Mutant Quail Research Articles

Research Note: Sexual dimorphic effect of myostatin mutation on fat accumulation in Japanese quail

As an anti-myogenic factor, the myostatin (MSTN) gene was mainly considered as a genetic marker to improve meat production. Moreover, an additional effect of the MSTN mutation on reducing fat deposition in various farm animals suggested a potential application of the MSTN gene on regulating fat deposition in poultry species. Although increase in muscle mass resulted from muscle hyperplasia in the MSTN mutant quail, cellular mechanism behind the decrease in fat deposition was not investigated in the quail model. In the current study, to investigate sexual dimorphic association between fat deposition and Mstn mutation in quail, leg and abdominal fat pads from 4-month-old male and female quail were histologically analyzed. Interestingly, abdominal and leg fat pad weights were significantly decreased by the MSTN mutation only in female quail, but not in male quail, showing sexual dimorphism in regulating fat deposition by the MSTN mutation in quail. Histological analysis also revealed that fat cell sizes of leg and abdominal fats were significantly reduced only in female groups aligning with the decreased fat pad weights. Sexual dimorphic effect of the MSTN mutation on fat cell hypotrophy and reduced fat pad weights in quail provided an important scientific finding to be considered on the usage of the MSTN gene as a genetic marker to reduce fat deposition in poultry species.

Effects of a myostatin mutation in Japanese quail (Coturnix japonica) on the physicochemical and histochemical characteristics of the pectoralis major muscle.

The aim of this study was to compare the carcass, meat quality, and histochemical characteristics of pectoralis major (PM) muscle between wild type (WT) and myostatin (Mstn) homozygous mutant (HO) quail lines. The HO quail line exhibited significantly heavier body weight (HO vs. WT, 115.7g vs. 106.2g, approximately 110%) and PM muscle weight (HO vs. WT, 18.0g vs. 15.2g, approximately 120%) compared to the WT (p < 0.001). However, the two groups had similar traits (pH, redness, yellowness, and drip loss) for meat quality, although slightly higher lightness and cooking loss were observed in the mutant quail (103% and 141%, respectively, p < 0.05). For histochemical traits of PM muscle, Mstn mutant quail exhibited lower type IIA and higher type IIB percentage in the deep region than WT quail (p < 0.05), indicating a fiber conversion from the type IIA to IIB. However, the two quail lines had comparable histochemical traits in the superficial region (p > 0.05). These data suggest that Mstn mutation greatly increases muscle mass without significantly affecting meat quality.

Increased sizes and improved qualities of tibia bones by myostatin mutation in Japanese quail.

Production of large amounts of meat within a short growth period from modern broilers provides a huge economic benefit to the poultry industry. However, poor bone qualities of broilers caused by rapid growth are considered as one of the problems in the modern broilers industry. After discovery and investigation of myostatin (MSTN) as an anti-myogenic factor to increase muscle mass by targeted knockout in various animal models, additional positive effects of MSTN mutation on bone qualities have been reported in MSTN knockout mice. Although the same beneficial effects on muscle gain by MSTN mutation have been confirmed in MSTN mutant quail and chickens, bone qualities of the MSTN mutant birds have not been investigated, yet. In this study, tibia bones were collected from MSTN mutant and wild-type (WT) quail at 4months of age and analyzed by Micro-Computed Tomography scanning to compare size and strength of tibia bone and quality parameters in diaphysis and metaphysis regions. Length, width, cortical thickness, and bone breaking strength of tibia bones in the MSTN mutant group were significantly increased compared to those of the WT group, indicating positive effects of MSTN mutation on tibia bone sizes and strength. Furthermore, bone mineral contents and bone volume of whole diaphysis, diaphyseal cortical bone, whole metaphysis, and metaphyseal trabecular and cortical bones were significantly increased in the MSTN mutant group compared to the WT group, indicating increased mineralization in the overall tibia bone by MSTN mutation. Especially, higher bone mineral density (BMD) of whole diaphysis, higher total surface of whole metaphysis, and higher BMD, trabecular thickness, and total volume of metaphyseal trabecular bones in the MSTN mutant group compared to the WT group suggested improvements in bone qualities and structural soundness of both diaphysis and metaphysis regions with significant changes in trabecular bones by MSTN mutation. Taken together, MSTN can be considered as a potential target to not only increase meat yield, but also to improve bone qualities that can reduce the incidence of leg bone problems for the broiler industry.

Research Note: Growth promoting potential in Mstn mutant quail dependent and independent of increased egg size

In avian species, positive relationships between egg weight (EW) and body weight (BW) have been reported. However, the correlation between the body growth rate and different weights of eggs from genetically mutated avian species was not studied yet. Myostatin (Mstn), an anti-myogenic factor, mutant quail were recently developed, and it was reported that EW produced from Mstn homozygous mutant quail (HO) was heavier compared to those from wild-type quail (WT). In the current study, distributions of pre-incubated EW and associations between EW and BW were compared between the Mstn mutant and WT quail lines. Average egg weight for the HO group was significantly heavier than the WT (P < 0.001) and the number of eggs having heavier EW (over 11 g) was higher in the HO compared to the WT (P < 0.01). BWs at wk (W) 0, 4, and 6 after hatch were also significantly greater in the HO (P < 0.001 in all groups). In addition, linear regression analyses revealed positive relationships between EW and BW from W0 to W6, regardless of sexes and genotypes. Furthermore, Mstn mutant quail were a heavier BW compared to the WT quail originated from eggs with similar weights. These data indicate that increased BW by Mstn mutation is contributed by increased EW and/or growth promoting activity of Mstn mutation independent of increasing egg sizes. These findings provide Mstn as a desirable genetic factor for selection of poultry breeds with superior growth. In addition, the knowledge gained from this study could inspire similar proof-of-concept studies involving standard and commercial lines of poultry.

Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quail.

The precursor of heme, protoporphyrin IX (PPIX), accumulates abundantly in the uteri of birds, such as Japanese quail, Coturnix japonica, which has brown-speckled eggshells; however, the molecular basis of PPIX production in the uterus remains largely unknown. Here, we investigated the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis. We also performed genetic linkage analysis to identify the causative genomic region of the white eggshell phenotype. We found that 11 genes, including 5'-aminolevulinate synthase 1 (ALAS1) and hephaestin-like 1 (HEPHL1), were specifically upregulated in the wild-type uterus and downregulated in the mutant. We mapped the 172 kb candidate genomic region on chromosome 6, which contains several genes, including a part of the paired-like homeodomain 3 (PITX3), which encodes a transcription factor. ALAS1, HEPHL1, and PITX3 were expressed in the apical cells of the luminal epithelium and lamina propria cells of the uterine mucosa of the wild-type quail, while their expression levels were downregulated in the cells of the mutant quail. Biochemical analysis using uterine homogenates indicated that the restricted availability of 5'-aminolevulinic acid is the main cause of low PPIX production. These results suggest that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail. Based on these findings, we discussed the molecular basis of PPIX production in the uteri of Japanese quails.

Myostatin Mutation in Japanese Quail Increased Egg Size but Reduced Eggshell Thickness and Strength.

Simple SummaryAvian eggs provide huge benefits to both science and society by providing an important model for developmental studies and a high-quality protein source for the human diet. Especially, the hard-shell layer existing at the outer part of eggs is a unique characteristic, which is exclusive in avian species compared to other egg-laying species. Among various avian models developed to investigate genetic factors for potential industrial application, myostatin (MSTN) mutations in quail and chickens were recently generated, resulting in improved meat yield. In addition to previously reported growth and egg production traits in MSTN mutant quail, eggshell quality of mutants was further investigated in this study. Although eggshell height, width, and weight were increased by the MSTN mutation, eggshell breaking strength (EBS) and eggshell thickness were decreased in mutant eggs compared to wild-type eggs. Although these data indicated that decreased eggshell thickness contributed to decreased EBS in mutant eggs, the cellular mechanism of thinner eggshell formation in uterus by MSTN mutation needs to be further investigated using MSTN mutant quail.Recently developed myostatin (MSTN) mutant quail and chickens demonstrated similar effects of MSTN on muscle and fat developments between avian and mammalian species. However, the effect of MSTN mutation on the quality of eggshells, an important avian specific characteristic, has not yet been investigated although egg production traits of mutant quail have been studied. In this study, several parameters for eggshell quality, including eggshell size, eggshell weight, eggshell breaking strength (EBS), and eggshell thickness, were all compared between MSTN mutant and wild-type (WT) eggs. MSTN mutant eggs had greater height and width along with heavier eggshell weight compared to WT eggs, which shows proportional improvement in egg size as affected by the MSTN mutation. However, EBS and eggshell thickness were decreased in mutant eggs compared to WT eggs. In addition, the palisade layer, the thickest and most important layer for the strength of an eggshell, was also decreased without a change in the number of vesicular holes. These data indicated that decreases in the thickness of the eggshell and the palisade layer would be a main factor contributing to a lower EBS in mutant eggs. MSTN mutant quail provide a useful model to better understand the function of MSTN on avian uterine cell development and eggshell biomineralization.

Effects of Myostatin Mutation on Onset of Laying, Egg Production, Fertility, and Hatchability.

Simple SummaryPoultry can be classified as broilers for meat production and layers for egg production. Modern poultry farming improved economically important traits of broilers and layers by breeding and genetic selection. Myostatin (MSTN) has gained attention as a potential selection marker for higher meat production in the poultry industry, because MSTN mutant chickens and quail showed increased muscle mass. In this study, the effect of MSTN mutation on egg production was investigated to evaluate potential use of MSTN for higher egg production in the layer industry. MSTN homozygous mutant quail showed a significantly delayed onset of egg laying, a higher egg weight, and a lower number of eggs produced during the active laying period compared to wild-type quail. However, there were no significant differences in total egg production for 20 days, percentage proportion of egg white and yolk in egg weight, and egg fertility, and hatchability between MSTN mutant and WT quail. Although a clear benefit on egg production by MSTN mutation in quail was not revealed, this study provided useful information to understand the productive performance of MSTN mutant hens. Increased body weight and muscle mass, along with improved feed efficiency, by myostatin (MSTN) mutation in quail, supports the potential use of MSTN as a selection marker for higher meat yield in the poultry industry. Although economically important traits of broilers have been studied using recently generated MSTN mutant quail, the effect of MSTN mutation on egg production has not yet been investigated. In this study, several economically important traits of layers, including egg production, reproduction, and body composition of hens, were compared between MSTN homozygous mutant, heterozygous mutant, and wild-type (WT) quail. In terms of egg production, MSTN homozygous mutant quail, showing significantly delayed onset of egg laying, laid significantly heavier eggs, but a significantly lower number of eggs compared to WT quail for 20 days after 3 months of age, resulting in similar total egg production among groups. In addition, the percentage proportion of egg white and yolk in egg weight were similar among groups. Furthermore, similar fertility and hatchability of eggs from MSTN homozygous mutant breeding pairs and WT breeding pairs indicated normal reproductive function of MSTN mutant quail. These findings will provide scientific rationales for the consideration of MSTN as a potential selection marker for layers in the poultry industry.

Research Note: Improved feed efficiency in quail with targeted genome editing in the myostatin gene

Increased growth rate and decreased cost of feed are main focuses to increase revenue of poultry farms. Myostatin (MSTN) is a negative regulator of muscle growth and mutation on MSTN results in increased muscle growth. Due to the antimyogenic function of MSTN, MSTN gains high attention as a potential target and genetic selection marker to increase meat yield in the livestock industry. In addition, MSTN can affect feed efficiencies and, thus decrease total feed requirement as shown in increased feed efficiencies in pigs and cattle with MSTN mutations. Although MSTN mutation in various animal species has been previously studied, MSTN mutation in avian species has only recently been generated to characterize its biological function. However, beneficial effects of MSTN mutation on poultry production need to be further investigated. In this study, using the MSTN mutant quail, feed efficiency related to interplay of changes in body weight gain (WG), feed intake (FI), and fat accretion were investigated. WG of mutant quail were significantly higher (P< 0.001) than those of wild-type (WT) from all time periods, 10-d interval from post-hatching day (D)10 to 40. Feed intake of mutant quail were significantly higher than those of WT from D 10 to 20 (P< 0.01) and D 20 to 30 (P< 0.001), but not from D 30 to 40, resulting in a significantly lower feed conversion ratio (FCR) of mutant quail compared to WT quail only from D 30 to 40 (P< 0.001). From those results, overall (D 10 to 40) FCR was significantly lower in mutant quail (P< 0.001) indicating improved feed efficiency by MSTN mutation. In addition, percentages of leg or abdominal fat compared to body weight in mutant quail at 8 wk were significantly lower than WT (P< 0.05). In combination to greater WG, less fat accretion might partially contribute to improved feed efficiency in MSTN mutant quail. As there is a current preference of meat with lower fat as a healthy food, MSTN can be used for the potential selection marker for not only bigger and leaner poultry, but also better feed efficiency that can satisfy both producers and consumers.

Muscle Hyperplasia in Japanese Quail by Single Amino Acid Deletion in MSTN Propeptide.

Mutation in myostatin (MSTN), a negative regulator of muscle growth in skeletal muscle, resulted in increased muscle mass in mammals and fishes. However, MSTN mutation in avian species has not been reported. The objective of this study was to generate MSTN mutation in quail and investigate the effect of MSTN mutation in avian muscle growth. Recently, a new targeted gene knockout approach for the avian species has been developed using an adenoviral CRISPR/Cas9 system. By injecting the recombinant adenovirus containing CRISPR/Cas9 into the quail blastoderm, potential germline chimeras were generated and offspring with three base-pair deletion in the targeted region of the MSTN gene was identified. This non-frameshift mutation in MSTN resulted in deletion of cysteine 42 in the MSTN propeptide region and homozygous mutant quail showed significantly increased body weight and muscle mass with muscle hyperplasia compared to heterozygous mutant and wild-type quail. In addition, decreased fat pad weight and increased heart weight were observed in MSTN mutant quail in an age- and sex-dependent manner, respectively. Taken together, these data indicate anti-myogenic function of MSTN in the avian species and the importance of cysteine 42 in regulating MSTN function.

The silkworm (Bombyx mori) neuropeptide orcokinin is involved in the regulation of pigmentation

The natural colorful cuticles of insects play important roles in many physiological processes. Pigmentation is a physiological process with a complex regulatory network whose regulatory mechanism remains unclear. Bombyx mori pigmentation mutants are ideal materials for research on pigmentation mechanisms. The purple quail-like (q-lp) and brown quail-like (q-lb) mutants originated from plain silkworm breeds 932VR and 0223JH respectively exhibit similar cuticle pigmentation to that of the quail mutant. The q-lp mutant also presents a developmental abnormality. In this study, genes controlling q-lp and q-lb mutants were located on chromosome 8 by positional cloning. Then the neuropeptide gene orcokinin (OK) was identified to be the major gene responsible for two quail-like mutants. The B. mori orcokinin gene (BommoOK) produces two transcripts, BommoOKA and BommoOKB, by alternative splicing. The CRISPR/Cas9 system and orcokinin peptides injection were used for further functional verification. We show a novel function of BommoOKA in inhibiting pigmentation, and one mature peptide of orcokinin A, OKA_type2, is the key factor in pigmentation inhibition. These results provide a reference for studying the function of orcokinin and are of theoretical importance for studying the regulatory mechanism of pigmentation.

Direct delivery of adenoviral CRISPR/Cas9 vector into the blastoderm for generation of targeted gene knockout in quail

Zygotes at the 1-cell stage have been genetically modified by microinjecting the CRISPR/Cas9 components for the generation of targeted gene knockout in mammals. In the avian species, genetic modification of the zygote is difficult because its unique reproductive system limits the accessibility of the zygote at the 1-cell stage. To date, only a few CRISPR/Cas9-mediated gene knockouts have been reported using the chicken as a model among avian species, which requires 3 major processes: isolation and culture of primordial germ cells (PGCs), modification of the genome of PGCs in vitro, and injection of the PGCs into the extraembryonic blood vessel at the early embryonic stages when endogenous PGCs migrate through circulation to the genital ridge. In the present study, the adenoviral CRISPR/Cas9 vector was directly injected into the quail blastoderm in newly laid eggs. The resulting chimeras generated offspring with targeted mutations in the melanophilin (MLPH) gene, which is involved in melanosome transportation and feather pigmentation. MLPH homozygous mutant quail exhibited gray plumage, whereas MLPH heterozygous mutants and wild-type quail exhibited dark brown plumage. In addition, the adenoviral vector was not integrated into the genome of knockout quail, and no mutations were detected in potential off-target regions. This method of generating genome-edited poultry is expected to accelerate avian research and has potential applications for developing superior genetic lines for poultry production in the industry.

Transcriptome analysis of the epidermis of the purple quail-like (q-lp) mutant of silkworm, Bombyx mori.

A new purple quail-like (q-lp) mutant found from the plain silkworm strain 932VR has pigment dots on the epidermis similar to the pigment mutant quail (q). In addition, q-lp mutant larvae are inactive, consume little and grow slowly, with a high death rate and other developmental abnormalities. Pigmentation of the silkworm epidermis consists of melanin, ommochrome and pteridine. Silkworm development is regulated by ecdysone and juvenile hormone. In this study, we performed RNA-Seq on the epidermis of the q-lp mutant in the 4th instar during molting, with 932VR serving as the control. The results showed 515 differentially expressed genes, of which 234 were upregulated and 281 downregulated in q-lp. BLASTGO analysis indicated that the downregulated genes mainly encode protein-binding proteins, membrane components, oxidation/reduction enzymes, and proteolytic enzymes, whereas the upregulated genes largely encode cuticle structural constituents, membrane components, transport related proteins, and protein-binding proteins. Quantitative reverse transcription PCR was used to verify the accuracy of the RNA-Seq data, focusing on key genes for biosynthesis of the three pigments and chitin as well as genes encoding cuticular proteins and several related nuclear receptors, which are thought to play key roles in the q-lp mutant. We drew three conclusions based on the results: 1) melanin, ommochrome and pteridine pigments are all increased in the q-lp mutant; 2) more cuticle proteins are expressed in q-lp than in 932VR, and the number of upregulated cuticular genes is significantly greater than downregulated genes; 3) the downstream pathway regulated by ecdysone is blocked in the q-lp mutant. Our research findings lay the foundation for further research on the developmental changes responsible for the q-lp mutant.

Development and Performances of a Japanese Quail Line Homozygous for the Diabetes Insipidus (di) Mutation

A strain of Japanese quail with the polyuria disorder (excessive urination) was developed from founders that regurgitated water spontaneously. A back-cross with a nonpolyuric quail line showed that the polyuric strain was fixed for an autosomal recessive mutation that also induced polydipsia (excessive drinking). Plasma levels and brain mRNA contents for avian Arg vasotocin were little affected by the mutation, but plasma avian Arg vasotocin was 13-fold higher and brain mRNA contents were significantly increased in both normal and mutant quail following a 24-h water deprivation. Affected and normal birds had similar performance traits (egg production and quality, feed intake, and gross carcass traits), but residual feed consumption was higher in polydipsic males. These results are consistent with the hypothesis that this strain was fixed for a mutation similar to the di gene described in the chicken and which induces nephrogenic diabetes insipidus. This new strain of Japanese quail might constitute a convenient model for the analysis of the underlying mechanisms of the disorder in birds and for comparative study with mammals.

Pigment pattern formation in the quail mutant of the silkworm, Bombyx mori: parallel increase of pteridine biosynthesis and pigmentation of melanin and ommochromes

The larval pigment pattern in the silkworm, Bombyx mori, is formed by melanin, ommochromes and pteridines. During development all these pigments are synthesized autonomously, and possibly also with mutual interaction between them, to yield unique pigment patterns. In order to find the key trigger for such pigment pattern formation, developmental changes in pteridine biosynthesis were studied using the quail mutant (q/q), which has darker larval marks formed by melanin and an abundance of ommochromes in the integument. In the current study, emphasis has been placed on the analysis of GTP-cyclohydrolase I (GTP-CH I), which is a key enzyme for the biosynthesis of pteridines, during the development of the silkworm. Results of Northern blotting showed that in the quail mutant strong signals of GTP-CH I mRNA appeared around each period of ecdysis, while no such signals appeared in the background strain (+q/q) used. Also, both GTP-CH I activities and pteridine content were higher in the quail mutant compared with the background strain. These results strongly suggest that pteridine biosynthesis is closely linked to the formation of melanin and ommochromes. It is also suggested here that in the silkworm a recessive gene (q) may be involved in the regulation of its pigment pattern formation.

Mapping of expressed sequence tag markers with a cDNA‐amplified fragment length polymorphism method in Japanese quail (Coturnix japonica)

ABSTRACTIn our previous study, a Kobe‐NIBS Japanese quail (KNQ) linkage map was constructed mainly using amplified fragment length polymorphism (AFLP) markers. In order to compare chicken and quail chromosomes, we developed expressed sequence tag (EST) markers derived from cDNA‐AFLP fragments and localized these markers on the linkage map. Using a total of 128 AFLP primer combinations, 24 polymorphic bands were obtained between a neurofilament‐deficient mutant quail line male and a muscular disorder quail line female, which were the parents of the KNQ resource family. Nine of the 24 markers were mapped by linkage analysis. These markers were mapped to seven linkage groups, namely 1, 2, 3, 6, 8, 15 and 42. A subsequent homology search using chicken genome sequences strongly suggests that these linkage groups correspond with chicken chromosomes 1, 2, 3, 5, 15, 23 and 26.

Epithelia-mesenchyme interaction plays an essential role in transdifferentiation of retinal pigment epithelium of silver mutant quail: localization of FGF and related molecules and aberrant migration pattern of neural crest cells during eye rudiment formation.

Homozygotes of the quail silver mutation, which have plumage color changes, also display a unique phenotype in the eye: during early embryonic development, the retinal pigment epithelium (RPE) spontaneously transdifferentiates into neural retinal tissue. Mitf is considered to be the responsible gene and to function similarly to the mouse microphthalmia mutation, and tissue interaction between RPE and surrounding mesenchymal tissue in organ culture has been shown to be essential for the initiation of the transdifferentiation process in which fibroblast growth factor (FGF) signaling is involved. The immunohistochemical results of the present study show that laminin and heparan sulfate proteoglycan, both acting as cofactors for FGF binding, are localized in the area of transdifferentiation of silver embryos much more abundantly than in wild-type embryos. More intense immunohistochemical staining with FGF-1 antibody, but not with FGF-2 antibody, is also found in the neural retina, RPE, and choroidal tissue of silver embryos than in wild-type embryos. HNK-1 immunohistochemistry revealed that clusters of HNK-1-positive cells (presumptive migrating neural crest cells) are frequently located around the developing eyes and in the posterior region of the silver embryonic eye. Finally, chick-quail chimerical eyes were made by grafting silver quail optic vesicles to chicken host embryos: in most cases, no transdifferentiation occurs in the silver RPE, but in a few cases, transdifferentiation occurs where silver quail cells predominate in the choroid tissue. These observations together with our previous in vitro study indicate that the silver mutation affects not only RPE cells but also cephalic neural crest cells, which migrate to the eye rudiment, and that these crest cells play an essential role in the transdifferentiation of RPE, possibly by modifying the FGF signaling pathway. The precise molecular mechanism involved in RPE-neural crest cell interaction is still unknown, and the quail silver mutation is considered to be a good experimental model for studying the role of neural crest cells in vertebrate eye development.

Electrophysiological measures of auditory function in the neurofilament-deficient mutant quail (Quv)

Auditory pathway electrophysiological studies were performed on the mutant quail ‘Quv’. This mutation is known to result in neurofilament deficiencies of both the peripheral and central nervous systems. Auditory evoked brainstem responses (ABRs), electrocochleograms (EcochGs) and middle latency responses (MLRs) were evaluated. ABRs in Quv quails demonstrated markedly altered waveforms exhibiting longer latencies, absence of the later peaks and lower amplitudes. The EcochG showed normal cochlear microphonics with no obvious abnormalities in amplitude or latency and normal latencies for peak N1. Quv quails had a mild threshold elevation with a normal latency for the first peak of the ABR (P1). The Quv MLRs showed no significant differences in amplitude but they revealed a latency prolongation for peaks N0, Pa and Na relative to the controls. We have discovered abnormal findings of auditory evoked potentials in the neurofilament-deficient quail (Quv). We suggest that the smaller axonal size and axonal hypotrophy due to altered neurofilament expression underlies these abnormal auditory evoked potential responses.

Occurrence of ommochrome-containing pigment granules in the central nervous system of the silkworm, Bombyx mori

Dark-red pigment granules were found in the brain and ganglion of the normal strain of the silkworm, Bombyx mori, by light microscopy. No other pigmentation was seen in the brain or ganglia. Electron microscopy showed that the granules were electron-dense. The granules were similar to the ommochrome-containing pigment granules that are present in the epidermal cells of the quail mutant, as previously reported. The pigment in the larval central nervous system (CNS) of the normal silkworm was identical to the ommin standard with respect to the absorption spectrum, the infrared spectrum, and the R f value in thin-layer chromatography (TLC). After acid hydrolysis of the pigment, 3-hydroxykynurenine was detected by TLC. The pigment granules in the CNS contained mainly ommin. An ommochrome-binding protein was also detected in the CNS by in vitro binding studies and Western blotting. The ommochrome granules may have an important function in the CNS of the silkworm.

Drosophila quail, a villin-related protein, bundles actin filaments in apoptotic nurse cells.

Drosophila Quail protein is required for the completion of fast cytoplasm transport from nurse cells to the oocyte, an event critical for the production of viable oocytes. The abundant network of cytoplasmic filamentous actin, established at the onset of fast transport, is absent in quail mutant egg chambers. Previously, we showed that Quail is a germline-specific protein with sequence homology to villin, a vertebrate actin-regulating protein. In this study, we combined biochemical experiments with observations in egg chambers to define more precisely the function of this protein in the regulation of actin-bundle assembly in nurse cells. We report that recombinant Quail can bind and bundle filamentous actin in vitro in a manner similar to villin at a physiological calcium concentration. In contrast to villin, Quail is unable to sever or cap filamentous actin, or to promote nucleation of new actin filaments at a high calcium concentration. Instead, Quail bundles the filaments regardless of the calcium concentration. In vivo, the assembly of nurse-cell actin bundles is accompanied by extensive perforation of the nurse-cell nuclear envelopes, and both of these phenomena are manifestations of nurse-cell apoptosis. To investigate whether free calcium levels are affected during apoptosis, we loaded egg chambers with the calcium indicator Indo-1. Our observations indicate a rise in free calcium in the nurse-cell cytoplasm coincident with the permeabilization of the nuclear envelopes. We also show that human villin expressed in the Drosophila germline could sense elevated cytoplasmic calcium; in nurse cells with reduced levels of Quail protein, villin interfered with actin-bundle stability. We conclude that Quail efficiently assembles actin filaments into bundles in nurse cells and maintains their stability under fluctuating free calcium levels. We also propose a developmental model for the fast phase of cytoplasm transport incorporating findings presented in this study.

Expression of gicerin, a cell adhesion molecule, in the abnormal retina in silver plumage color mutation of Japanese quail ( Coturnix japonica)

Silver plumage color mutant ( B/B) quail has an abnormal retina characterizing the transdifferentiation of retinal pigment epithelium (RPE) following the retinal separation in the early developmental stage. In the present study; (i) the expression of gicerin, an immunoglobulin-superfamily cell adhesion molecule, was examined in the retina of B/B quail. In the wild-type quail, gicerin protein was enriched in the apical membrane (facing the neural retina, NR) of RPE cells on embryonic day (E) 4 and then appeared also in NR cells from E5. However, in the B/B retina, no gicerin expression was found in the transdifferentiation area of RPE prior to the retinal separation. (ii) In addition to this, microinjection of anti-gicerin polyclonal antibody into the eyeball of wild-type quail on E3 caused the retinal separation and induced the transdifferentiation of RPE into new NR. These observations suggest that the decrease of gicerin expression might participate in the retinal separation and RPE-transdifferentiation in B/B quail.