Requirement for mast cell growth factor for primordial germ cell survival in culture.

Mast-cell growth factor (MGF) is encoded by the murine steel (Sl) locus and is a ligand for the tyrosine kinase receptor protein encoded by the proto-oncogene c-kit at the murine dominant white spotting (W) locus. Mutations at both these loci affect mast cells, primordial germ cells (PGCs), haemopoietic stem cells and melanocytes. In many Sl and W mutants, the rapid proliferation of PGC that normally occurs between day 7 and 13.5 of embryonic development fails to occur. As c-kit is expressed in PGCs while MGF is expressed in the surrounding mesenchyme, MGF might promote the proliferation of PGCs. Here we report that MGF is essential for PGC survival in culture, but does not stimulate PGC proliferation. Moreover, whereas both the transmembrane and soluble proteolytic cleavage forms of MGF stimulate mast-cell proliferation, soluble MGF has a relatively limited ability to support survival of PGCs in culture, thus explaining the sterility in mice carrying the steel-dickie (Sld) mutation, which encodes only a soluble form of MGF, and providing a functional role for a transmembrane growth factor.     

Embryonic expression of a haematopoietic growth factor encoded by the Sl locus and the ligand for c-kit.

Mice carrying mutations at the W (Dominant white spotting) and Sl (Steel) loci develop abnormalities in three independent systems: neural crest-derived melanocytes, primordial germ cells and haematopoietic stem cells. Consequently, homozygotes of viable mutant alleles have white coats and are sterile and severely anaemic. Tissue recombination studies predict that the W gene is expressed cell autonomously, whereas the product of the Sl locus affects the microenvironment in which the stem cells migrate, proliferate and differentiate. The W locus encodes the protoncogene c-kit, a member of the tyrosine kinase receptor family. The haematopoietic growth factor SCF (stem cell factor) has been identified as the product of the Sl locus and a ligand for c-kit. Here, we report that SCF is expressed during embryogenesis in cells associated with both the migratory pathways and homing sites of melanoblasts, germ cells and haematopoietic stem cells. Both SCF and c-kit are also expressed in a variety of other tissues, including the brain and spinal cord, suggesting that the receptor-ligand system has additional roles in embryogenesis.     

Long-term proliferation of mouse primordial germ cells in culture.

Primordial germ cells (PGCs) are first identifiable as a population of about eight alkaline phosphatase-positive cells in the 7.0 days postcoitum mouse embryo. During the next 6 days of development they proliferate to give rise to the 25,000 cells that will establish the meiotic population. Steel factor is required for PGC survival both in vivo and in vitro and together with leukaemia inhibitory factor stimulates PGC proliferation in vitro. In feeder-dependent culture, PGCs will proliferate for up to 7 days, but their numbers eventually decline and their proliferative capacity is only a fraction of that seen in vivo. Here we report a further factor that stimulates PGC proliferation in vitro, basic fibroblast growth factor (bFGF). Furthermore, bFGF, in the presence of steel factor and leukaemia inhibitory factor, stimulates long-term proliferation of PGCs, leading to the derivation of large colonies of cells. These embryonic germ cells resemble embryonic stem cells, pluripotent cells derived from preimplantation embryos, or feeder-dependent embryonal carcinoma cells, pluripotent stem cells of PGC-derived tumours (teratomas and teratocarcinomas). To our knowledge, these results provide the first system for long-term culture of PGCs.     

Effects of the steel gene product on mouse primordial germ cells in culture.

Mutations at the steel (sl) and dominant white spotting (W) loci in the mouse affect primordial germ cells (PGC), melanoblasts and haemopoietic stem cells. The W gene encodes a cell-surface receptor of the tyrosine kinase family, the proto-oncogene c-kit. In situ analysis has shown c-kit messenger RNA expression in PGC in the early genital ridges. The Sl gene encodes the ligand for this receptor, a peptide growth factor, called here stem cell factor (SCF). SCF mRNA is expressed in many regions of the early mouse embryo, including the areas of migration of these cell types. It is important now to identify the role of the Sl-W interaction in the development of these migratory embryonic stem cell populations. Using an in vitro assay system, we show that SCF increases both the overall numbers and colony sizes of migratory PGC isolated from wild-type mouse embryos, and cultured on irradiated feeder layers of STO cells (a mouse embryonic fibroblast line). In the absence of feeder cells, SCF causes a large increase in the initial survival and apparent motility of PGC in culture. But labelling with bromodeoxyuridine shows that SCF is not, by itself, a mitogen for PGC. SCF does not exert a chemotropic effect on PGC in in vitro assays. These results suggest that SCF in vivo is an essential requirement for PGC survival. This demonstrates the control of the early germ-line population by a specific trophic factor.     

Soma-germline interactions coordinate homeostasis and growth in the Drosophila gonad

The ability of organs such as the liver or the lymphoid system to maintain their original size or regain it after injury is well documented. However, little is known about how these organs sense that equilibrium is breached, and how they cease changing when homeostasis is reached. Similarly, it remains unclear how, during normal development, different cell types within an organ coordinate their growth. Here we show that during gonad development in the fruitfly Drosophila melanogaster the proliferation of primordial germ cells (PGCs) and survival of the somatic intermingled cells (ICs) that contact them are coordinated by means of a feedback mechanism composed of a positive signal and a negative signal. PGCs express the EGF receptor (EGFR) ligand Spitz, which is required for IC survival. In turn, ICs inhibit PGC proliferation. Thus, homeostasis and coordination of growth between soma and germ line in the larval ovary is achieved by using a sensor of PGC numbers (EGFR-mediated survival of ICs) coupled to a correction mechanism inhibiting PGC proliferation. This feedback loop ensures that sufficient numbers of PGCs exist to fill all the stem-cell niches that form at the end of larval development. We propose that similar feedback mechanisms might be generally used for coordinated growth, regeneration and homeostasis.     

The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus

Mice carrying mutations at the W locus located on chromosome 5 are characterized by severe macrocytic anaemia, lack of hair pigmentation and sterility. Mutations at this locus appear to affect the proliferation and/or migration of cells during early embryogenesis and result in an intrinsic defect in the haematopoietic stem cell hierarchy. An understanding of the molecular basis of the complex and pleiotropic phenotype in W mutant mice would thus provide insights into the important developmental processes of gametogenesis, melanogenesis and haematopoiesis. Here we show that the mouse mutant W has a deletion of the c-kit proto-oncogene. Interspecific backcross analysis demonstrates that the W locus is very tightly linked to c-kit and that the two loci cannot be segregated at this level of analysis. c-kit is the cellular homologue of the oncogene v-kit of the HZ4 feline sarcoma virus and encodes a transmembrane protein tyrosine kinase receptor that is structurally similar to the receptors for colony-stimulating factor-1 (CSF-1) and platelet derived growth factor. The co-localization of c-kit with W provides a molecular entry into this important region of the mouse genome. In addition, these observations provide the first example of a germ-line mutation in a mammalian proto-oncogene and implicate the c-kit gene as a candidate for the W locus.     

保护剂浓度对第19期鸡胚原始生殖细胞存活率的影响

采用Ficoll密度梯度离心 ,提取第 19期 (孵化 72h)性腺中的原始生殖细胞 (PGCs) ,用不同浓度的冷冻保护液进行冷冻保存。复苏后的PGCs用台盼蓝染色检测其存活率 ,并进行体外培养。结果发现 :在同一浓度下 ,不同的冷冻保护液之间存在显著 (P <0 0 5 )或极显著 (P <0 0 1)差异。在同一种冷冻保护液下 ,不同的浓度之间存在显著(P <0 0 5 )或极显著 (P <0 0 1)差异 ,具体表现为冷冻保护液浓度越大 ,复苏后细胞的存活率越低。PGCs复苏后于体外培养 ,可增殖形成细胞克隆 ,并可传代培养     

Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo.

Clinical and experimental studies suggest that angiogenesis is a prerequisite for solid tumour growth. Several growth factors with mitogenic or chemotactic activity for endothelial cells in vitro have been described, but it is not known whether these mediate tumour vascularization in vivo. Glioblastoma, the most common and most malignant brain tumour in humans, is distinguished from astrocytoma by the presence of necroses and vascular proliferations. Here we show that expression of an endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), is induced in astrocytoma cells but is dramatically upregulated in two apparently different subsets of glioblastoma cells. The high-affinity tyrosine kinase receptor for VEGF, flt, although not expressed in normal brain endothelium, is upregulated in tumour endothelial cells in vivo. These observations strongly support the concept that tumour angiogenesis is regulated by paracrine mechanisms and identify VEGF as a potential tumour angiogenesis factor in vivo.     

Chromatin dynamics during epigenetic reprogramming in the mouse germ line

A unique feature of the germ cell lineage is the generation of totipotency. A critical event in this context is DNA demethylation and the erasure of parental imprints in mouse primordial germ cells (PGCs) on embryonic day 11.5 (E11.5) after they enter into the developing gonads. Little is yet known about the mechanism involved, except that it is apparently an active process. We have examined the associated changes in the chromatin to gain further insights into this reprogramming event. Here we show that the chromatin changes occur in two steps. The first changes in nascent PGCs at E8.5 establish a distinctive chromatin signature that is reminiscent of pluripotency. Next, when PGCs are residing in the gonads, major changes occur in nuclear architecture accompanied by an extensive erasure of several histone modifications and exchange of histone variants. Furthermore, the histone chaperones HIRA and NAP-1 (NAP111), which are implicated in histone exchange, accumulate in PGC nuclei undergoing reprogramming. We therefore suggest that the mechanism of histone replacement is critical for these chromatin rearrangements to occur. The marked chromatin changes are intimately linked with genome-wide DNA demethylation. On the basis of the timing of the observed events, we propose that if DNA demethylation entails a DNA repair-based mechanism, the evident histone replacement would represent a repair-induced response event rather than being a prerequisite.     

鸟类原生殖细胞的实验观察

本文系统地报导了鸟类原生殖细胞(PGC_s)的实验观察。初步提出了关于使用简易方法制备鸟类PGC_s标本的一系列技术(包括取胚、取胚血、制作胚血涂片、活胚标本、整胚装片和胚组织切片等)。实验观察表明,鸟类PGC_s可以在光镜下借助普通细胞学和组织化学相结合的方法进行鉴别。     

Autophosphorylation sites on the epidermal growth factor receptor

The epidermal growth factor (EGF) receptor is a tyrosine-specific protein kinase with autophosphorylating activity. A 300 amino acid-long region of the receptor's cytoplasmic domain matches (35-90% homology) sequences of transforming proteins from the src family and includes a putative nucleotide binding site. Several of the src transforming proteins have tyrosine kinase activity, but v-erb-B, which appears to be a truncated EGF receptor, is virtually identical to the receptor over this region and yet lacks detectable kinase activity. To locate possible acceptor sites in the v-erb-B protein, we have mapped these sites in the human EGF receptor. We report here that three tyrosine sites near the C-terminus are phosphorylated in vitro. In intact cells, we find that EGF stimulates phosphorylation of several sites, the tyrosine 14 residues from the C-terminus being modified the most extensively. The equivalent site is absent in the v-erb-B protein of avian erythroblastosis virus (AEV) and may influence tyrosine kinase activity.     

鸭胚胎血液中PGCs数量及其外源DNA的体内转染

抽取了麻鸭和骡鸭胚胎发育第14－20期的血液，制作血涂片，PAS染色，观察血液中的原生殖细胞的含量，并对各个时期血液中原生殖细胞的含量进行了统计，最高含量都出现在第16期,分别为8．9752％和2．1871％，用In vivo Gene Delivery Systern和pEGFP-N对PGCs进行了外源DNA的体内转染。结果在6只胚胎中的3只中发现GFP报告基因的表达。     

ATP-stimulated interaction between epidermal growth factor receptor and supercoiled DNA

The receptor for epidermal growth factor (EGF) has been identified as a transmembrane glycoprotein that has tyrosine-specific kinase activity. The kinase activity of the receptor is enhanced in the presence of EGF (or related peptides), and the phosphorylation of a number of substrates, as well as autophosphorylation of the receptor, has been reported. Analogous findings have been described for the insulin receptor and the receptor for platelet-derived growth factor (PDGF). Thus, a number of hormone receptors and several viral transforming proteins appear to share the highly unusual property of tyrosine-specific kinase activity. Nevertheless, the specific relationship between tyrosine kinase activity and the control of cell growth and replication is unknown. It is known that after the initial binding of EGF to the plasma membrane, the hormone together with its receptor is rapidly internalized in endocytic vesicles and the hormone is eventually degraded in lysosomes. It is possible that the function of EGF is simply to stimulate internalization of its receptor, and that as a result of its altered location the receptor is able to phosphorylate a cytoplasmic component or even interact directly with a nuclear component. We now report that the purified receptor for EGF is able to interact with and nick supercoiled double-stranded DNA in an ATP-stimulated manner.     

Epidermal growth factor-dependent phosphorylation of lipocortin

Lipocortin-like proteins are a family of steroid-induced inhibitors of phospholipase activity with potential anti-inflammatory activity. Related proteins have been detected in a variety of tissues and species. The best characterized form is a protein of relative molecular mass (Mr) approximately 40,000 (40K), which is phosphorylated in vivo by protein tyrosine kinases and by protein serine-threonine kinases. It has been proposed that the phospholipase inhibitory activity of lipocortin can be regulated by its phosphorylation. In the A431 cell line, a protein of approximately 35K is phosphorylated by the protein tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Here we report that human lipocortin is phosphorylated near its amino terminus by the EGF receptor/kinase. By peptide mapping and immunological analyses, we show that lipocortin and the endogenous 35K substrate for the EGF receptor/kinase from A431 cells are the same protein.     

Abnormal gonad development in Kit W-2Bao mice caused by a Kit gene missense mutation

Kit ^W-2Bao mice are single-gene autosomal dominant mutation mice with a B6 background that were bred in our laboratory. Heterozygotes had morphological characteristics including albinism of the abdomen, extremities, and tail, whereas the homozygotes had albinism of the body, black eyes, and infertility. The homozygous mutants showed small, structurally abnormal gonads, and lacked germ cells. Heterozygous male mice lacked germ cells in some contorted seminiferous tubules. This mutation has been mapped at 43.8 cM from the centromere in chromosome 5 by linkage analysis and Kit has been identified as the candidate gene. After Kit full-length mRNA amplification, it was found that a G to T conversion at position 1228 in the ORF changed the 410th amino acid from V to F. This amino acid change could affect the protein’s secondary structure. Heterozygous mutant mice were intercrossed and homozygous mutant mice were bred and genotyped. We found that no primordial germ cells (PGCs) appeared in the urogenital ridge area at fetus day 11.5 in the homozygotes. The number of PGCs also significantly decreased in heterozygotes. At fetus day 15.5, the differentiation of the testis tubule structure was unclear; as well, they contained no spermatogonia. Female homozygotes contained no primordial follicles in the ovary. The numbers of PGCs and primordial follicles were significantly decreased in heterozygous mice. W ^?2Bao is the only mutated site in the extracellular 4th Ig-like domain and this mutant mouse model provides new material for the study of the mechanism of reproductive system development.     

实验用五指山小型猪胚胎生殖细胞的分离培养

目的对从猪的原始生殖细胞分离获得胚胎干细胞的方法进行初步研究。方法胎儿取自怀孕26～28d的实验用五指山母猪。分离胎儿生殖嵴获得PGCs,接种于STO饲养层上,以DMEM+2mmol/L谷氨酰胺+0.1mmol/L非必需氨基酸+0.1mmol/Lβ-巯基乙醇+100IU/mL青霉素+100μg/mL链霉素+15%FCS作为培养基,对细胞进行培养传代。结果培养的细胞传至4～5代,形态多样,经AKP染色、SSEA-1免疫荧光染色、Oct-4免疫荧光标记等方法鉴定为阳性,具有胚胎干细胞的特征。     

Testis determination requires insulin receptor family function in mice

In mice, gonads are formed shortly before embryonic day 10.5 by the thickening of the mesonephros and consist of somatic cells and migratory primordial germ cells. The male sex-determining process is set in motion by the sex-determining region of the Y chromosome (Sry), which triggers differentiation of the Sertoli cell lineage. In turn, Sertoli cells function as organizing centres and direct differentiation of the testis. In the absence of Sry expression, neither XX nor XY gonads develop testes, and alterations in Sry expression are often associated with abnormal sexual differentiation. The molecular signalling mechanisms by which Sry specifies the male pathway and models the undifferentiated gonad are unknown. Here we show that the insulin receptor tyrosine kinase family, comprising Ir, Igf1r and Irr, is required for the appearance of male gonads and thus for male sexual differentiation. XY mice that are mutant for all three receptors develop ovaries and show a completely female phenotype. Reduced expression of both Sry and the early testis-specific marker Sox9 indicates that the insulin signalling pathway is required for male sex determination.     

The Ter mutation in the dead end gene causes germ cell loss and testicular germ cell tumours

In mice, the Ter mutation causes primordial germ cell (PGC) loss in all genetic backgrounds. Ter is also a potent modifier of spontaneous testicular germ cell tumour (TGCT) susceptibility in the 129 family of inbred strains, and markedly increases TGCT incidence in 129-Ter/Ter males. In 129-Ter/Ter mice, some of the remaining PGCs transform into undifferentiated pluripotent embryonal carcinoma cells, and after birth differentiate into various cells and tissues that compose TGCTs. Here, we report the positional cloning of Ter, revealing a point mutation that introduces a termination codon in the mouse orthologue (Dnd1) of the zebrafish dead end (dnd) gene. PGC deficiency is corrected both with bacterial artificial chromosomes that contain Dnd1 and with a Dnd1-encoding transgene. Dnd1 is expressed in fetal gonads during the critical period when TGCTs originate. DND1 has an RNA recognition motif and is most similar to the apobec complementation factor, a component of the cytidine to uridine RNA-editing complex. These results suggest that Ter may adversely affect essential aspects of RNA biology during PGC development. DND1 is the first protein known to have an RNA recognition motif directly implicated as a heritable cause of spontaneous tumorigenesis. TGCT development in the 129-Ter mouse strain models paediatric TGCT in humans. This work will have important implications for our understanding of the genetic control of TGCT pathogenesis and PGC biology.