Regional distribution of alpha9beta1 integrin within the limbus of the mouse ocular surface.

The epithelial basal cells of the corneal limbus are known to contain adult corneal epithelial stem cells, but the properties of these cells are not well understood. In addition, how and when the limbal epithelium forms during postnatal development in mammals is not clear. To better understand the anatomy and cell biology of the limbus, a whole-mount procedure was used to show that the nasal, inferior, temporal, and superior regions of the mouse limbus contain different numbers of alpha9 integrin-positive cells most of which are observed in the nasal region. We also show that this pattern develops progressively over time from 1 to 8 weeks after birth. High magnification image projections and three-dimensional reconstructions of the limbal region were generated from confocal images obtained after tissues were dual stained with alpha9 integrin and propidium iodide (PI) or triple stained with alpha9 integrin, E-cadherin, and PI. Data show that alpha9 integrin is present on the adult mouse cornea in the limbal basal cells and is more abundant in the apical-most cytoplasm of the limbal basal cells, where it can be found colocalized within the plasma membrane with E-cadherin. These studies are an important step toward improving our understanding of the development and cell biology of limbal basal cells.     

Morphological and Autoradiographic Studies on the Corneal and Limbal Epithelium of Rabbits

The investigation was centered on the morphological features of the conjunctiva–cornea transition (limbus) of the rabbit eye and the proliferative behavior of its epithelium. The eyes were processed for examination with light and electron microscopy, as well as for autoradiography after intravitreal injection of [³H]thymidine ([³H]TdR). At the sites of extraocular muscle insertion, the vascularization of the stroma extended to the peripheral cornea, and the limbal epithelium was thin with its basal stratum made up by clear cuboidal cells. In between the muscle insertions, the cuboidal clear cells, as well as the stroma blood vessels, were scarce. At the light microscope level, the basement membrane was distinct in the cornea but not in the limbus or the conjunctiva. Autoradiographs demonstrated that, at the limbus, the basal cells migrated very quickly to the suprabasal region and remained there up to the 28‐day interval. Labeled cells were identified in all epithelial layers of the cornea, including the basal one, at 21 and 28 days but not in the limbal basal clear cells. The rate of renewal of conjunctival epithelium was similar to that observed for the transition with scarce clear cells. The high‐resolution autoradiographs demonstrated that the basal cuboidal clear limbal cells exhibit a quick renewal and that they are not label‐retaining cells. These latter ones were detected all over the corneal epithelium and in the suprabasal layers of the limbus up to 28 days, in physiological conditions, without the need of stimulation by damage to the corneal epithelium. Anat Rec, 291:191–203, 2008. © 2008 Wiley‐Liss, Inc.     

Fibromodulin interactions with type I and II collagens

Fibromodulin is a keratan-sulfate small leucine-rich proteoglycan (SLRP) regulating collagen I and II fibril formation. In vivo studies suggest that, alongside decorin, fibromodulin plays an important role in the maintenance of mature tissues. To characterize fibromodulin/decorin differences in binding to type I and II collagen, we tested the collagen CNBr peptides in solid-phase assays. Only one peptide from collagen II and several peptides from collagen I interacted with fibromodulin, pointing to multiple binding sites in the collagen I molecule. By Scatchard-type analysis, the fibromodulin molecule showed only one class of binding sites for collagen I and both low and high affinity (classes of) binding sites for collagen II. Lys/Hyl residues in both collagens are essential for the interaction. Fibril formation assays showed the concomitant presence of fibromodulin and decorin in fibrils and a cumulative inhibitory effect. In solid-phase assays decorin seems to inhibit fibromodulin binding, whereas the contrary does not occur. We found fibromodulin and decorin have similarities and differences that may represent the biochemical basis of redundancy in SLRP function with compensation between different (classes of) SLRPs.     

Endothelial–Stromal Communication in Murine and Human Corneas

The purpose of this study is to identify and characterize interactions of corneal endothelial cells with the posterior stroma. Corneal endothelial–stromal interactions were examined in developing postnatal day 3 (P3) and mature postnatal day 30 (P30) C57BL/6 mice and adult human corneas. Flat mounts and cross-sections were studied using immunofluorescence microscopy. F-actin was labeled with phalloidin to evaluate cell processes traversing Descemet's membrane (DM). Dynamic cell–cell communication was evaluated with fluorescence recovery after photobleaching (FRAP) using calcein acetoxymethyl dye. Endothelial–stromal interactions were observed across the whole cornea transversing DM during early postnatal development (P3), while these interactions became restricted to the periphery in the mature murine cornea (P30). In adult human corneas, endothelial extensions through the DM were observed in the peripheral cornea. The pattern of FRAP in both mature mice and human central corneas demonstrated endothelial–endothelial cell communication. In contrast, in the human cornea 2, distinct patterns were observed consistent with endothelial–endothelial and stromal–endothelial communication. Endothelial–stromal interactions were observed in the entire cornea during early postnatal mouse corneas. This evidence of endothelial–posterior stromal contact contradicts the hypothesis that corneal endothelial cells are isolated from the stroma by the DM and provides direct data to support endothelial–stromal comunication that may directly influence posterior corneal structure and function. Anat Rec, 2020. © 2020 American Association for Anatomy     

A direct role for Wnt8 in ventrolateral mesoderm patterning

Vertebrate dorsoventral patterning requires both Wnt8 and BMP signaling. Because of their multiple interactions, discerning roles attributable specifically to Wnt8 independent of BMP has been a challenge. For example, Wnt8 represses the dorsal organizer that negatively regulates ventral BMP signals, thus Wnt8 loss‐of‐function phenotypes may reflect the combined effects of reduced Wnt8 and BMP signaling. We have taken a loss‐of‐function approach in the zebrafish to generate embryos lacking expression of both Wnt8 and the BMP antagonist Chordin. wnt8;chordin loss‐of‐function embryos show rescued BMP signaling, thereby allowing us to identify Wnt8‐specific requirements. Our analysis shows that Wnt8 is uniquely required to repress prechordal plate specification but not notochord, and that Wnt8 signaling is not essential for specification of tailbud progenitors but is required for normal expansion of posterior mesoderm cell populations. Thus, Wnt8 and BMP signaling have independent roles during vertebrate ventrolateral mesoderm development that can be identified through loss‐of‐function analysis. Developmental Dynamics 239:2828–2836, 2010. © 2010 Wiley‐Liss, Inc.     

Collagen fibril assembly during postnatal development and dysfunctional regulation in the lumican-deficient murine cornea.

The transparent cornea is the outer barrier of the eye and is its major refractive surface. Development of a functional cornea requires a postnatal maturation phase involving development, growth and organization of the stromal extracellular matrix. Lumican, a leucine-rich proteoglycan, is implicated in regulating assembly of collagen fibrils and the highly organized extracellular matrix essential for corneal transparency. We investigated the regulatory role(s) of lumican in fibril assembly during postnatal corneal development using wild type (Lum+/+) and lumican-null (Lum-/-) mice. In Lum+/+ mice, a regular architecture of small-diameter fibrils is achieved in the anterior stroma by postnatal day 10 (P10), while the posterior stroma takes longer to reach this developmental maturity. Thus, the anterior and the posterior stroma follow distinct developmental timelines and may be under different regulatory mechanisms. In Lum-/- mice, it is the posterior stroma where abnormal lateral associations of fibrils and thicker fibrils with irregular contours are evident as early as P10. In contrast, the anterior stroma is minimally perturbed by the absence of lumican. In Lum+/+ mice, lumican is expressed throughout the developing stroma at P10, with strong expression limited to the posterior stroma in the adult. Therefore, the posterior stroma, which is most vulnerable to lumican-deficiency, demonstrates an early developmental defect in fibril structure and architecture in the Lum-/- mouse. These defects underlie the reported increased light scattering and opacity detectable in the adult. Our findings emphasize the early regulation of collagen structure by lumican during postnatal development of the cornea.     

Effect of adenoviral mediated overexpression of fibromodulin on human dermal fibroblasts and scar formation in full-thickness incisional wounds

Fibromodulin, a member of the small leucine-rich proteoglycan family, has been recently suggested as a biologically significant mediator of fetal scarless repair. To assess the role of fibromodulin in the tissue remodeling, we constructed an adenoviral vector expressing human fibromodulin cDNA. We evaluated the effect of adenovirus-mediated overexpression of fibromodulin in vitro on transforming growth factors and metalloproteinases in fibroblasts and in vivo on full-thickness incisional wounds in a rabbit model. In vitro, we found that Ad-Fibromodulin induced a decrease of expression of TGF-β₁ and TGF-β₂ precursor proteins, but an increase in expression of TGF-β₃ precursor protein and TGF-β type II receptor. In addition, fibromodulin overexpression resulted in decreased MMP-1 and MMP-3 protein secretion but increased MMP-2, TIMP-1, and TIMP-2 secretion, whereas MMP-9 and MMP-13 were not influenced by fibromodulin overexpression. In vivo evaluation by histopathology and tensile strength demonstrated that Ad-Fibromodulin administration could ameliorate wound healing in incisional wounds. In conclusion, although the mechanism of scar formation in adult wounds remains incompletely understood, we found that fibromodulin overexpression improves wound healing in vivo, suggesting that fibromodulin may be a key mediator in reduced scarring.

Distribution of Amelotin in Mouse Tooth Development

Amelotin is expressed and secreted by ameloblasts in tooth development, but amelotin distribution during enamel development is not clear. In this report, we first investigated amelotin expression in developing teeth by immunohistochemistry. Amelotin was detected in the enamel matrix at the secretion and maturation stages of enamel development. Amelotin was also observed at Tomes' processes on the apical ends of secretory ameloblasts. We then compared amelotin gene expression with those of amelogenin, enamelin, and ameloblastin in the mandibles of postnatal mice by RT‐PCR. The expression of amelotin was detected as early as in postnatal day 0 mandibles and amelotin was coexpressed with amelogenin, ameloblastin, and enamelin during tooth development. These data strongly suggest that amelotin is an enamel matrix protein expressed at the secretion and maturation stages of enamel development. Anat Rec, 2010. © 2009 Wiley‐Liss, Inc.     

Non‐core subunit eIF3h of translation initiation factor eIF3 regulates zebrafish embryonic development

Eukaryotic translation initiation factor eIF3, which plays a central role in translation initiation, consists of five core subunits that are present in both the budding yeast and higher eukaryotes. However, higher eukaryotic eIF3 contains additional (non‐core) subunits that are absent in the budding yeast. We investigated the role of one such non‐core eIF3 subunit eIF3h, encoded by two distinct genes—eif3ha and eif3hb, as a regulator of embryonic development in zebrafish. Both eif3h genes are expressed during early embryogenesis, and display overlapping yet distinct and highly dynamic spatial expression patterns. Loss of function analysis using specific morpholino oligomers indicates that each isoform has specific as well as redundant functions during early development. The morphant phenotypes correlate with their spatial expression patterns, indicating that eif3h regulates development of the brain, heart, vasculature, and lateral line. These results indicate that the non‐core subunits of eIF3 regulate specific developmental programs during vertebrate embryogenesis. Developmental Dynamics 239:1632–1644, 2010. © 2010 Wiley‐Liss, Inc.     

Effect of N-methyl-D-aspartate receptor blockade on caspase activation and neuronal death in the developing rat cerebellum

In vitro studies have demonstrated that N-methyl-D-aspartate (NMDA) receptor activation rescue cerebellar granule neurons (CGN) from apoptotic death. It has been suggested that this effect mimics the transient glutamate receptors activation by mossy fibers during cerebellar development. We reported previously that CGN from postnatal days 2-4 (P2-P4) rats increased cell survival in response to NMDA treatment. In this study, we evaluated the effect of dizocilpine (MK-801) administrated for three consecutive days on the apoptotic death of CGN during development. MK-801 treatment decreased the large number of CGN condensed nuclei found at P8, but this drug increased the proportion of condensed nuclei at P16. We also found a high activity of caspases during the first postnatal week that decreased during development. MK-801 treatment did not modify the activity of caspase-8 at any age, but decreased caspase-9 activity at P8 and increased the activity of caspase-1 (76%) at P8, caspase-3 (160%) at P16 and caspase-9 (50%) at P12. These results suggest that NMDA receptor stimulation regulates the activity of caspases in a differential way and plays an important role in the in vivo CGN death during postnatal development.     

K14 + Compound niches are present on the mouse cornea early after birth and expand after debridement wounds

Background: We previously identified compound niches (CNs) at the limbal:corneal border of the mouse cornea that contain corneal epithelial progenitor cells, express Keratin 8 (K8), and goblet cell mucin Muc5AC. During re‐epithelialization after 2.5 mm epithelial debridement wounds, CNs migrate onto the cornea and expand in number mimicking conjunctivalization. When CNs form during development and whether they express corneal epithelial progenitor cell enriched K14 was not known. Results: To provide insight into corneal epithelial homeostasis, we quantify changes in expression of simple (K8, K18, K19) and stratified squamous epithelial keratins (K5, K12, K14, and K15) during postnatal development and in response to 2.5 mm wounds using quantitative polymerase chain reaction (Q‐PCR), confocal imaging and immunoblots. K14 + CNs are present 7 days after birth. By 21 days, when the eyelids are open, K8, K19, and Muc5AC are also expressed in CNs. By 28 days after wounding, the corneal epithelium shows enhanced mRNA and protein expression for K14 and retains mRNA and protein for corneal epithelial specific K12. Conclusions: The keratin phenotype observed in corneal epithelial cells before eyelid opening is similar to that seen during wound healing. Data show K14 + corneal epithelial progenitor cells expand in number after 2.5 mm wounds. Developmental Dynamics 245:132–143, 2016. © 2015 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc.     

The development of Kv4.2 expression in the retina

Throughout the brain, the potassium channel Kv4.2 regulates signal propagation in dendrites and action potential properties in subtypes of neurons. In adult rodents Kv4.2 is expressed predominantly in two bands in the inner plexiform layer (IPL) and in retinal ganglion cell (RGC) somas (Klumpp et al. [15]; Pinto and Klumpp [20]), suggesting a role regulating the activity of specific subtypes of RGCs. To understand the role of Kv4.2 in the regulation of the activity of RGCs during development we determined the developmental expression pattern of Kv4.2 immunoreactivity (Kv4.2-IR). At P4-6 Kv4.2-IR appeared diffusely throughout the IPL in cross-sectioned retinas. From postantal day 10 (P10) through adult there was an additional pair of brighter Kv4.2-IR bands between the ChAT bands that had a reticular pattern in flat-mounted retinas. Kv4.2-IR was not present in somas at P4–6, but appeared in ganglion cell layer (GCL) somas beginning at P10. The fraction of somas expressing Kv4.2 in the GCL was about 8% at P10–11, decreased to 5% at P20–21, then increased to 9% in adult retinas. The restriction of Kv4.2 expression to less than 10% of the GCL somas and the specificity of expression in the IPL suggest that Kv4.2 regulates activity in one or a few functional subtypes of RGCs. The pattern of Kv4.2-IR through postnatal development indicates that Kv4.2-mediated currents are important for development in a subset of RGCs, especially around P10 as the bipolar cells mature.     

Rapid remodeling of airway vascular architecture at birth

Recent advances have documented the development of lung vasculature before and after birth, but less is known of the growth and maturation of airway vasculature. We sought to determine whether airway vasculature changes during the perinatal period and when the typical adult pattern develops. On embryonic day 16.5 mouse tracheas had a primitive vascular plexus unlike the adult airway vasculature, but instead resembling the yolk sac vasculature. Soon after birth (P0), the primitive vascular plexus underwent abrupt and extensive remodeling. Blood vessels overlying tracheal cartilage rings regressed from P1 to P3 but regrew from P4 to P7 to form the hierarchical, segmented, ladder‐like adult pattern. Hypoxia and HIF‐1α were present in tracheal epithelium over vessels that survived but not where they regressed. These findings reveal the plasticity of airway vasculature after birth and show that these vessels can be used to elucidate factors that promote postnatal vascular remodeling and maturation. Developmental Dynamics 239:2354–2366, 2010. © 2010 Wiley‐Liss, Inc.     

Small leucine‐rich proteoglycans exhibit unique spatiotemporal expression profiles during cardiac valve development

Background: Small Leucine Rich Proteoglycans (SLRPs) play a role in collagen fiber formation and also function as signaling molecules. Given the importance of collagen synthesis to the cardiovascular extracellular matrix (ECM), we examined the spatiotemporal expression of SLRPs, not previously investigated in the murine heart. Results: Cardiac expression using antibodies specific for biglycan (BGN), decorin (DCN), fibromodulin (FMOD), and lumican (LUM) revealed distinct patterns among the SLRPs in mesenchymal‐derived tissues. DCN showed the most intense localization within the developing valve cusps, while LUM was evident primarily in the hinge region of postnatal cardiac valves. BGN, DCN, and FMOD were immunolocalized to regions where cardiac valves anchor into adjacent tissues. Medial (BGN) and adventitial (BGN, DCN, FMOD and LUM) layers of the pulmonary and aortic arteries also showed intense staining of SLRPs but this spatiotemporal expression varied with developmental age. Conclusions: The unique expression patterns of SLRPs suggest they have adapted to specialized roles in the cardiovascular ECM. SLRP expression patterns overlap with areas where TGFβ signaling is critical to the developing heart. Therefore, we speculate that SLRPs may not only be required to facilitate collagen fiber formation but may also regulate TGFβ signaling in the murine heart. Developmental Dynamics 243:601–611, 2014. © 2013 Wiley Periodicals, Inc.     

Reiterative use of the notch signal during zebrafish intrahepatic biliary development

The Notch signaling pathway regulates specification of zebrafish liver progenitor cells towards a biliary cell fate. Here, using staged administration of a pharmacological inhibitor of Notch receptor processing, we show that activation of the Notch pathway is also important for growth and expansion of the intrahepatic biliary network in zebrafish larvae. Biliary expansion is accompanied by extensive cell proliferation and active remodeling of the nascent ductal network, as revealed by time lapse imaging of living zebrafish larvae that express a Notch responsive fluorescent reporter transgene. Together, these data support a model in which the Notch signal functions reiteratively during biliary development; first to specific biliary cells and then to direct remodeling of the nascent biliary network. As the Notch pathway plays a comparable role during mammalian biliary development, including humans, these studies also indicate broad conservation of the molecular mechanisms directing biliary development in vertebrates. Developmental Dynamics 239:855–864, 2010. © 2010 Wiley‐Liss, Inc.     

Differential maturational patterns of nitric oxide synthase-I and NADPH diaphorase in functionally distinct cortical areas of the mouse cerebral cortex

Nitric oxide (NO) regulates several functions both in the developing and the adult central nervous systems (CNS). During development, NO is assumed to contribute to the histogenetic differentiation of the CNS especially through the modulation of programmed neuronal death. The embryonal and postnatal changes in the distribution of the cortical NO producing system were studied in Balb/c mice using immunocytochemistry for nitric oxide synthase-I (NOS-I) and NADPH-diaphorase (NADPH-d) enzyme histochemistry. NOS-I reactive neurons (RN) appeared first at embryonic day 14 (E14) in the spinal cord in the vicinity of the central canal, and later, at E16-18, in the thalamus and striatum. The first cortical region to present NOS-I reactivity was the parietal cortex, which happened at E18-20. After E20 the number of NOS-I RN increased in every cortical area, plateauing at postnatal day 4 (P4). In parietal regions, however, the highest density of NOS-I RN was observed already at P1. The neuronal packing density (PD) of NOS-I RN declined until adulthood, interrupted by a transient increase in some cortical areas at the onset of puberty. The heterochronous appearance of NOS-I during pre- and postnatal development of different brain regions and the sequence of up- and downregulation of expression until adult stages points to an important role of NO in brain development and functional differentiation.     

Collagen fibrillogenesis and mRNA levels in the maturing rabbit medial collateral ligament and patellar tendon

This study compared collagen fibril diameter and mRNA changes in a subset of molecules involved in collagen fibrillogenesis during postnatal development and at maturity of rabbit medial collateral ligament (MCL) and patellar tendon (PT). Tissue was analyzed by RT-PCR for mRNA levels and collagen fibril diameters were measured using transmission electron microscopy. Collagen fibril diameters increased from 3 to 14 weeks with mean fibril diameters of PT significantly greater than MCL at 9, 12, and 14 weeks and maturity. RT-PCR analysis showed decorin and lumican mRNA levels were significantly higher in PT than MCL at all ages. Type I collagen, MMP-11, and procollagen C proteinase enhancer mRNA levels also were higher in the PT than the MCL between 3 and 14 weeks but not at maturity. Further understanding of collagen fibrillogenesis by studying protein synthesis and matrix turnover during maturation may provide insight into the mechanism(s) by which fibrils accrete in maturing connective tissues and how they are altered during healing following injury.     

Vagal Fibers Form Associations With Interstitial Cells of Cajal During Fetal Development

Vagal intramuscular arrays (IMAs) have been shown to form complexes with intramuscular interstitial cells of Cajal (ICC). We tested the hypothesis that associations between vagal nerve endings and ICC arise in fetal development. Intraganglionic laminar endings (IGLEs) and IMAs were identified by applying 1,1’‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanineperchlorate (DiI) to vagal nerve trunks and myenteric plexus (MP) and intramuscular (IM) ICC were immunolabeled with antibodies to c‐Kit in fetal and early postnatal mice (E16‐P7). At E16, c‐Kit immunoreactive cells were abundant in the primordial smooth muscle, with early ICC networks discernable by E18 and ongoing organization at P1 and P7. The distribution of vagal endings was found to change during the course of development, with significantly more putative IGLEs in the prenatal compared to the postnatal period and less IMAs in the prenatal compared to postnatal period. Associations of ICC with both IGLEs and IMAs were detected as early as E16 and were maintained into postnatal life. These findings suggest that vagal fibers begin to associate with ICC during prenatal development. Future studies will be needed to determine the mechanisms through which vagal endings and ICC interact. Anat Rec, 298:1780–1785, 2015. © 2015 Wiley Periodicals, Inc.     

Lectin binding patterns in nonsensory regions of rat cochlea during postnatal development

The distribution of glycoconjugates was examined in the nonsensory regions of the rat cochlea during postnatal development using biotin-conjugated lectins. Temporal bones of rats at postnatal d 1 and at wk 2, 4 and 6 were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde and processed for paraffin wax embedding. The dewaxed sections were incubated with 7 biotinylated lectins, followed by avidin-biotin-peroxidase complex. A different staining pattern was observed in the stria vascularis, spiral ligament and spiral limbus in the age groups examined. The staining intensity varied between lectins and the reaction product exhibited limited disparity. The staining intensity for WGA increased with age in all the 3 nonsensory regions. The staining patterns for the other lectins differed in the various nonsensory regions examined indicating tissue specificity. The limited variations in the lectin binding patterns after 2nd wk of postnatal life also indicate that the changes in the carbohydrate moieties are established during the fetal period of cochlear development and limited changes take place during postnatal maturation of the nonsensory regions.