Hwei-Jan Hsu

Insulin levels control female germline stem cell maintenance via the niche in Drosophila

Stem cell maintenance depends on local signals provided by specialized microenvironments, or niches, in which they reside. The potential role of systemic factors in stem cell maintenance, however, has remained largely unexplored. Here, we show that insulin signaling integrates the effects of diet and age on germline stem cell (GSC) maintenance through the dual regulation of cap cell number (via Notch signaling) and cap cell–GSC interaction (via E-cadherin) and that the normal process of GSC and niche cell loss that occurs with age can be suppressed by increased levels of insulin-like peptides. These results underscore the importance of systemic factors for the regulation of stem cell niches and, thereby, of stem cell numbers.

Parallel early development of zebrafish interrenal glands and pronephros: differential control by wt1 and ff1b

Steroids are synthesized mainly from the adrenal cortex. Adrenal deficiencies are often associated with problems related to its development, which is not fully understood. To better understand adrenocortical development, we studied zebrafish because of the ease of embryo manipulation. The adrenocortical equivalent in zebrafish is called the interrenal, because it is embedded in the kidney. We find that interrenal development parallels that of the embryonic kidney (pronephros). Primordial interrenal cells first appear as bilateral intermediate mesoderm expressing ff1b in a region ventral to the third somite. These cells then migrate toward the axial midline and fuse together. The pronephric primordia are wt1-expressing cells located next to the interrenal. They also migrate to the axial midline and fuse to become glomeruli at later developmental stages. Our gene knockdown experiments indicate that wt1 is required for its initial restricted expression in pronephric primordia, pronephric cell migration and fusion. wt1 also appears to be involved in interrenal development and ff1b expression. Similarly, ff1b is required for interrenal differentiation and activation of the differentiated gene, cyp11a1. Our results show that the zebrafish interrenal and pronephros are situated close together and go through parallel developmental processes but are governed by different signaling events.

Specific roles of Target of rapamycin in the control of stem cells and their progeny in the Drosophila ovary

Stem cells depend on intrinsic and local factors to maintain their identity and activity, but they also sense and respond to changing external conditions. We previously showed that germline stem cells (GSCs) and follicle stem cells (FSCs) in the Drosophila ovary respond to diet via insulin signals. Insulin signals directly modulate the GSC cell cycle at the G2 phase, but additional unknown dietary mediators control both G1 and G2. Target of rapamycin, or TOR, is part of a highly conserved nutrient-sensing pathway affecting growth, proliferation, survival and fertility. Here, we show that optimal TOR activity maintains GSCs but does not play a major role in FSC maintenance, suggesting differential regulation of GSCs versus FSCs. TOR promotes GSC proliferation via G2 but independently of insulin signaling, and TOR is required for the proliferation, growth and survival of differentiating germ cells. We also report that TOR controls the proliferation of FSCs but not of their differentiating progeny. Instead, TOR controls follicle cell number by promoting survival, independently of either the apoptotic or autophagic pathways. These results uncover specific TOR functions in the control of stem cells versus their differentiating progeny, and reveal parallels between Drosophila and mammalian follicle growth.

Pregnenolone stabilizes microtubules and promotes zebrafish embryonic cell movement

Embryonic cell movement is essential for morphogenesis and the establishment of body shapes, but little is known about its mechanism. Here we report that pregnenolone, which is produced from cholesterol by the steroidogenic enzyme Cyp11a1 (cholesterol side-chain cleavage enzyme, P450scc), functions in promoting cell migration during epiboly. Epiboly is a process in which embryonic cells spread from the animal pole to cover the underlying yolk. During epiboly, cyp11a1 is expressed in an extra-embryonic yolk syncytial layer. Reducing cyp11a1 expression in zebrafish using antisense morpholino oligonucleotides did not perturb cell fates, but caused epibolic delay. This epibolic defect was partially rescued by the injection of cyp11a1 RNA or the supplementation of pregnenolone. We show that the epibolic delay is accompanied by a decrease in the level of polymerized microtubules, and that pregnenolone can rescue this microtubule defect. Our results indicate that pregnenolone preserves microtubule abundance and promotes cell movement during epiboly.

Expression of zebrafish cyp11a1 as a maternal transcript and in yolk syncytial layer.

Cyp11a1 (P450scc, cholesterol side-chain cleavage enzyme) is the first enzyme for the synthesis of all steroid hormones. The regulation of steroid synthesis has been extensively investigated, except during embryogenesis. To study steroidogenesis in embryos, we have isolated the zebrafish cyp11a1 gene, which consists of 11 exons. Reverse transcription-polymerase chain reaction analysis indicates that zebrafish cyp11a1 is expressed temporally in two waves during embryonic stages and when sexual differentiation begins. It is expressed in adult brain, testicular Leydig cells, and the granulosa/theca layer of the ovary. In addition, zebrafish cyp11a1 is expressed in oocytes, and is inherited as a maternal transcript in early embryos. Throughout zebrafish epiboly and segmentation stages, cyp11a1 is expressed in the yolk syncytial layer. At 36 h post fertilization, cyp11a1 transcript is located ventral to the third somite, where the primordial interrenal gland is located. In summary, zebrafish cyp11a1 is expressed in the cytoplasm of oocytes, as a maternal transcript, and in yolk syncytial layer during early embryogenesis.

Zebrafish monosex population reveals female dominance in sex determination and earliest events of gonad differentiation

The zebrafish is a popular model for genetic analysis and its sex differentiation has been the focus of attention for breeding purposes. Despite numerous efforts, very little is known about the mechanism of zebrafish sex determination. The lack of discernible sex chromosomes and the difficulty of distinguishing the sex of juvenile fish are two major obstacles that hamper the progress in such studies. To alleviate these problems, we have developed a scheme involving methyltestosterone treatment followed by natural mating to generate fish with predictable sex trait. Female F1 fish that gave rise to all-female offspring were generated. This predictable sex trait enables characterization of gonadal development in juvenile fish by histological examination and gene expression analysis. We found the first sign of zebrafish sex differentiation to be ovarian gonocyte proliferation and differentiation at 10 to 12 days post-fertilization (dpf). Somatic genes were expressed indifferently at 10 to 17 dpf, and then became sexually dimorphic at three weeks. This result indicates clear distinction of male and female gonads derived independently from primordial gonads. We classified the earliest stages of zebrafish sex determination into the initial preparation followed by female germ cell growth, oocyte differentiation, and somatic differentiation. Our genetic selection scheme matches the prediction that female-dominant genetic factors are required to determine zebrafish sex.

Function of Cyp11a1 in animal models.

CYP11A1 encodes the P450scc enzyme that catalyzes the first and rate-limiting step of steroid biosynthesis. It is expressed in the adrenals and gonads under the control of pituitary peptide hormones. Transcription factor SF-1, which binds to the CYP11A1 promoter, plays an important role in the tissue-specific and hormonally regulated expression. Transgenic mouse experiments linking the CYP11A1 promoter to a reporter gene demonstrate the importance of the SF-1-binding site in directing balanced CYP11A1 gene expression. In addition, targeted mutagenesis of Cyp11a1 also reveals the importance of this gene in steroid secretion, gene regulation, and development of genitalia. Zebrafish cyp11a1 is expressed in the gonads and interrenal glands in the adults, similar to mammals. In addition, it is expressed in yolk syncytial layer during early embryogenesis. These two animal models complement each other for the understanding of gene regulatory pathways in human diseases related to steroid imbalance.

Steroidogenesis in zebrafish and mouse models

Steroid hormones regulate physiological homeostasis for salt, sugar, and sex differentiation. All steroids are synthesized from a common precursor, cholesterol, in a step that converts cholesterol to pregnenolone. The enzyme carrying out this first conversion step is CYP11A1. To further investigate the importance of steroid biosynthesis, animal models with defects in the Cyp11a1 gene are used. Mice with targeted disruption of the Cyp11a1 gene produce no steroids with severe adrenal defects. These mice survive during embryogenesis, but die after birth. Zebrafish with a block in cyp11a1 gene function has an earlier defect, presumably because it lacks adequate maternal steroid supply. When cyp11a1 activity was compensated by the injection of antisense morpholino oligos, the embryos have shortened axis and a defect of epibolic cell movement during early embryogenesis. The discovery of steroid function in cell movement is novel, and should provide new insights into our understanding of diverse functions of steroids.

Overexpression of a Zebrafish ARNT2-like Factor Represses CYP1A Transcription in ZLE Cells

Abstract: Aryl hydrocarbon receptor nuclear translocator (ARNT) factors belong to a novel basic-helix-loop-helix–PAS (bHLH-PAS) transcription factor family that controls a variety of physiological and developmental processes. In a previous study, we obtained a partial complementary DNA fragment of an ARNT2-like factor from zebrafish embryo, liver, and other tissues by reverse transcription–polymerase chain reaction. In an effort to characterize the function of this factor, we screened an embryonic complementary DNA library and obtained a complete cDNA of this ARNT2-like factor, zARNT2A. The deduced protein sequence of zARNT2A encompasses the basic-helix-loop-helix and PAS-A/B motifs and shares highest sequence similarity with the amino-terminal half of mouse ARNT2 factor. However, it lacks a carboxy-terminal transactivation motif following the PAS-A/B motifs. Transient expression of zARNT2A in cultured cells resulted in repression of TCDD-dependent CYP1A transcription. Whole-mount in situ hybridization revealed that zARNT2A is expressed in brain and pronephros at prime-5 stages. In adult fish, zARNT2A messenger RNA is transcribed in a wide range of tissues, which indicates that zARNT2A and its corresponding signal transduction mechanisms have important roles in fish development and other physiological aspects.

Zebrafish cyp11a1 and hsd3b genes: structure, expression and steroidogenic development during embryogenesis.

Zebrafish has been used increasingly as a good animal model for a number of studies. To facilitate the use of this zebrafish model, the current report put emphasis on the study of two steroidogenic genes: cyp11a1 and hsd3b. These two genes encode enzymes that catalyze the first two steps of the steroidogenic pathway, and both enzymes are important for the synthesis of all steroids. Zebrafish cyp11a1 and hsd3b genes are expressed in the same cells in the gonads and interrenal gland. The interrenal gland is the counterpart of mammalian adrenal; it is located inside the head kidney and is developed parallel to the development of the pronephros. In addition, cyp11a1 and hsd3b are also expressed in the blastomeres of the early embryos before gastrulation, and in the extra-embryonic yolk syncytial layer during gastrulation. This early expression implies a novel role of steroids at gastrulation.