Control of Germline Stem Cell Division Frequency – A Novel, Developmentally Regulated Role for Epidermal Growth Factor Signaling

Exploring adult stem cell dynamics in normal and disease states is crucial to both better understanding their in vivo role and better realizing their therapeutic potential. Here we address the division frequency of Germline Stem Cells (GSCs) in testes of Drosophila melanogaster. We show that GSC division frequency is under genetic control of the highly conserved Epidermal Growth Factor (EGF) signaling pathway. When EGF signaling was attenuated, we detected a two-fold increase in the percentage of GSCs in mitotic division compared to GSCs in control animals. Ex vivo and in vivo experiments using a marker for cells in S-phase of the cell cycle showed that the GSCs in EGF mutant testes divide faster than GSCs in control testes. The increased mitotic activity of GSCs in EGF mutants was rescued by restoring EGF signaling in the GSCs, and reproduced in testes from animals with soma-depleted EGF-Receptor (EGFR). Interestingly, EGF attenuation specifically increased the GSC division frequency in adult testes, but not in larval testes. Furthermore, GSCs in testes with tumors resulting from the perturbation of other conserved signaling pathways divided at normal frequencies. We conclude that EGF signaling from the GSCs to the CySCs normally regulates GSC division frequency. The EGF signaling pathway is bifurcated and acts differently in adult compared to larval testes. In addition, regulation of GSC division frequency is a specific role for EGF signaling as it is not affected in all tumor models. These data advance our understanding concerning stem cell dynamics in normal tissues and in a tumor model

RNA-seq analysis of the gonadal transcriptome during Alligator mississippiensis temperature-dependent sex determination and differentiation

Annotation of development-dependent dimorphic genes in gonad during Day 0–12. Annotation of development-dependent significantly up- and down- regulated DEGs at FDR < 0.01 in gonadal regions incubated under MPT and FPT conditions during Day 0 to Day 12. Ordered by decreasing fold change. (XLSX 196 kb

A Comprehensive Clinical Genetics Approach to Critical Congenital Heart Disease in Infancy

Objective: To investigate the frequency of genetic diagnoses among infants with critical congenital heart disease (CHD) using a comprehensive cardiovascular genetics approach and to identify genotype-phenotype correlations. Study design: A retrospective chart review of patients evaluated by cardiovascular genetics in a pediatric cardiac intensive care unit from 2010 to 2015 was performed. Infants with CHD who were <1 month of age were included. CHD was classified using structured phenotype definitions. Cardiac and noncardiac phenotypes were tested for associations with abnormal genetic testing using χ1 and Fisher exact tests. Results: Genetic evaluation was completed in 293 infants with CHD, of whom 213 had isolated congenital heart disease (iCHD) and 80 had multiple congenital anomalies. Overall, the yield of abnormal genetic testing was 26%. The multiple congenital anomalies cohort had a greater yield of genetic testing (39%) than the iCHD cohort (20%) (OR 2.7). Using a non-hierarchical CHD classification and excluding 22q11.2 deletion and common aneuploidies, right ventricular obstructive defects were associated with abnormal genetic testing (P = .0005). Extracardiac features associated with abnormal genetic testing included ear, nose, and throat (P = .003) and brain (P = .0001) abnormalities. A diagnosis of small for gestational age or intrauterine growth retardation also was associated with abnormal genetic testing (P = .0061), as was presence of dysmorphic features (P = .0033, OR 3.5). Infants without dysmorphia with iCHD or multiple congenital anomalies had similar frequencies of abnormal genetic testing. Conclusions: The present study provides evidence to support a comprehensive cardiovascular genetics approach in evaluating infants with critical CHD while also identifying important genotype-phenotype considerations

Nucleoporin98-96 Function Is Required for Transit Amplification Divisions in the Germ Line of Drosophila melanogaster

Production of specialized cells from precursors depends on a tightly regulated sequence of proliferation and differentiation steps. In the gonad of Drosophila melanogaster, the daughters of germ line stem cells (GSC) go through precisely four rounds of transit amplification divisions to produce clusters of 16 interconnected germ line cells before entering a stereotypic differentiation cascade. Here we show that animals harbouring a transposon insertion in the center of the complex nucleoporin98-96 (nup98-96) locus had severe defects in the early steps of this developmental program, ultimately leading to germ cell loss and sterility. A phenotypic analysis indicated that flies carrying the transposon insertion, designated nup98-962288, had dramatically reduced numbers of germ line cells. In contrast to controls, mutant testes contained many solitary germ line cells that had committed to differentiation as well as abnormally small clusters of two, four or eight differentiating germ line cells. This indicates that mutant GSCs rather differentiated than self-renewed, and that these GSCs and their daughters initiated the differentiation cascade after zero, or less than four rounds of amplification divisions. This phenotype remained unaffected by hyper-activation of signalling pathways that normally result in excessive proliferation of GSCs and their daughters. Expression of wildtype nup98-96 specifically in the germ line cells of mutant animals fully restored development of the GSC lineage, demonstrating that the effect of the mutation is cell-autonomous. Nucleoporins are the structural components of the nucleopore and have also been implicated in transcriptional regulation of specific target genes. The nuclear envelopes of germ cells and general nucleocytoplasmic transport in nup98-96 mutant animals appeared normal, leading us to propose that Drosophila nup98-96 mediates the transport or transcription of targets required for the developmental timing between amplification and differentiation

A temporal signature of epidermal growth factor signaling regulates the differentiation of germline cells in testes of Drosophila melanogaster.

Tissue replenishment from stem cells follows a precise cascade of events, during which stem cell daughters first proliferate by mitotic transit amplifying divisions and then enter terminal differentiation. Here we address how stem cell daughters are guided through the early steps of development. In Drosophila testes, somatic cyst cells enclose the proliferating and differentiating germline cells and the units of germline and surrounding cyst cells are commonly referred to as cysts. By characterizing flies with reduced or increased Epidermal Growth Factor (EGF) signaling we show that EGF triggers different responses in the cysts dependent on its dose. In addition to the previously reported requirement for EGF signaling in cyst formation, a low dose of EGF signaling is required for the progression of the germline cells through transit amplifying divisions, and a high dose of EGF signaling promotes terminal differentiation. Terminal differentiation was promoted in testes expressing a constitutively active EGF Receptor (EGFR) and in testes expressing both a secreted EGF and the EGFR in the cyst cells, but not in testes expressing either only EGF or only EGFR. We propose that as the cysts develop, a temporal signature of EGF signaling is created by the coordinated increase of both the production of active ligands by the germline cells and the amount of available receptor molecules on the cyst cells

The EGF repression of GSC divisions is developmentally regulated.

<p>(A, B) Testes from (A) <i>w¹¹¹⁸</i> or (B) <i>w^-; spi^77-20/spi^77-20</i> 3^rd instar larvae stained with anti-Vasa (green) and DAPI (blue). Arrows: spermatocytes, arrowheads: early stage germline cells, scale bars: 50 µm. (C) M-phase index for the GSCs of each genotype. No significant difference was detected, all p<b>-</b>values>0.30. (D) A model depicting the requirement for EGF signaling. EGF is required in both larvae and adults for promoting germline differentiation. In contrast, EGF is not required in larvae for the repression of GSC division frequency, demonstrating a developmental uncoupling of EGF-function.</p

GSCs in <i>spi^77-20</i> testes cycle faster than GSCs in control testes.

<p>(A) Cartoon depicting the organization of germ line cells and somatic cells at the tip of wildtype testes. GSCs (light green) are organized around the hub (red). CySCs (light pink) encase GSCs and are also in contact with the hub. The gonialblast (dark green) is displaced away from the hub and encased by two cyst cells (dark pink). (B) The apical tip of a <i>w¹¹¹⁸</i> testis stained with antibodies labeling the cytoplasm of the germline cells (anti-Vasa, green), the membrane of the hub cells (anti-Fasciclin III, red), and mitotic chromatin (anti-pHH3, red). Arrowheads: GSCs, arrow: GSC in mitosis, scale bar: 10 µm. The inset shows the pHH3-positive GSC next to the hub (circle). (C–H) Genotypes as indicated. >500 stem cells were scored for each genotype. (C–E) The percentage of pHH3-positive GSCs (M-phase index). (C) ***p-value<0.0001. (D) Conditions as indicated. p-value = 0.18 (E) Conditions as indicated. ***p-value<0.0001; No significant difference was noted between 18°C and 26.5°C, p-value = 0.22. (F) The percentage of pHH3-positive CySCs. No significant difference was noted. p = 0.28. (G,H) GSC S-phase indices. (G) Ex vivo labeling of testes with BrdU, ***p-value = 0.0004. (H) Flies fed a continuous diet of BrdU for 36 hours or 48 hours, ***p-value<0.0001, **p-value = 0.0074.</p