Shh mRNA Research Articles

Loss of glypican-3 function causes growth factor-dependent defects in cardiac and coronary vascular development.

Glypican-3 (Gpc3) is a heparan sulfate proteoglycan (HSPG) expressed widely during vertebrate development. Loss-of-function mutations cause Simpson-Golabi-Behmel syndrome (SGBS), a rare and complex congenital overgrowth syndrome with a number of associated developmental abnormalities including congenital heart disease. We found that Gpc3-deficient mice display a high incidence of congenital cardiac malformations like ventricular septal defects, common atrioventricular canal and double outlet right ventricle. In addition we observed coronary artery fistulas, which have not been previously reported in SGBS. Coronary artery fistulas are noteworthy because little is known about the molecular basis of this abnormality. Formation of the coronary vascular plexus in Gpc3-deficient embryos was delayed compared to wild-type, and consistent with GPC3 functioning as a co-receptor for fibroblast growth factor-9 (FGF9), we found a reduction in Sonic Hedgehog (Shh) mRNA expression and signaling in embryonic mutant hearts. Interestingly, we found an asymmetric reduction in SHH signaling in cardiac myocytes, as compared with perivascular cells, resulting in excessive coronary artery formation in the Gpc3-deficient animals. We hypothesize that the excessive development of coronary arteries over veins enables the formation of coronary artery fistulas. This work has broad significance to understanding the genetic basis of coronary development and potentially to molecular mechanisms relevant to revascularization following ischemic injury to the heart.

Differential Shh, Bmp and Wnt gene expressions during craniofacial development in mice

In this study, Bmp-4, Wnt-5a and Shh gene expressions were compared during early craniofacial development in mice by comparative non-isotopic in situ hybridization. Wild-type C57BL/6J mice were studied at various stages of embryonic development (from 8.5- to 13.5-day-old embryos--E8.5-13.5). During early odontogenesis, transcripts for Bmp-4, Shh and Wnt-5a were co-localised at the tooth initiation stage. At E8.5, Shh mRNA expression was restricted to diencephalon and pharyngeal endoderm. Before maxillae and mandible ossification, Bmp-4 and Wnt-5a signals were detected in the mesenchymal cells and around Meckel's cartilage. During palatogenesis, Shh was expressed only in the epithelium and Wnt-5a only in the mesenchyme of the elevating palatal shelves. During tongue development, Shh expression was found in mesenchyme, probably contributing to tongue miogenesis, while Wnt-5a signal was in the epithelium, possibly during placode development and papillae formation. Taken together, these findings suggest that Bmp-4, Shh and Wnt-5a gene expressions may act together on the epithelial-mesenchymal interactions occurring in several aspects of the early mouse craniofacial development, such as odontogenesis, neuronal development, maxillae and mandible ossification, palatogenesis and tongue formation.

Inducing Effects of Zhichan Soup on Neuron Stem Cell Differentiation

Abstract This is an experiment to observe the inducing effects of Zhichan Soup on neuron stem cell (NSC) differentiation. NSCs were divided into three groups. The normal group was treated with the normal medium. The herb group was treated with the Zhichan Soup-medicated sera. The control group was treated with the normal rat sera. After the 21-day treatment, TH and DAT proteins were checked by immunocytochemistry, and Shh, Ptx3 and Nurrl mRNA were checked by RT-PCR. Results of this experiment indicated that neither TH nor DAT proteins were found in any groups. Compared with other groups, the Zhichan Soup group had registered a significantly enhanced expression of Shh, Ptx3 and Nurrl mRNA. It is believed that the Zhichan Soup-medicated serum may be able to improve the expression of Shh, Ptx3 and Nurrl in a micro-environment to facilitate NSCs to differentiate into dopamine neurons.

M1604 Transgenic Expression of PKD Stimulates Intestinal Epithelial Cell Proliferation

intestinal crypts and villi. Since defective Hh signaling causes IEC hyperproliferation, we hypothesized that the increased growth factor receptor expression and signaling may modulate Hh expression. Inhibitors of EGFR, MEK-1 and PI3K caused increased Shh & Ihh mRNA and protein levels, while EGF had the opposite effects. CONCLUSIONS: Our studies suggest that Hh's are major effectors of β1 integrin signaling during intestinal epithelial development. This β1 integrin-Hh signaling pathway represents a novel mechanism by which the extracellular matrix regulates IEC proliferation and intestinal development. β1 integrins may regulate Hh expression through crosstalk with growth factor signaling pathways in IECs.

Bone Marrow Stromal Cells Increase Oligodendrogenesis after Stroke

Oligodendrocytes are sensitive to ischemic damage. The Sonic hedgehog (Shh) pathway is critical in oligodendrogenesis; Gli1 is the principal effector of Shh signaling. We investigated oligodendrogenesis and Shh/Gli1 pathway activation after bone marrow stromal cell (BMSC) treatment of stroke in rats. Rats were subjected to the middle cerebral artery occlusion (MCAo). BMSCs have been shown to promote functional recovery post stroke. A therapeutic dose of BMSC (3 x 10(6) cells) treatment was initiated 1 day after MCAo. Immunohistochemistry was carried out to measure the oligodendrocyte progenitor cells, oligodendrocytes, myelin, and expressions of Shh and Gli1 at 14 days after MCAo. Gene expression of Shh and Gli1 was tested at 2 days after MCAo. An in vitro study was used to investigate the effects of BMSC on a premature oligodendrocyte cell line (N20.1 cells). BMSC treatment significantly increased O4(+) oligodendrocytes, MBP(+) area, and bromodeoxyuridine (BrdU)(+), NG2(+), BrdU(+)-NG2(+) cells, and mRNA and protein expressions of Shh and Gli1 in the ipsilateral brain of the MCAo rats than that in phosphate buffered saline (PBS)-treated rats. BMSCs promoted N20.1 cell proliferation and Gli1 mRNA expression, and these effects were abolished by the Shh pathway inhibitor cyclopamine. These data indicate that the BMSC treatment stimulates oligodendrogenesis by activation of the Shh/Gli1 pathway post stroke.

Sonic hedgehog signaling regulates reciprocal epithelial-mesenchymal interactions controlling palatal outgrowth.

The mammalian secondary palate arises by outgrowth from the oral side of the paired maxillary processes flanking the primitive oral cavity. Palatal growth depends on reciprocal interactions between the oral ectoderm and the underlying neural-crest-derived mesenchyme. Previous studies have implicated sonic hedgehog (Shh) as an important epithelial signal for regulating palatal growth. However, the cellular and molecular mechanisms through which Shh regulates palatal development in vivo have not been directly analyzed, due in part to early embryonic lethality of mice lacking Shh or other essential components of the Shh signaling pathway. Using Cre/loxP-mediated tissue-specific inactivation of the smoothened (Smo) gene in the developing palatal mesenchyme, we show that the epithelially expressed Shh signals directly to the palatal mesenchyme to regulate palatal mesenchyme cell proliferation through maintenance of cyclin D1 (Ccnd1) and Ccnd2 expression. Moreover, we show that Shh-Smo signaling specifically regulates the expression of the transcription factors Foxf1a, Foxf2 and Osr2 in the developing palatal mesenchyme. Furthermore, we show that Shh signaling regulates Bmp2, Bmp4 and Fgf10 expression in the developing palatal mesenchyme and that specific inactivation of Smo in the palatal mesenchyme indirectly affects palatal epithelial cell proliferation. Together with previous reports that the mesenchymally expressed Fgf10 signals to the palatal epithelium to regulate Shh mRNA expression and cell proliferation, these data demonstrate that Shh signaling plays a central role in coordinating the reciprocal epithelial-mesenchymal interactions controlling palatal outgrowth.

Sonic hedgehog ligand partners with caveolin-1 for intracellular transport

Prenatal alcohol exposure is the most common environmental factor leading to congenital birth defects in the United States. Although significant progress has been made in this field, the detailed molecular pathology of fetal alcohol syndrome (FAS) remains to be determined. Previously, we have shown that alcohol exposure perturbs hedgehog signal transduction in zebrafish embryos by inhibiting the post-translational cholesterol modification of Sonic hedgehog (Shh), leading to decreased levels of mature Shh ligand that is associated with the plasma membrane, and causing transient loss of Hh signaling, resulting in permanent FAS-related morphological abnormalities. In the present study, we further elucidate the mechanisms that regulate the intracellular transportation and secretion of Shh using the hepatic stellate cell line HSC8B. We have found that Shh is associated with caveolin-1 in the Golgi apparatus to form protein complexes and that these complexes are packaged as large punctuate structures (transport vesicles) that are transported to the plasma membrane in lipid raft microdomains. Alcohol exposure does not significantly interrupt translation of shh mRNA in endoplasmic reticulum (ER) or the trafficking of Shh from the ER to the Golgi apparatus. However, alcohol does prevent the entry of Shh into transport vesicles from the Golgi to the plasma membrane and specifically decreases the amount of caveolin-1/Shh complex found in lipid rafts, causing cytoplasmic accumulation of Shh and leading to a deficiency of Shh ligand secretion into the extracellular matrix.

Influence of retinoic acid on TBX1 expression in myocardial cells induced by Shh and Fgf8

The aim of this study was to explore the regulatory mechanism of retinoic acid (RA) on the TBX1 gene expression in myocardial cells. Ventricular cardiocytes were isolated from neonatal rats and cultured, and then treated with different concentrations of retinoic acid. The expression of Shh and Fgf8 at mRNA and protein levels in neonatal rat myocardial cells were measured by using RT-PCR and Western blot technique, respectively. There was basal expression of Shh and Fgf8 in the control group. When treated with 3 × 10−7 mol/L RA, we observed that the expression of Shh mRNA and protein in neonatal rat myocardial cells were up-regulated by 1.51 (P < 0.05) and 1.10 times (P < 0.05), respectively. In comparison with the control group, under the concentration of 5 × 10−7 mol/L RA, they were up-regulated by 2.21 (P < 0.05) and 2.38 times (P < 0.05) individually. Meanwhile, we could detect that the expression of Fgf8 mRNA and protein were up-regulated by 2.50 times (P < 0.05) and 80% (P < 0.05) separately compared with the control group after stimulation of 3 × 10−7 mol/L RA, and they were up-regulated by 3.48 (P < 0.05) and 2.04 times (P < 0.05) individually after stimulation of 5 × 10−7 mol/L RA. The results indicated that RA could induce the expression of Shh and Fgf8 in neonatal rat myocardial cells. At the same time, it has shown that Shh and Fgf8 were involved in the regulation process of RA on TBX1 expression.

Deciphering the role of Shh signaling in axial defects produced by ethanol exposure

The phenotype of embryos exposed to ethanol is complex and likely due to multiple alterations in developmental pathways. We have previously demonstrated that Sonic hedgehog signaling (Shh-s) was reduced in both chicken and zebrafish embryos when exposed to ethanol. There are many tissues affected by embryonic ethanol exposure, and in this article we explore the development of axial tissues, using zebrafish embryos. We then compare these effects to the phenotypes produced by exposure to two drugs that also inhibit Shh-s: cyclopamine and forskolin. We found alterations in the development of the notochord and somites produced by all three compounds, although only ethanol produced developmental delay of epiboly. Upon observation of early developing embryos, muscle pioneer cells were completely lost in cyclopamine-treated embryos, and reduced, but less so, in embryos treated with forskolin and ethanol. Ethanol treatment produced a dose-dependent reduction in total body length that may be linked to epiboly delay seen earlier during development. Despite the differences between cyclopamine and forskolin, we found that shh mRNA injection rescued the short body length, the alteration in somite shape, and the cyclopia produced by ethanol exposure. Taken together, each teratogen produced a unique set of phenotypic changes in the body axis, suggesting that each compound affects Shh-s and also produces a distinctive set of molecular alterations. However, addition of exogenous Shh to ethanol treated zebrafish prevented many of the gross physical phenotypes, suggesting that the suppression of Shh-s is one of the major effects of ethanol exposure.

The role of Hedgehog pathway in hepatic fibrosis and hepatic stellate cell activation

Objective To investigate the role of Hedgehog pathway in hepatic fibrosis and its association with activation of hepatic stellate cells. Methods Twenty male Spragur-Dawley rats were divided into control and model groups with 10 each. The animal models were induced by injection with CCl4 and fed with fat-rich diet. The rats in both groups were sacrified at the 8 week with 5 each and the liver tissues were removed for HSC-T6 culture. The deposition of collagen fiber in liver was detected with HE and Masson staining. RT-PCR was used to detect the expressions of Sonic hedgehog (Shh), smoothened (Smo), patched (Ptc), Gli-1 and α-smooth muscle action (α-SMA) mRNA in HSC-T6 and liver tissues. The influence of cyclopamine (Cyc) and lipopolysaccharide (LPS) on HSC-T6 proliferation were assayed by MTT. The expressions of Shh, Smo, Ptc, Gli-1 and α-SMA mRNA after intervention with Cyc (100μmol/L) and LPS were measured by real-time PCR. Results A lot of lipo and collagen deposited in liver of model rats. The Shh,Smo,Gli-1 and α-SMA mRNA were highly expressed in model rats than those in control group (2-△△Ct were 20.45±3.31 vs. 1, 12.78 ± 0. 53 vs. 1, 10.88 ± 2.41 vs. 1, 4.91 ± 2. 59 vs. 1, respectively, all P value <0. 05). In vitro Cyc inhibited HSC-T6 proliferation in dose dependant manner (F=636.81, P<0.01). Compared to the control group, the mRNA expressions of Smo, Ptc, Gli-1,α-SMA in HSC-T6 were significantly reduced after Cyc intervention (2△△Ct, were 0. 20±0. 11, 0. 21 ± 0. 08, 0. 28 ± 0. 05,0. 27±0.10,respectively, all P values<0.01). Conclusion The expression of members of Hedgehog pathway are increased in the progress of hepatic fibrosis, which may accelerate the hepatiee fibrosis by activating HSC. Key words: Hedgehog proteins: Liver cirrhosis, experimental: Hepatic stellate cells

140. THE DEVELOPMENT OF THE PHALLUS IN A MARSUPIAL

The phallus, the limbs and the tail are all considered appendages in the developing mammal. In mice, several key genes including FGF8, BMP4, SHH, DLX5 and DLX6 are known to control the precise pattering of the limb and phallus in the fetus (reviewed in Yamada et al. 2006). The signalling cascade in both appendages begins with SHH interacting with FGF8. In humans, disruptions to these gene pathways result in malformations of both limbs and phallus because these appendages share conserved elements in patterning and development (Yamada et al. 2003). However, this is a poorly researched area so additional models are needed to provide a greater perspective into mammalian embryonic patterning especially of the external genitalia. In marsupials, since most sexual differentiation occurs after birth, the developing phallus and limbs are accessible at stages that occur in utero in humans and other eutherian mammals. We have used the tammar as an alternative model to understand the differentiation of the phallus and limb. FGF8, SHH and megalin mRNA and protein are all expressedin the limb and phallus from the early embryo until post-natal stages. SHH and megalin were co-localised in the urethral epithelium of the tammar phallus. This is the first study to detect megalin in the developing mammalian phallus. SHH is a critical gene in patterning the appendages, and megalin is a transport protein that ferries steroids into the cell where they can bind to their cognate receptor. It has a high affinity for the potent androgen androstanediol, an androgen critical for virilisation of the tammar phallus (Leihy et al. 2004). Further examination is underway to confirm whether the expression of the genes patterning the phallus in the mouse are conserved in the marsupial and whether megalin plays any role in the patterning of the phallus, possibly mediated by SHH.

Sonic Hedgehog Expression and Its Role in Form-Deprivation Myopia in Mice

Purpose: To investigate whether sonic hedgehog (Shh) plays a role in postnatal eye development and the development of experimentally induced myopia. Methods: Expression of Shh, Patched-1 (Ptc-1), and Gli3 was evaluated in the eyes of 13- to 14-day-old C57B/L6 mice with form-deprivation myopia (FDM) (n = 100) and controls (n = 100) using real-time PCR and Western blot analysis. In a second experiment, 336 mice were divided into two groups: the first wore a unilateral translucent diffuser to induce myopia and the second served as a control. Both groups received four intravitreal injections of either Shh-N (Sonic hedgehog amino-terminal peptide) or cyclopamine (a specific inhibitor of the Shh pathway) every other day. Retinoscopic refraction and axial length measurements were performed on the 11th day of form deprivation. Sections of the eyes were observed using a light microscope. Results: Inducing myopia caused a significant increase in expression of Shh mRNA (7 days: t = 6.09, p = 0.004; 14 days: t = 3.48, p = 0.025) and protein (7 days: t = 4.06, p = 0.015; 14 days: t = 4.25, p = 0.013). Expression of both Gli3 mRNA (t = 7.61, p = 0.002) and protein (t = 2.89, p = 0.045) increased after 7 days of occlusion. Administration of Shh-N stimulated the development of myopia and axial growth in both occluded (refraction: F = 7.49, p = 0.001; axial length: F = 9.89, p < 0.001) and non-occluded eyes (refraction: F = 14.20, p < 0.001; axial length: F = 20.37, p < 0.001). Cyclopamine reduced myopic refractive error and axial elongation in occluded eyes (refraction: F = 27.91, p < 0.001; axial length: F = 15.18, p < 0.001). It also influenced non-occluded eyes, reducing axial growth and shifting the refraction toward hyperopia (refraction: F = 14.81, p < 0.001; axial length: F = 3.99, p = 0.024). No difference in retinal thickness was found between experimental and control eyes. Conclusions: The Shh signaling pathway may influence both form-deprivation myopia and the postnatal growth of eyes with normal visual input.

Chromosomal Dynamics at the Shh Locus: Limb Bud-Specific Differential Regulation of Competence and Active Transcription

The expression of Sonic hedgehog (Shh) in mouse limb buds is regulated by a long-range enhancer 1 Mb upstream of the Shh promoter. We used 3D-FISH and chromosome conformation capture assays to track changes at the Shh locus and found that long-range promoter-enhancer interactions are specific to limb bud tissues competent to express Shh. However, the Shh locus loops out from its chromosome territory only in the posterior limb bud (zone of polarizing activity or ZPA), where Shh expression is active. Notably, while Shh mRNA is detected throughout the ZPA, enhancer-promoter interactions and looping out were only observed in small fractions of ZPA cells. In situ detection of nascent Shh transcripts and unstable EGFP reporters revealed that active Shh transcription is likewise only seen in a small fraction of ZPA cells. These results suggest that chromosome conformation dynamics at the Shh locus allow transient pulses of Shh transcription.

Neuroprotective effect of sonic hedgehog up‐regulated in Schwann cells following sciatic nerve injury

The physiological roles of sonic hedgehog (Shh) have been intensively characterized in development of various organs. However, their functions in adult tissues have not been fully elucidated. We investigated the expression and the potential function of Shh in crush-injured adult rat sciatic nerves. The Shh expression was up-regulated in Schwann cells adjacent to the injured site. The time-course analyses of various neurotrophic factors revealed the up-regulation of Shh mRNA followed by that of brain-derived neurotrophic factor (BDNF) mRNA. The continuous administration of cyclopamine, a hedgehog signal inhibitor, to the injured site suppressed the increase of BDNF expression and deteriorated the survival of motor neurons in lumbar spinal cord. Treatment of exogenous Shh in cultured Schwann cells enhanced the BDNF expression. The BDNF promoter activity (exon I and II) was increased in IMS32 cells co-transfected with Shh and its receptor Smoothened. These findings imply that the up-regulated expression of Shh in Schwann cells may play an important role in injured motor neurons through the induction of BDNF.

Characterization of sonic hedgehog as a novel NF‐κB target gene that promotes NF‐κB‐mediated apoptosis resistance and tumor growth in vivo

To explore mechanisms controlling sonic hedgehog (Shh) expression in human cancers, we investigated regulation of Shh by the transcription factor NF-kappaB. We identify putative NF-kappaB binding sites in the human Shh promoter region that specifically bind NF-kappaB complexes. Further, NF-kappaB activation by tumor necrosis factor alpha (TNF-alpha) or p65 overexpression stimulates Shh promoter activity and p65 binds to Shh promoter in vivo. NF-kappaB-mediated transcriptional activation of Shh is mapped to a minimal NF-kappaB consensus site at position +139 of Shh promoter. NF-kappaB activation results in increased Shh mRNA and protein expression in vitro and, notably, also in vivo in a genetic mouse model of inducible NF-kappaB activity. Specific NF-kappaB inhibition by inhibitory NF-kappaBalpha (Ikappa-Balpha) superrepressor or p65 knockdown inhibits NF-kappaB-induced Shh promoter activation and Shh expression. NF-kappaB-mediated Shh expression promotes proliferation and confers resistance to TRAIL-induced apoptosis. Silencing of Shh prevents NF-kappaB-stimulated proliferation, while the addition of Shh rescues the proliferation defect imposed by NF-kappaB inhibition. Notably, NF-kappaB-stimulated tumor growth is significantly impaired by Shh knockdown in an in vivo model of pancreatic cancer. By demonstrating that NF-kappaB regulates Shh expression, which contributes to NF-kappaB-mediated proliferation and apoptosis resistance in vitro and in vivo, our findings have important implications to target aberrant Shh expression in human cancers.

Hedgehog signaling is involved in development of the neocortex

Sonic hedgehog (Shh) function is essential for patterning and cell fate specification, particularly in ventral regions of the central nervous system. It is also a crucial mitogen for cerebellar granule neuron precursors and is important in maintenance of the stem cell niche in the postnatal telencephalon. Although it has been reported that Shh is expressed in the developing dorsal telencephalon, functions of Shh in this region are unclear, and detailed characterization of Shh mRNA transcripts in situ has not been demonstrated. To clarify the roles of Shh signaling in dorsal pallium (neocortex primordium) development, we have knocked out the Shh and Smo genes specifically in the early developing dorsal telencephalon by using Emx1cre mice. The mutants showed a smaller dorsal telencephalon at E18.5, which was caused by cell cycle kinetics defects of the neural progenitor/stem cells. The cell cycle length of the progenitor/stem cells was prolonged, and the number of cycle-exiting cells and neurogenesis were decreased. Birth-date analysis revealed abnormal positioning of neurons in the mutants. The characteristics of the subventricular zone, ventricular zone and subplate cells were also affected. Weak immunoreactivity of Shh was detected in the dorsal telencephalon of wild types. Reduced Shh immunoreactivity in mutant dorsal telencephalons supports the above phenotypes. Our data indicate that Shh signaling plays an important role in development of the neocortex.

Detailed analysis of formation of chicken pituitary primordium in early embryonic development

The primordium of the mammalian adenohypophysis derived from Rathke's pouch (RP) is known to be formed by oral ectoderm invagination. However, in the early phase of pituitary development, the detailed process by which the oral ectoderm develops into the adenohypophysis remains largely unknown. Using high-resolution non-radiolabeled in situ hybridization and the BrdU and TUNEL methods, we have examined the detailed expression pattern of factors involved in the formation of RP of chicken and the changes in the mitotic and apoptotic cell regions in RP. In the chicken embryo, Sonic hedgehog (Shh) mRNA was initially expressed in the stomodeal plate but not in the oral ectoderm. After prospective diencephalon had detached from the oral ectoderm, another Shh-expressing region appeared in the most rostral part of the recess. LIM homeobox gene 3 (Lhx3) mRNA first appeared in the anterior area of Rathke's recess, and expression then spread to the caudal region. alphaGSU mRNA-expressing cells were observed at both ends of the Lhx3-expressing region, and thereafter the expression area moved to the posterior region. Furthermore, a close overlap was found between the proliferating region and Lhx3 mRNA-expressing area, and TUNEL-positive cells appeared in Seessel's pouch derived from the foregut. Thus, the primordium of the pituitary gland corresponding to the Lhx3-expressing region is surrounded by the Shh-expressing region, which appears in two steps, and the mass growth and invagination of RP of chicken result from the coordination of the dorsal extension of the anterior region and the ventral extension of the posterior region of RP.

Age-Related Cone Abnormalities in Zebrafish with Genetic Lesions in Sonic Hedgehog

Sonic hedgehog (Shh) signaling is essential for photoreceptor differentiation and retinal cell survival in embryonic zebrafish. The study was conducted to determine whether adult heterozygous carriers of mutant alleles for the shh gene display retinal abnormalities. Retinal cryosections from young, middle-aged, and senescent wild-type and sonic-you(+/-) (syu(+/-)) zebrafish were probed with retinal cell type-specific markers. Contralateral retinal flatmounts from these fish, and from adult albino zebrafish subjected to light-induced photoreceptor damage followed by regeneration, were hybridized with blue cone opsin cRNA for quantitative analysis of the blue cone pattern. Retinal expression of shh mRNA was measured by quantitative RT-PCR. Regions of cone loss and abnormal cone morphology were observed in the oldest syu(+/-) zebrafish, although no other retinal cell type was affected. This phenotype was age-related and genotype-specific. Cone distribution in the oldest syu(+/-) zebrafish was predominantly random, as assessed by measuring the short-range pattern, whereas that of wild-type fish and the younger syu(+/-) zebrafish was statistically regular. A measure of long-range pattern revealed atypical cone aggregation in the oldest syu(+/-) zebrafish. The light-treated albino zebrafish displayed random cone patterns immediately after light toxicity, but showed cone aggregation on regeneration. Retinas from the syu(+/-) fish showed reduced expression of shh mRNA compared with those of wild-type siblings. The syu(+/-) zebrafish presents a model for the study of hereditary age-related cone abnormalities. The syu(+/-) retinas most likely experience progressive cone photoreceptor loss, accompanied by cone regeneration. Shh signaling may be required to maintain cone viability throughout life.

Facilitated sprouting in a peripheral nerve injury

During regeneration of injured peripheral nerves, local conditions may influence how regenerative axon sprouts emerge from parent axons. More extensive lesions might be expected to disrupt such growth. In this work, we discovered instead that long segmental crush injuries facilitate the growth and maturation of substantially more axon sprouts than do classical short crush injuries (20 mm length vs. 2 mm). At identical distances from the proximal site of axon interruption there was a 45% rise in the numbers of neurofilament labeled axons extending through a long segmental crush zone by 1 week. By 2 weeks, there was a 35% greater density of regenerating myelinated axons in long compared with short crush injuries just beyond (5 mm) the proximal injury site. Moreover, despite the larger numbers of axons, their maturity was identical and they were regular, parallel, associated with Schwann cells (SCs) and essentially indistinguishable between the injuries. Backlabeling with Fluorogold indicated that despite these differences, the axons arose from similar numbers of parent motor and sensory neurons. Neither injury was associated with ischemia. Both injuries were associated with rises in GFAP (glial acidic fibrillary protein) and p75 mRNAs, markers of SC plasticity but p75, GFAP and brain-derived neurotrophic factor mRNAs did not differ between the injuries. There was a higher local mRNA level of GAP43/B50 at 7 days following injury and a higher sonic hedgehog protein (Shh) mRNA at 24 h in long crush zones. GAP43/B50 protein and SHH protein both had prominent localization within regenerating axons.Long segmental nerve trunk crush injuries do not impair regeneration but instead generate greater axon plasticity that results in larger numbers of mature myelinated axons. The changes occur without apparent change in SC activation, overall nerve architecture or nerve blood flow. While the mechanism is uncertain, the findings indicate that manipulation of the nerve microenvironment can induce substantial changes in regenerative sprouting.

Expression of Hedgehog Family Genes in the Rat Uterus During Early Pregnancy

Hedgehog (Hh) plays a pivotal role in various tissues during embryonic development, tissue homeostasis and tumorigenesis. In mammals, Hh exists in three homologs: Desert hedgehog (Dhh), Indian hedgehog (Ihh) and Sonic hedgehog (Shh). In this study, we cloned full-length cDNAs encoding Dhh and Ihh from the rat uterus. Their amino acid sequences have a high homology with those of the mouse and human. In addition, the changes of Hh gene expression in the rat uterus during early pregnancy were analyzed. The results showed that all three hedgehog mRNAs were detected in the rat uterus at the proestrus stage and during early pregnancy (1.5, 3.5, 5.5 and 7.5 days post coitus: dpc). Ihh mRNA expression varied and peaked at 3.5 dpc in the luminal and glandular epithelium. Expression was decreased on 5.5 dpc with the exception of sustained expression in the glandular epithelium. Despite such Ihh variability, the expressions of Dhh and Shh mRNA remained unchanged. This indicated that Ihh was mainly expressed in the rat uterus during early pregnancy. Moreover, the Hh target gene (glioma-associated oncogene homolog 1; Gli1) was also highly expressed at 3.5 dpc in the epithelium and periepithelial stroma in a manner similar to the temporal pattern of Ihh expression. This suggests that Ihh signaling axis play a role in the rat uterus during early pregnancy. In summary, our results elucidate that Ihh is a predominant Hh protein in the rat uterus during early pregnancy and that other Hhs have the potential to be expressed. This observation will help to elucidate the basic molecular mechanism of rat uterus during early pregnancy.