scholarly journals A conserved role of the duplicated Masculinizer gene in sex determination of the Mediterranean flour moth, Ephestia kuehniella

PLoS Genetics ◽  
2021 ◽  
Vol 17 (8) ◽  
pp. e1009420
Author(s):  
Sander Visser ◽  
Anna Voleníková ◽  
Petr Nguyen ◽  
Eveline C. Verhulst ◽  
František Marec
Keyword(s):  
Sex Determination ◽  
Zinc Finger ◽  
Molecular Mechanisms ◽  
Ephestia Kuehniella ◽  
Close Relative ◽  
Z Chromosome ◽  
Zinc Finger Domain ◽  
The Mediterranean ◽  
Mediterranean Flour Moth

Sex determination in the silkworm, Bombyx mori, is based on Feminizer (Fem), a W-linked Fem piRNA that triggers female development in WZ individuals, and the Z-linked Masculinizer (Masc), which initiates male development and dosage compensation in ZZ individuals. While Fem piRNA is missing in a close relative of B. mori, Masc determines sex in several representatives of distant lepidopteran lineages. We studied the molecular mechanisms of sex determination in the Mediterranean flour moth, Ephestia kuehniella (Pyralidae). We identified an E. kuehniella Masc ortholog, EkMasc, and its paralog resulting from a recent duplication, EkMascB. Both genes are located on the Z chromosome and encode a similar Masc protein that contains two conserved domains but has lost the conserved double zinc finger domain. We developed PCR-based genetic sexing and demonstrated a peak in the expression of EkMasc and EkMascB genes only in early male embryos. Simultaneous knock-down experiments of both EkMasc and EkMascB using RNAi during early embryogenesis led to a shift from male- to female-specific splicing of the E. kuehniella doublesex gene (Ekdsx), their downstream effector, in ZZ embryos and resulted in a strong female-biased sex-ratio. Our results thus confirmed the conserved role of EkMasc and/or EkMascB in masculinization. We suggest that the C-terminal proline-rich domain, we have identified in all functionally confirmed Masc proteins, in conjunction with the masculinizing domain, is important for transcriptional regulation of sex determination in Lepidoptera. The function of the Masc double zinc finger domain is still unknown, but appears to have been lost in E. kuehniella.

scholarly journals A conserved role of the duplicated Masculinizer gene in sex determination of the Mediterranean flour moth, Ephestia kuehniella

2021 ◽  
Author(s):  
Sander Visser ◽  
Anna Voleníková ◽  
Petr Nguyen ◽  
Eveline C. Verhulst ◽  
František Marec
Keyword(s):  
Sex Determination ◽  
Early Embryogenesis ◽  
Ephestia Kuehniella ◽  
Z Chromosome ◽  
Female Development ◽  
Biased Sex Ratio ◽  
The Mediterranean ◽  
Female Specific ◽  
Mediterranean Flour Moth

AbstractSex determination in the silkworm, Bombyx mori, is based on Feminizer (Fem), a W-linked Fem piRNA that triggers female development in WZ individuals, and the Z-linked Masculinizer (Masc), which initiates male development and dosage compensation in ZZ individuals. While Fem piRNA is missing in a close relative of B. mori, Masc determines sex in several representatives of distant lepidopteran lineages. We studied the molecular mechanisms of sex determination in the Mediterranean flour moth, Ephestia kuehniella (Pyralidae). We identified an E. kuehniella Masc ortholog, EkMasc, and its paralog resulting from a recent duplication, EkMascB. Both genes are located on the Z chromosome and encode a similar Masc protein that contains two conserved domains but has lost the conserved double zinc finger domain. We developed PCR-based genetic sexing and demonstrated a peak in the expression of EkMasc and EkMascB genes only in early male embryos. Simultaneous knock-down experiments of both EkMasc and EkMascB using RNAi during early embryogenesis led to a shift from male- to female-specific splicing of the E. kuehniella doublesex gene (Ekdsx), their downstream effector, in ZZ embryos and resulted in a strong female-biased sex-ratio. Our results thus confirmed the conserved role of both EkMasc and EkMascB genes in masculinization. We suggest that the C-terminal proline-rich domain, we have identified in all functionally confirmed Masc proteins, in conjunction with the masculinizing domain, is important for transcriptional regulation of sex determination in Lepidoptera. The function of the Masc double zinc finger domain is still unknown, but appears to have been lost in E. kuehniella.Author summaryThe sex-determining cascade in the silkworm, Bombyx mori, differs greatly from those of other insects. In B. mori, female development is initiated by Fem piRNA expressed from the W chromosome during early embryogenesis. Fem piRNA silences Masculinizer (Masc) thereby blocking the male pathway resulting in female development. It is currently unknown whether this cascade is conserved across Lepidoptera. In the Mediterranean flour moth, Ephestia kuehniella, we identified an ortholog of Masc and discovered its functional duplication on the Z chromosome, which has not yet been found in any other lepidopteran species. We provide two lines of evidence that both the EkMasc and EkMascB genes play an essential role in masculinization: (i) they show a peak of expression during early embryogenesis in ZZ but not in WZ embryos and (ii) their silencing by RNAi results in female-specific splicing of the E. kuehniella doublesex gene (Ekdsx) in ZZ embryos and in a female-biased sex ratio. Our results suggest a conserved role of the duplicated Masc gene in sex determination of E. kuehniella.

Essential Role of Kindlin 3 in Zebrafish Thrombopoiesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1166-1166
Author(s):  
James J. Mann ◽  
Nathaniel B. Langer ◽  
Andrew Woo ◽  
Tyler B. Moran ◽  
Yocheved Schindler ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Zinc Finger ◽  
Essential Role ◽  
Molecular Mechanisms ◽  
Fetal Liver ◽  
Affinity Purification ◽  
Transgenic Zebrafish ◽  
Zinc Finger Transcription Factor ◽  
Stage Of Development

Abstract The zinc finger transcription factor GATA-1 is required for proliferative inhibition and terminal maturation of megakaryocytes, and is mutated in Down Syndrome Transient Myeloproliferative Disorder (TMD) and Acute Megakaryoblastic Leukemia (DS-AMKL). Yet the molecular mechanisms that regulate GATA-1 activity in megakaryopoiesis remain incompletely understood. Many transcription factors, in addition to binding DNA, make important protein-protein interactions that modulate their activity. In order to further understand GATA-1’s function, and possibly identify new factors involved in megakaryopoiesis, we purified GATA-1 containing multiprotein complexes from the murine L8057 megakaryocytic cell line. We generated stable L8057 cell lines expressing metabolically biotinylated and FLAG epitope tagged GATA-1, and then performed a tandem anti-FLAG immunoaffinity and streptavidin affinity purification. Using mass spectrometry (LC/MS/MS), we identified the known GATA-1 associated proteins Friend of GATA-1 (FOG-1), SCL, Ldb1, Runx-1/Cbf-β. SP1 and all components of the NuRD complex (which binds FOG-1) as co-purifying proteins. In addition, we reproducibly obtained several novel proteins. We previously reported the identification of the kruppel-type zinc finger transcription factor zfp148 (also called ZBP-89), and showed that it plays an essential role in megakaryopoiesis and definitive erythropoiesis. Here we report the identification of Kindlin 3 (also called URP2 for UNC-112 related protein 2), a member of a family of PH and FERM domain containing proteins that are thought to play a role in integrin-mediated processes. Expression of Kindlin 3 is restricted to hematopoietic cells, principally megakaryocytes and lymphocytes. It is first expressed at ~E9.5 during murine embryogenesis, and is abundant in fetal liver megakaryocytes by day E14.5. In order to begin to assess the role of Kindlin 3 in megakaryopoiesis in vivo, we performed morpholino-mediated knockdown of Kindlin 3 expression in CD41-GFP transgenic zebrafish embryos. In contrast to control embryos, embryos injected with Kindlin 3 specific morpholinos exhibited nearly complete loss of GFP+ thrombocytes (equivalent to mammalian megakaryocyte/platelets). Erythroid development (equivalent to mammalian primitive erythropoiesis at this stage of development) was not significantly affected, similar to embryos injected with zfp148-specific morpholinos. Given the role of integrin outside-to-inside signaling in megakaryopoiesis, we propose that Kindlin 3 may play a role linking extracellular signals to megakaryocyte maturation and growth control via GATA-1 transcription complexes. Further analysis in murine systems is underway to test this hypothesis.

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