin is required for the formation of focal and fibrillar adhesions in mPFBs. Our results suggest that periostin modulates myofibroblast differentiation and contraction via integrinß1/RhoA pathway, and fibronectin synthesis in an ECM stiffness dependent manner in palatal healing.

Armadillo-like helical domain containing-4 is dynamically expressed in both the first and second heart fields.

Armadillo repeat and Armadillo-like helical domain containing proteins form a large family with diverse and fundamental functions in many eukaryotes. Herein we investigated the spatiotemporal expression pattern of Armadillo-like helical domain containing 4 (or Armh4) as an uncharacterized protein coding mouse gene, within the mouse embryo during the initial stages of heart morphogenesis. We found Armh4 is initially expressed in both first heart field as well as the second heart field progenitors and subsequently within predominantly their cardiomyocyte derivatives. Armh4 expression is initially cardiac-restricted in the developing embryo and is expressed in second heart field subpharyngeal mesoderm prior to cardiomyocyte differentiation, but Armh4 diminishes as the embryonic heart matures into the fetal heart. Armh4 is subsequently expressed in craniofacial structures and neural crest-derived dorsal root and trigeminal ganglia. Whereas lithium chloride-induced stimulation of Wnt/ß-catenin signaling elevated Armh4 expression in both second heart field subpharyngeal mesodermal progenitors and outflow tract, right ventricle and atrial cardiomyocytes, neither a systemic loss of Islet-1 nor an absence of cardiac neural crest cells had any effect upon Armh4 expression. These results confirm that Wnt/ß-catenin-responsive Armh4 is a useful specific biomarker of the FHF and SHF cardiomyocyte derivatives only.

Analysis of Uncharacterized mKiaa1211 Expression during Mouse Development and Cardiovascular Morphogenesis.

Mammalian Kiaa1211 and Kiaa1211-like are a homologous pair of uncharacterized, highly conserved genes cloned from fetal and adult brain cDNA libraries. Herein we map the in utero spatiotemporal expression of mKiaa1211 and mKiaa1211L mRNA and their expression patterns in postnatal testis, skin, gastrointestinal, and adipose progenitor tissues. Significantly, mKiaa1211 is present throughout the early stages of mouse heart development, particularly in the second heart field (SHF) lineage as it differentiates from mesenchymal cells into cardiomyocytes. We also show that mKiaa1211 is expressed within several early neuronal tissues destined to give rise to central, peripheral, and sympathetic nervous system structures. Expression profiling revealed that the paralog mKiaa1211L is not expressed during the normal developmental process and that mKiaa1211 expression was noticeably absent from most adult terminally differentiated tissues. Finally, we confirm that a previously uncharacterized CRISPR/CAS-generated mKiaa1211 mouse mutant allele is hypomorphic.

Proteomic profiling of TGFBI-null mouse corneas reveals only minor changes in matrix composition supportive of TGFBI knockdown as therapy against TGFBI-linked corneal dystrophies.

TGFBIp is a constituent of the extracellular matrix in many human tissues including the cornea, where it is one of the most abundant proteins expressed. TGFBIp interacts with Type I, II, IV, VI, and XII collagens as well as several members of the integrin family, suggesting it plays an important role in maintaining structural integrity and possibly corneal transparency as well. Significantly, more than 60 point mutations within the TGFBI gene have been reported to result in aberrant TGFBIp folding and aggregation in the cornea, resulting in severe visual impairment and blindness. Several studies have focused on targeting TGFBIp in the cornea as a therapeutic approach to treat TGFBI-linked corneal dystrophies, but the effect of this approach on corneal homeostasis and matrix integrity remained unknown. In the current study, we evaluated the histological and proteomic profiles of corneas from TGFBI-deficient mice as well as potential redundant functions of the paralogous protein POSTN. The absence of TGFBIp in mouse corneas did not grossly affect the collagen scaffold, and POSTN is unable to compensate for loss of TGFBIp. Proteomic comparison of wild-type and TGFBI-/- mice revealed 11 proteins were differentially regulated, including Type VI and XII collagens. However, as these alterations did not manifest at the macroscopic and behavioral levels, these data support partial or complete TGFBI knockdown as a potential therapy against TGFBI-linked corneal dystrophies. Lastly, in situ hybridization verified TGFBI mRNA in the epithelial cells but not in other cell types, supportive of a therapy directed specifically at this lineage.

Periostin contributes to epidermal hyperplasia in psoriasis common to atopic dermatitis.

BACKGROUND: Epidermal hyperplasia is a histological hallmark observed in both atopic dermatitis (AD) and psoriasis, although the clinical features and the underlying immunological disorders of these diseases are different. We previously showed that periostin, a matricellular protein, plays a critical role in epidermal hyperplasia in AD, using a mouse model and a 3-dimensional organotypic coculture system. In this study, we explore the hypothesis that periostin is involved in epidermal hyperplasia in psoriasis. METHODS: To examine expression of periostin in psoriasis patients, we performed immunohistochemical analysis on skin biopsies from six such patients. To investigate periostin's role in the pathogenesis of psoriasis, we evaluated periostin-deficient mice in a psoriasis mouse model induced by topical treatment with imiquimod (IMQ). RESULTS: Periostin was substantially expressed in the dermis of all investigated psoriasis patients. Epidermal hyperplasia induced by IMQ treatment was impaired in periostin-deficient mice, along with decreased skin swelling. However, upon treatment with IMQ, periostin deficiency did not alter infiltration of inflammatory cells such as neutrophils; production of IL-17, -22, or -23; or induction/expansion of IL-17- and IL-22-producing group 3 innate lymphoid cells. CONCLUSIONS: Periostin plays an important role during epidermal hyperplasia in IMQ-induced skin inflammation, independently of the IL-23-IL-17/IL-22 axis. Periostin appears to be a mediator for epidermal hyperplasia that is common to AD and psoriasis.

Persistent Noggin arrests cardiomyocyte morphogenesis and results in early in utero lethality.

BACKGROUND: Multiple bone morphogenetic protein (BMP) genes are expressed in the developing heart from the initiation to late-differentiation stages, and play pivotal roles in cardiovascular development. In this study, we investigated the requirement of BMP activity in heart development by transgenic over-expression of extracellular BMP antagonist Noggin. RESULTS: Using Nkx2.5-Cre to drive lineage-restricted Noggin within cardiomyocyte progenitors, we show persistent Noggin arrests cardiac development at the linear heart stage. This is coupled with a significantly reduced cell proliferation rate, subsequent cardiomyocyte programmed cell death and reduction of downstream intracellular pSMAD1/5/8 expression. Noggin mutants exhibit reduced heartbeat which likely results in subsequent fully penetrant in utero lethality. Significantly, confocal and electron micrographic examination revealed considerably fewer contractile elements, as well as a lack of maturation of actin-myosin microfilaments. Molecular analysis demonstrated that ectopic Noggin-expressing regions in the early heart's pacemaker region, failed to express the potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 (Hcn4), resulting in an overall decrease in Hcn4 levels. CONCLUSIONS: Combined, our results reveal a novel role for BMP signaling in the progression of heart development from the tubular heart stage to the looped stage by means of regulation of proliferation and promotion of maturation of the in utero heart's contractile apparatus and pacemaker.

In situ hybridization (both radioactive and nonradioactive) and spatiotemporal gene expression analysis.

Section in situ hybridization using either radioactive or nonradioactive labeled cDNA probes is an invaluable technique that enables the investigator to detect and localize mRNA expression within tissue sections and cells. Here, we describe the labeling of (35)S-UTP radioactive and nonradioactive digoxigenin probes, preparation of tissue sections, hybridization, and washing of non-hybridized probes, followed by the detection of radioactive signals via dipping in nuclear emulsion and the immunohistochemical and subsequent colorimetric detection of nonradioactive signals.

Ectopic Noggin in a Population of Nfatc1 Lineage Endocardial Progenitors Induces Embryonic Lethality.

The initial heart is composed of a myocardial tube lined by endocardial cells. The TGFß superfamily is known to play an important role, as BMPs from the myocardium signal to the overlying endocardium to create an environment for EMT. Subsequently, BMP and TGFß signaling pathways synergize to form primitive valves and regulate myocardial growth. In this study, we investigated the requirement of BMP activity by transgenic over-expression of extracellular BMP antagonist Noggin. Using Nfatc1Cre to drive lineage-restricted Noggin within the endocardium, we show that ectopic Noggin arrests cardiac development in E10.5-11 embryos, resulting in small hearts which beat poorly and die by E12.5. This is coupled with hypoplastic endocardial cushions, reduced trabeculation and fewer mature contractile fibrils in mutant hearts. Moreover, Nfatc1Cre -mediated diphtheria toxin fragment-A expression in the endocardium resulted in genetic ablation and a more severe phenotype with lethality at E11 and abnormal linear hearts. Molecular analysis demonstrated that endocardial Noggin resulted in a specific alteration of TGFß/BMP-mediated signal transduction, in that, both Endoglin and ALK1 were downregulated in mutant endocardium. Combined, these results demonstrate the cell-autonomous requirement of the endocardial lineage and function of unaltered BMP levels in facilitating endothelium-cardiomyocyte cross-talk and promoting endocardial cushion formation.

Periostin controls keratinocyte proliferation and differentiation by interacting with the paracrine IL-1α/IL-6 loop.

Proliferation and differentiation of keratinocytes are normally well balanced, but this balance can be perturbed in wound healing and is dysregulated in pathological conditions such as atopic dermatitis. Epithelial-mesenchymal interaction affects this event via the cross-talk of cytokines and growth factors. Periostin, a matricellular protein, has an important role during reepithelialization in wound healing and is critical for hyperproliferation of keratinocytes in atopic dermatitis. Here we investigated how periostin regulates proliferation and differentiation of keratinocytes in the epithelial-mesenchymal interactions using a three-dimensional organotypic air-liquid interface coculture system. The release of IL-1α from keratinocytes and subsequent IL-6 production from fibroblasts were critical for keratinocyte proliferation and differentiation. Periostin secreted from fibroblasts was required for IL-1α-induced IL-6 production and enhanced IL-6 production by activation of the NF-κB pathway synergistically with IL-1α. Thus, the combination of an autocrine loop of periostin and a paracrine loop composed of IL-1α and IL-6 regulates keratinocyte proliferation and differentiation in the epithelial-mesenchymal interactions, and periostin tunes the magnitude of keratinocyte proliferation and differentiation by interacting with the paracrine IL-1α/IL-6 loop.

Notochordal and foregut abnormalities correlate with elevated neural crest apoptosis in Patch embryos.

Although Patch mutants show severe abnormalities in many neural crest-derived structures including the face and the heart, there is a paucity of information characterizing the mechanisms underlying these congenital defects. Via manipulating the genetic background to circumvent early embryonic lethality, our results revealed that Patch phenotypes are most likely due to a significant decrease in migratory neural crest lineage due to diminished neural crest survival and elevated apoptosis. Homozygous mutant neural crest precursors can undergo typical expansion within the neural tube, epithelial-to-mesenchymal transformation, and initiate normal neural crest emigration. Moreover, in vitro explant culture demonstrated that when isolated from the surrounding mesenchyme, Patch mutant neural crest cells (NCCs) can migrate appropriately. Additionally, Patch foregut, notochord and somitic morphogenesis, and Sonic hedgehog expression profiles were all perturbed. Significantly, the timing of lethality and extent of apoptosis correlated with the degree of severity of Patch mutant foregut, notochord, and somite dysfunction. Finally, analysis of Balb/c-enriched surviving Patch mutants revealed that not all the neural crest subpopulations are affected and that Patch mutant neural crest-derived sympathetic ganglia and dorsal root ganglia were unaffected. We hypothesize that loss of normal coordinated signaling from the notochord, foregut, and somites underlies the diminished survival of the neural crest lineage within Patch mutants resulting in subsequent neural crest-deficient phenotypes.