Cyclopamine and jervine in embryonic rat tongue cultures demonstrate a role for Shh signaling in taste papilla development and patterning: fungiform papillae double in number and form in novel locations in dorsal lingual epithelium

From time of embryonic emergence, the gustatory papilla types on the mammalian tongue have stereotypic anterior and posterior tongue locations. Furthermore, on anterior tongue, the fungiform papillae are patterned in rows. Among the many molecules that have potential roles in regulating papilla location and pattern, Sonic hedgehog (Shh) has been localized within early tongue and developing papillae. We used an embryonic, tongue organ culture system that retains temporal, spatial, and molecular characteristics of in vivo taste papilla morphogenesis and patterning to study the role of Shh in taste papilla development. Tongues from gestational day 14 rat embryos, when papillae are just beginning to emerge on dorsal tongue, were maintained in organ culture for 2 days. The steroidal alkaloids, cyclopamine and jervine, that specifically disrupt the Shh signaling pathway, or a Shh-blocking antibody were added to the standard culture medium. Controls included tongues cultured in the standard medium alone, and with addition of solanidine, an alkaloid that resembles cyclopamine structurally but that does not disrupt Shh signaling. In cultures with cyclopamine, jervine, or blocking antibody, fungiform papilla numbers doubled on the dorsal tongue with a distribution that essentially eliminated inter-papilla regions, compared with tongues in standard medium or solanidine. In addition, fungiform papillae developed on posterior oral tongue, just in front of and beside the single circumvallate papilla, regions where fungiform papillae do not typically develop. The Shh protein was in all fungiform papillae in embryonic tongues, and tongue cultures with standard medium or cyclopamine, and was conspicuously localized in the basement membrane region of the papillae. Ptc protein had a similar distribution to Shh, although the immunoproduct was more diffuse. Fungiform papillae did not develop on pharyngeal or ventral tongue in cyclopamine and jervine cultures, or in the tongue midline furrow, nor was development of the single circumvallate papilla altered. The results demonstrate a prominent role for Shh in fungiform papilla induction and patterning and indicate differences in morphogenetic control of fungiform and circumvallate papilla development and numbers. Furthermore, a previously unknown, broad competence of dorsal lingual epithelium to form fungiform papillae on both anterior and posterior oral tongue is revealed.     

Sonic hedgehog regulates prostatic growth and epithelial differentiation

The Sonic hedgehog (SHH)-signalling pathway mediates epithelial-mesenchymal interactions in several tissues during development and disease, and we have investigated its role in rat ventral prostate (VP) development. We have demonstrated that Shh and Ptc expression correlates with growth and development of the prostate and that their expression is not regulated by androgens in the VP. Prostatic budding was induced in response to testosterone in Shh null mouse urogenital sinus (UGS) explants grown in vitro and in rat UGS explants cultured with cyclopamine, suggesting that SHH-signalling is not critical for prostatic induction. SHH-signalling was disrupted at later stages of VP development (in vitro), resulting in a reduction in organ size, an increase in ductal tip number, and reduced proliferation of ductal tip epithelia. The addition of recombinant SHH to VPs grown in vitro caused a decrease in ductal tip number and expansion of the mesenchyme. In the presence of testosterone, inhibition of SHH-signalling accelerated the canalisation of prostatic epithelial ducts and resulted in ducts that showed morphological similarities to cribiform prostatic intraepithelial neoplasia (PIN). The epithelia of these ducts also demonstrated precocious and aberrant differentiation, when examined by immunohistochemistry for p63 and cytokeratin 14. In conclusion, we show that SHH-signalling is not essential for prostatic induction, but is important for prostatic growth, branching, and proliferation, and that androgen-stimulated growth in the absence of signalling from the SHH pathway results in aberrant epithelial differentiation.     

Sonic hedgehog signaling during digit pattern duplication after application of recombinant protein and expressing cells

HoxD expression and cartilage pattern formation were compared after application of a recombinant amino-terminal peptide of Sonic hedgehog protein (Shh-N) and implantation of cells expressing the Sonic hedgehog (Shh) gene. During digit duplication after implantation of a Shh-N-soaked bead, BMP-2 and Patched expression was transiently induced in the anterior limb mesenchyme 20 h after grafting, but was reduced to the basal level 48 h after grafting. On the contrary, when Shh-expressing cells were grafted to the anterior limb bud, expression domains of the BMP-2 and Patched genes were initially induced in the restricted region in close proximity to the grafted cells. Induced expression of BMP-2 and Patched was maintained in the anterior-peripheral region of the limb bud for 42 h after grafting. In either case, HoxD12 and HoxD13 were consistently induced in the anterior-distal limb mesenchyme, accompanying mirror-image duplication of the digit pattern. Induction and maintenance of HoxD expression were consistent with the resultant digit pattern. A steep gradient of Shh activity provided by Shh-expressing cells is most adequate to induce complete digit pattern, as compared to the shallow gradient provided by Shh-N protein released from a bead. These results suggest that positional identity is respecified by Shh-N activity within the first 24 h during digit duplication, and that Shh-N on its own is not acting as a long-range signaling molecule to determine positional identity at a distance in the limb bud.     

Sonic hedgehog regulates otic capsule chondrogenesis and inner ear development in the mouse embryo

Development of the cartilaginous capsule of the inner ear is dependent on interactions between otic epithelium and its surrounding periotic mesenchyme. During these tissue interactions, factors endogenous to the otic epithelium influence the differentiation of the underlying periotic mesenchyme to form a chondrified otic capsule. We report the localization of Sonic hedgehog (Shh) protein and expression of the Shh gene in the tissues of the developing mouse inner ear. We demonstrate in cultures of periotic mesenchyme that Shh alone cannot initiate otic capsule chondrogenesis. However, when Shh is added to cultured periotic mesenchyme either in combination with otic epithelium or otic epithelial-derived fibroblast growth factor (FGF2), a significant enhancement of chondrogenesis occurs. Addition of Shh antisense oligonucleotide (AS) to cultured periotic mesenchyme with added otic epithelium decreases levels of endogenous Shh and suppresses the chondrogenic response of the mesenchyme cells, while supplementation of Shh AS-treated cultures with Shh rescues cultures from chondrogenic inhibition. We demonstrate that inactivation of Shh by targeted mutation produces anomalies in the developing inner ear and its surrounding capsule. Our results support a role for Shh as a regulator of otic capsule formation and inner ear development during mammalian embryogenesis.     

Pax9 is required for filiform papilla development and suppresses skin-specific differentiation of the mammalian tongue epithelium

The epidermis is a derivative of the surface ectoderm. It forms a protective barrier and specific appendages including hair, nails, and different eccrine glands. The surface ectoderm also forms the epithelium of the oral cavity and tongue, which develop a slightly different barrier and form different appendages such as teeth, filiform papillae, taste papillae, and salivary glands. How this region-specific differentiation is genetically controlled is largely unknown. We show here that Pax9, which is expressed in the epithelium of the tongue but not in skin, regulates several aspects of tongue-specific epithelial differentiation. In Pax9-deficient mice filiform papillae lack the anterior-posterior polarity, a defect that is associated with temporal-spatial changes in Hoxc13 expression. Barrier formation is disturbed in the mutant tongue and genome-wide expression profiling revealed that the expression of specific keratins (Krt), keratin-associated proteins, and members of the epidermal differentiation complex is significantly down-regulated. In situ hybridization demonstrated that several 'hard' keratins, Krt1-5, Krt1-24, and Krt2-16, are not expressed in the absence of Pax9. Notably, specific 'soft' keratins, Krt2-1 and Krt2-17, normally weakly expressed in the tongue but present at high levels in skin and in orthokeratinized oral dysplasia are up-regulated in the mutant tongue epithelium. This result indicates a partial trans-differentiation to an epithelium with skin-specific characteristics. Together, our findings show that Pax9 regulates appendage formation in the mammalian tongue and identify Pax9 as an important factor for the region-specific differentiation of the surface ectoderm.     

Thyroid hormone-induced expression of Sonic hedgehog correlates with adult epithelial development during remodeling of the Xenopus stomach and intestine

Sonic hedgehog (Shh) was isolated from the Xenopus laevis intestine as an early thyroid hormone (TH) response gene. To investigate possible roles of TH-upregulated expression of Shh during metamorphosis, we raised a polyclonal antibody against Xenopus Shh and immunohistochemically examined the relationship between Shh expression and the larval-to-adult intestinal remodeling at the cellular level. Our results indicate that the epithelial-specific expression of Shh in the intestine spatiotemporally correlates well with active proliferation and/or initial differentiation of the secondary (adult) epithelial primordia that originate from stem cells, but not with apoptosis of the primary (larval) epithelium. Given the similar transformations of the stomach during metamorphosis, we also analyzed Shh expression in this organ and found similar correlations in the stomach, although the position of the adult epithelial primordia and their final differentiation in the stomach are different from those in the intestine. Furthermore, we show here that Shh expression is organ-autonomously induced by TH and its correlation with the adult epithelial development is reproduced in vitro in both the intestine and the stomach. More importantly, addition of recombinant Shh protein to the culture medium results in developmental anomalies of both organs. However, differentiation of the adult epithelium is more severely inhibited by exogenous Shh in the intestine than in the stomach. These results suggest that TH-upregulated expression of Shh plays important roles in the postembryonic gastrointestinal remodeling, but its roles are at least partially different between the intestine and the stomach.     

Endoderm-derived Sonic hedgehog and mesoderm Hand2 expression are required for enteric nervous system development in zebrafish

The zebrafish enteric nervous system (ENS), like those of all other vertebrate species, is principally derived from the vagal neural crest cells (NCC). The developmental controls that govern the migration, proliferation and patterning of the ENS precursors are not well understood. We have investigated the roles of endoderm and Sonic hedgehog (SHH) in the development of the ENS. We show that endoderm is required for the migration of ENS NCC from the vagal region to the anterior end of the intestine. We show that the expression of shh and its receptor ptc-1 correlate with the development of the ENS and demonstrate that hedgehog (HH) signaling is required in two phases, a pre-enteric and an enteric phase, for normal ENS development. We show that HH signaling regulates the proliferation of vagal NCC and ENS precursors in vivo. We also show the zebrafish hand2 is required for the normal development of the intestinal smooth muscle and the ENS. Furthermore we show that endoderm and HH signaling, but not hand2, regulate gdnf expression in the intestine, highlighting a central role of endoderm and SHH in patterning the intestine and the ENS.     

Sonic hedgehog signalling inhibits palatogenesis and arrests tooth development in a mouse model of the nevoid basal cell carcinoma syndrome

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant or spontaneous disorder characterized by multiple cutaneous basal cell carcinomas, odontogenic keratocysts, skeletal anomalies and facial dysmorphology, including cleft lip and palate. Causative mutations for NBCCS occur in the PTCH1 gene on chromosome 9q22.3-q31, which encodes the principle receptor for the Hedgehog signalling pathway. We have investigated the molecular basis of craniofacial defects seen in NBCCS using a transgenic mouse model expressing Shh in basal epithelium under a Keratin-14 promoter. These mice have an absence of flat bones within the skull vault, hypertelorism, open-bite malocclusion, cleft palate and arrested tooth development. Significantly, increased Hedgehog signal transduction in these mice can influence cell fate within the craniofacial region. In medial edge epithelium of the palate, Shh activity prevents apoptosis and subsequent palatal shelf fusion. In contrast, high levels of Shh in odontogenic epithelium arrests tooth development at the bud stage, secondary to a lack of cell proliferation in this region. These findings illustrate the importance of appropriately regulated Hedgehog signalling during early craniofacial development and demonstrate that oro-facial clefting and hypodontia seen in NBCCS can occur as a direct consequence of increased Shh signal activity within embryonic epithelial tissues.     

Sonic hedgehog signaling regulates Gli3 processing, mesenchymal proliferation, and differentiation during mouse lung organogenesis

Lack of Sonic hedgehog (Shh) signaling, mediated by the Gli proteins, leads to severe pulmonary hypoplasia. However, the precise role of Gli genes in lung development is not well established. We show Shh signaling prevents Gli3 proteolysis to generate its repressor forms (Gli3R) in the developing murine lung. In Shh(-/-) or cyclopamine-treated wild-type (WT) lung, we found that Gli3R level is elevated, and this upregulation appears to contribute to defects in proliferation and differentiation observed in the Shh(-/-) mesenchyme, where Gli3 is normally expressed. In agreement, we found Shh(-/-);Gli3(-/-) lungs exhibit enhanced growth potential. Vasculogenesis is also enhanced; in contrast, bronchial myogenesis remains absent in Shh(-/-);Gli3(-/-) compared with Shh(-/-) lungs. Genes upregulated in Shh(-/-);Gli3(-/-) relative to Shh(-/-) lung include Wnt2 and, surprisingly, Foxf1 whose expression has been reported to be Shh-dependent. Cyclins D1, D2, and D3 antibody labelings also reveal distinct expression patterns in the normal and mutant lungs. We found significant repression of Tbx2 and Tbx3, both linked to inhibition of cellular senescence, in Shh(-/-) and partial derepression in Shh(-/-); Gli3(-/-) lungs, while Tbx4 and Tbx5 expressions are less affected in the mutants. Our findings shed light on the role of Shh signaling on Gli3 processing in lung growth and differentiation by regulating several critical genes.     

Function and regulation of Alx4 in limb development: complex genetic interactions with Gli3 and Shh

The role of the aristaless-related homeobox gene Alx4 in antero-posterior (AP-) patterning of the developing vertebrate limb has remained somewhat elusive. Polydactyly of Alx4 mutant mice is known to be accompanied by ectopic anterior expression of genes like Shh, Fgf4 and 5'Hoxd. We reported previously that polydactyly in Alx4 mutant mice requires SHH signaling, but we now show that in early Alx4-/- limb buds the anterior ectopic expression of Fgf4 and Hoxd13, and therefore disruption of AP-patterning, occurs independently of SHH signaling. To better understand how Alx4 functions in the pathways that regulate AP-patterning, we also studied genomic regulatory sequences that are capable of directing expression of a reporter gene in a pattern corresponding to endogenous Alx4 expression in anterior limb bud mesenchyme. We observed, as expected for authentic Alx4 expression, expansion of reporter construct expression in a Shh-/- background. Total lack of reporter expression in a Gli3-/- background confirms the existence of Gli3-dependent and -independent Alx4 expression in the limb bud. Apparently, these two modules of Alx4 expression are linked to dissimilar functions.     

Light-quality effects on Arabidopsis development. Red, blue and far-red regulation of flowering and morphology

Arabidopsis thaliana (L.) Heynh. race Columbia plants were grown in red. blue, red + far-red, blue + far-red and various light mixtures of red + blue + far-red light under 14 h light/10 h dark photoperiods. Each single light source and light mixture maintained a constant irradiance (50 μmol m⁻² s⁻¹) and the mixtures of red + blue + far-red maintained a constant ratio of red/far-red light, but varied in the ratio of blue to red + far-red light. Depending on the method used for calculation, values of the fraction of phytochrome in the far-red absorbing form (P_fr/P_tot) for these light mixtures were either constant or decreased slightly with increasing percentage of blue light in the mixtures. Arabidopsis flowered early (20 days) in blue, blue + far-red and red + far-red light and late (55 days) in red light. In mixtures of red + blue + far-red light, each of which established a nearly constant P_fr/P_tot flowering was in direct relation to time and irradiance level of blue light. Leaf area and petiole length were also correlated with blue light irradiance levels.     

氰氟虫腙亚致死剂量对甜菜夜蛾生长发育和繁殖力的影响

本文研究了氰氟虫腙的亚致死剂量LC15(28.423μg/mL)和LC25(41.363μg/mL)对甜菜夜蛾Spodoptera exigua(Hübner)亲代和子1代的一些生物学特性的影响。结果表明:LC15和LC25处理组与对照组相比,甜菜夜蛾亲代的化蛹率,羽化率和单雌产卵量分别降低了49.3%,60.0%;♀(12.65%,25.09%)和♂(13.14%,28.91%);25.6%,65.7%,畸形蛹率分别提高了10.5%,14.4%。子1代的化蛹率,羽化率和单雌产卵量分别降低了11.8%,24.6%;♀(9.48%,21.54%)和♂(11.61%,24.08%);14.4%,54.3%,畸形蛹率分别提高了4.1%,5.8%。另外子1代的孵化率、幼虫的存活率降低,蛹期和成虫期缩短,产卵前期延长,产卵期显著缩短。总体来说,LC15和LC25剂量的氰氟虫腙对甜菜夜蛾亲代与子1代的生物特性都有不同程度的影响,并且LC25剂量对甜菜夜蛾的影响要比LC15剂量大。     

Thermal and thermoperiodic effects on larval development and diapause in the European corn borer, Ostrinia nubilalis

Effects of temperature and thermoperiod on larval development and the induction of diapause were investigated in the European corn borer, Ostrinia nubilalis. Developmental threshold temperatures characteristic of the first four larval instars were estimated under both thermoperiods and constant temperatures. Threshold values were similar under the two conditions, but the 4th-larval instar was shown to display a significantly lower developmental threshold temperature than was characteristic of the earlier instars. Although developmental times (days per instar) were not greatly affected by fluctuating temperature regimes, the quantity of growth (weight, head width) during the 4th and 5th (last) stadia was found to be much greater under thermoperiodic regimes than under comparable constant temperature conditions. By means of thermoperiodic regimes having identical mean temperatures but different cryophase durations, it was demonstrated that the induction of diapause is dependent on the duration of the cryophase rather than on the mean temperature of the thermoperiod. To be effective, the cryophase must be colder than the insect's thermoperiodic response threshold. It was also demonstrated that thermoperiodic responses that are readily produced under continuously dark rearing conditions are not manifested under continuous light.     

The effects of temperature and food on naupliar development,growth and metamorphosis in three species of Boeckella (Copepoda:Calanoida)

Three species of Boeckella (B. triarticulata, B. dilatata and B. hamata) were reared from hatching to copepodite I (CI) at three naturally fluctuating food levels and three temperatures in a 3 × 3 × 3 factorial design. Development times, lengths and mortality were measured for each species in nine treatments. Temperature had the major effect on development times but food level had the major effect on CI lengths. Mortality varied interspecifically with treatment. The combined effect of temperature and food on development times and lengths was species-specific. There were trade-offs between development time and growth to meet constraints imposed by naupliar metamorphosis. The three species varied in the timing of metamorphosis to CI. B. triarticulata nauplii were age determined, B. dilatata nauplii were size determined and B. hamata nauplii were flexibly age and size determined depending on treatment. Differences in life history parameters and the timing of naupliar metamorphosis are discussed in relation to the distributions of the species in ponds (B. triarticulata), glacial lakes (B. dilatata) and coastal lakes (B. hamata) in the South Island of New Zealand.     

Differential effects of light quantity and spectral light quality on growth, morphology and development of two stoloniferous Potentilla species

Plant species from open habitats often show pronounced responses to shading. Apart from a reduction in growth, shading can lead to marked changes in morphology and architecture, and it may affect the rate of plant development. Natural shade comprises two basically different features, a reduction in light quantity (amount of radiation) and changes in the spectral light quality. The first aspect represents changes in resource availability, while the latter acts as a source of information for plants and can prompt morphogenetic responses. A greenhouse experiment was carried out to study the effects of changes in light quality and quantity on the growth, morphology and development of two stoloniferous Potentilla species. Individual plants were subjected to three light treatments: (1) full daylight (control); and two shade treatments, in which (2) light quantity (photon flux density) and (3) light spectral quality (red/far-red ratio) were changed independently. Plant development was followed throughout the study. Morphological parameters, biomass and clonal offspring production were measured at the end of the experiment. Morphological traits such as petiole length, leaf blade characteristics and investment patterns into spacers showed high degrees of shade-induced plasticity in both species. With a few exceptions, light quality mainly affected morphological variables, while production parameters were most responsive to changes in light quantity. Potentilla anserina allocated resources preferentially to established rosettes at the cost of stolon growth and branching, while in P. reptans, all parameters related to development and allocation were slowed down to the same extent by light limitation. Light quality changes also positively affected biomass production via changes in leaf allocation. Changes in the spectral light quality had major effects on the size of modular structures (leaves, ramets), whereas changes in light quantity mainly affected their numbers. Received: 12 December 1997 / Accepted: 8 July 1998     

The effects of short−term incubation (aging) of mouse oocytes on in vitro fertilization,zona solubility,and embryonic development

The effects of in vitro aging of cumulus?intact versus cumulus?free metaphase II mouse oocytes were studied with respect to zona solubility and fertilization rates. Furthermore, zygotes from the in vitro fertilization studies were incubated and their developmental progress was recorded. The zona pellucida showed a gradual increase in resistance to dissolution by α?chymotrypsin with in vitro aging over a period of 6 hr. This effect was greater in cumulus?free as compared to cumulus?intact ova, but it was not nearly as profound as that seen in the control in vivo fertilized eggs. The fertilization rate of in vitro aging cumulus?intact ova compared favorably with the control in vivo aging group over a 6?hr time period. This was in sharp contrast to the decreased fertilization rate of in vitro aging cumulus?free ova over the same period of time. Lastly, development of zygotes to the blastocyst stage was also evaluated. The rate of first cleavage was similar in all experimental groups and compared favorably with the in vivo controls. However, further development to blastocysts of in vitro aged cumulus?free ova showed a marked decrease when compared to the cumulus?intact group and the in vivo fertilized controls. Thus we established a direct relationship between zona digestion time of in vitro aged cumulus?free oocytes and a decrease of fertilization rates in the mouse.     

Joint effects of temperature, food quality, and season on the development of the cerambycid Morimus funereus under laboratory conditions

The effects of constant temperature (23 °C), food quality (B, natural diet i.e. powdered oak bark; B+C, natural diet enriched with powdered sweet crackers) and season on larval development of the cerambycid Morimus funereus L. were studied. The temperature of 23 °C exerted an unfavourable effect on larvae collected from oak stumps in September and bred on B; i.e. the larvae died within 30 days. When the larvae consumed B+C the unfavourable effect of temperature was abolished and the larvae increased their weight by 543%, 897% and 1179% in 1, 2 and 3 months, respectively. Newly hatched summerand winter-larvae developed successfully on B+C, while all the winter-larvae died within 40 days on B. Larvae of an identical parentage hatched in different phases of the annual cycle and showed seasonal differences in the rate of development and survival when reared on B+C at 23 °C. The fastest development and the highest survival rate were observed in larvae which emerged in early- and midsummer, whereas the slowest development and the lowest survival rate were in those hatched in late summer. The synchronized seasonal changes of adults and their offspring, as expressed at different levels of biological organization, suggest the existence of an endogenous annual rhythm which is dependent upon the expression of different genes in the course of the annual cycle.

---

Résumé L'étude a porté sur les effets d'une température constante (23 °C), de la qualité de l'aliment,-B = aliment naturel, c'est-à-dire de l'écorce de chêne pulvérisée; B+C = aliment naturel enrichi avec de la poudre de biscuits sucrés-, et de la saison sur le développement larvaire de M. funereus L. La température de 23 °C a été défavorable aux larves récoltées en septembre dans des souches de chênes et élevées sur aliment B: les larves étaient mortes en 30 jours. Avec aliment B+C, l'effet défavorable a été neutralisé et le poids des larves augmenté de 543%, 897% et 1179% en 1, 2 et 3 mois. Des larves néonates d'été ou d'hiver se sont bien développées sur B+C, mais toutes les larves d'hiver étaient mortes sur B en 40 j. Des larves de mêmes parents, écloses à différentes époques de l'année, ont présenté des différences saisonnières du taux de survie et de la vitesse de développement lors de leur élevage à 23 °C sur B+C. Le développement le plus rapide et la meilleure survie ont été obtenus avec des larves écloses au début ou au milieu de l'été; tandis que le développement le plus lent et la plus mauvaise survie ont été obtenus avec les larves écloses à la fin de l'été. Les changements saisonniers synchrones des adultes et de leurs descendants, exprimés à différents niveaux d'organisation biologique, suggèrent l'existence d'un rythme annuel endogène qui dépend de l'expression de différents gènes au cours du cycle annuel.