Specification and spatial arrangement of cells in the germline stem cell niche of the <i>Drosophila</i> ovary depend on the Maf transcription factor Traffic jam

<div><p>Germline stem cells in the <i>Drosophila</i> ovary are maintained by a somatic niche. The niche is structurally and functionally complex and contains four cell types, the escort, cap, and terminal filament cells and the newly identified transition cell. We find that the large Maf transcription factor Traffic jam (Tj) is essential for determining niche cell fates and architecture, enabling each niche in the ovary to support a normal complement of 2–3 germline stem cells. In particular, we focused on the question of how cap cells form. Cap cells express Tj and are considered the key component of a mature germline stem cell niche. We conclude that Tj controls the specification of cap cells, as the complete loss of Tj function caused the development of additional terminal filament cells at the expense of cap cells, and terminal filament cells developed cap cell characteristics when induced to express Tj. Further, we propose that Tj controls the morphogenetic behavior of cap cells as they adopted the shape and spatial organization of terminal filament cells but otherwise appeared to retain their fate when Tj expression was only partially reduced. Our data indicate that Tj contributes to the establishment of germline stem cells by promoting the cap cell fate, and controls the stem cell-carrying capacity of the niche by regulating niche architecture. Analysis of the interactions between Tj and the Notch (N) pathway indicates that Tj and N have distinct functions in the cap cell specification program. We propose that formation of cap cells depends on the combined activities of Tj and the N pathway, with Tj promoting the cap cell fate by blocking the terminal filament cell fate, and N supporting cap cells by preventing the escort cell fate and/or controlling the number of cap cell precursors.</p></div

Partial reduction of Tj affects the number but not maintenance of GSCs.

<p>Images of the GSC niche in <i>tj</i>^<i>39</i><i>/tj</i>^<i>eo2</i> mutant (<i>tj</i>^hypo) and control (wt) germaria. Vasa labels germline cells, Tj labels cap cells, and LamC labels TF and cap cells. Hts highlights the spectrosome (Sp) of GSCs and pre-cystoblasts/cystoblasts (CB), and the fusome (Fu) of germline cysts, and outlines all cells. A yellow arrowhead marks the TF/germarium boundary. Red and blue arrows mark the posterior-and anterior-most cap cell, respectively (A-C'), and asterisks mark GSCs (F-K). (<b>A-C'</b>) In contrast to a wild-type niche (A) a <i>tj</i>^hypo niche often contains only a single GSC (B), but sometimes has two GSCs (C,C' are two sections of the same germarium). A dividing GSC that produces a cystoblast is seen in panels A and C'. (<b>D</b>) The graph compares the number of GSCs in control (<i>bam-GFP</i>) and <i>tj</i>^hypo germaria (<i>tj</i>^<i>39</i><i>/tj</i>^<i>eo2</i>; <i>bam-GFP)</i> from females aged for the indicated time interval. Mean + s.d. are indicated. <i>n</i>, sample size. The difference in GSC numbers between the two genotypes is significant for each time point (P<0.0001; Student T-test, unpaired, two-tailed, equal variance). Within each genotype, differences in GSC numbers between time points were not significant (P>0.05; ANOVA one-way). For each genotype, a colored number and horizontal bar indicate the mean and differences in the mean between time points. (<b>E</b>) The graph shows the relationship between the number of GSCs and the number of cap cells that were located in the germarium of <i>tj</i>^hypo ovarioles. Among the analyzed ovarioles (n = 72), germaria with 3 GSCs were rare. (<b>F,G</b>) Protrusions of GSCs (open arrowheads) partially wrap the cap cells (white) in a wild-type germarium (F). The single GSC of a <i>tj</i>^hypo germarium has an unusually long protrusion that contacts ectopically positioned cap cells outside the germarium (G). In both genotypes, GSCs have anteriorly located spectrosomes. (<b>H-K</b>) Similar to GSCs in the control (H,J), the anterior-most germline cell in the <i>tj</i>^hypo germarium shows nuclear pMad (I), and lacks <i>bam</i>-GFP (K). <i>bam</i>-GFP is prominent in dividing cystocytes (CC) in both genotypes (J,K). Anterior is up in all images. Scale bars: 10 μm in A-C, F-K.</p

Tj controls the morphogenetic behavior and specification of cap cells.

<p>Yellow arrowheads mark the germarium/TF boundary. (<b>A-G</b>) GSC niches of adult ovaries. LamC identifies TF and cap cells. In the control (wt) (A,C,E), cap cells form a cluster inside the germarium and TF cells form a stalk outside the germarium. In ovaries of <i>tj</i>^<i>39</i>/<i>tj</i>^<i>eo2</i> (<i>tj</i>^hypo) (B) and <i>tj</i>^null mutants (<i>tj</i>^<i>eo2</i><i>/tj</i>^<i>eo2</i> in D, <i>tj</i>^<i>eo2</i><i>/tj</i>^<i>Df1</i> in F,G), the cluster is missing and the stalk is abnormally long. (B) In a <i>tj</i>^hypo ovary, the basal cells of the elongated stalk (between arrow and arrowhead) express markers in a cap cell-specific manner (strong Bab2 and <i>1444-lacZ</i> signals), in contrast to distal cells in the stalk. (D,F,G) In <i>tj</i>^null ovaries, all cells in the elongated stalks express markers similar to normal TFs (weak and strong expression of Bab2 (D,F) and <i>LB27-lacZ</i> (F,G), respectively, and no detection of <i>1444-lacZ</i> (D). (<b>H,I</b>) Adult GSC niches with <i>tj</i>^<i>z4735</i> mutant cell clones. <i>tj</i>^<i>z4735</i> mutant cap cells, which only express the non-functional isoform of Tj (red) and lack GFP (green), are located in the TF (above the arrowhead) and display the flat shape of TF cells (asterisks), whereas control cap cells, which co-express Tj and GFP, are located in the germarium (below the arrowhead). (<b>J-M</b>) Prepupal ovaries. TFs are longer in <i>tj</i>^null ovaries (<i>tj</i>^<i>eo2</i><i>/tj</i>^<i>eo2</i> in K, <i>tj</i>^<i>z4735</i><i>/tj</i>^<i>eo2</i> in M) than in control ovaries (J,L). (J,K) <i>B1-lacZ</i> (red) labels cap and TF cells. LamC (green), which is only seen in the distal half of TFs in the control ovary, is present in all <i>B1-lacZ</i>-positive cells of the <i>tj</i>^null ovary. The germ cell population, marked by Vasa (blue), is not divided into ovarioles in the <i>tj</i>^null ovary in contrast to the control. (L,M) <i>1444-lacZ</i> (red), which labels nascent cap cells in the control ovary is not detected in the <i>tj</i>^null ovary. Tj (blue) is seen in cap cells but not in TFs in the control ovary. In the <i>tj</i>^null ovary, which expresses a mutant Tj isoform, Tj is seen in the basal portion of the elongated TFs (between arrowhead and arrow). Genotypic markers: <i>1444-lacZ/+</i> (A,B,L,M) or <i>1444-lacZ</i> (C,D), <i>LB27-lacZ/+</i> (E-G), <i>Ubi-GFP</i> (H,I), and <i>B1-lacZ</i> (J,K). Anterior is up. Scale bars: 10 μm in A-F,H-M; 50 μm in G.</p

Loss of Tj affects the structure of the stem cell niche.

<p>(<b>A,B</b>) Adult ovaries. <i>bab-lacZ</i> highlights TFs and adjacent cap cells. (A) In a control (wt) ovary, a bundle of ovarioles is present, each with a TF at the tip, followed by a cap cell cluster (Cap), germarium (Gm) and series of follicles (Fl). (B) The rudimentary <i>tj</i>^<i>eo2</i>/<i>tj</i>^<i>eo2</i> (<i>tj</i>^null) mutant ovary (Ov) has irregularly distributed and abnormally long TFs, and germarium and follicles are missing. Od, oviduct; Fb, fatbody. (<b>C</b>) Comparison of the number of TF and cap cells in control (+), <i>tj</i>^<i>39</i><i>/tj</i>^<i>eo2</i> (<i>tj</i>^hypo), and <i>tj</i>^<i>eo2</i><i>/tj</i>^<i>eo2</i> (<i>tj</i>^null) ovarioles. (<b>D-F</b>) Niches of 2-day-old pupal ovaries. (D) In the control (wt), Tj-positive (white) cap cells are round and form a cluster at the base of the TF. TC, transition cell. (E,F) In a <i>tj</i>^<i>z4735</i><i>/tj</i>^<i>eo2</i> (<i>tj</i>^null) ovary, cells expressing non-functional Tj protein are disc-shaped and either arranged in a single file, forming a stalk similar to the adjacent TF cells (E, F #1), or form a branched stalk (F #2). (<b>G-I</b>) Tip of an adult ovariole. Hts (red) outlines all cells, Vasa (green) marks germline cells, a yellow arrowhead marks the germarium/TF boundary, and red and blue arrows mark the posterior-and anterior-most cap cell, respectively. (G) In wild type (wt), all Tj-positive cells (white) are located inside the germarium, and the transition cell marks the boundary towards the TF. OS, ovariole sheath. (H,I) In a <i>tj</i>^<i>39</i><i>/tj</i>^<i>eo2</i> (<i>tj</i>^hypo) ovary, a variable number of Tj-positive cells have become part of the TF (see anterior shift of the blue and red arrows). The Tj signal is weaker in the <i>tj</i>^hypo mutant (H,I) than in wild type (G). (<b>J</b>) Location of cap cells in <i>tj</i>^<i>39</i><i>/tj</i>^<i>eo2</i> (<i>tj</i>^hypo) ovarioles. (<b>K,L</b>) Quantification of anterior niche cells, which include cap and TF cells, in <i>tj</i>^<i>eo2</i>/<i>tj</i>^<i>eo2</i> (<i>tj</i>^null) (K) and <i>tj</i>^<i>39</i><i>/tj</i>^<i>eo2</i> (<i>tj</i>^hypo) ovarioles (L). Graphs (C,J-L) are 'box' (25–75 percentile) and 'whisker' (maxima/minima) diagrams, showing the median (bar) and mean (plus sign). <i>n</i>, sample size; n.s., not significant (P≥0.05). Genotypic markers: <i>bab-lacZ</i> (A,B), <i>1444-lacZ/+</i> (D-F), <i>B1-lacZ</i> or <i>B1-lacZ/+</i> (C,K). Anterior is up in all images. Scale bars: 50 μm in A,B; 10 μm in D-I.</p

Cap cells form a cluster in the germarium in the absence of germline cells.

<p>Images show the GSC niche. TF cells display LamC (green), escort cells express Tj (magenta), and the cap cells, which are highlighted by a stippled line, are positive for both markers. Cap cells form a cluster posterior to the stack of TF cells and are part of the germarium in ovarioles of a <i>tud</i>^<i>1</i><i>/tud</i>^<i>b45</i> maternal effect mutant ovary that lacks the germline (B) similar to wild type (wt) (A). Anterior is up. Scale bars: 10 μm.</p

Ectopic expression of Tj in TF cells induces cap cell-like characteristics.

<p>All images show a single TF. Tj-expressing TF cells were induced in the larval ovary before TFs form and are marked by an asterisk. An arrowhead marks the TF/germarium boundary. (<b>A,B</b>) In mosaic TFs, Tj-positive cells strongly express the cap cell marker <i>1444-lacZ</i> in contrast to adjacent control cells. Also, the LamC signal appears weaker in Tj-positive cells than in normal Tj-negative TF cells. (<b>C-E</b>) Expression of the TF cell-specific marker <i>LB27-lacZ</i> is substantially reduced in Tj-positive cells (C,D) when compared to control TF cells (C,E). Note the rounded shape of Tj-expressing TF cells when compared to the flat shape of control TF cells in mosaic stalks (A-C). Genotypic markers: <i>1444-lacZ/+</i> (A,B), <i>LB27-lacZ/+</i> (C-E). Anterior is up in all panels. Scale bars: 10 μm.</p

Ectopic expression of Tj affects the shape of TF cells.

<p>Ectopic expression of Tj affects the shape of TF cells.</p

Loss of Tj function impairs GSC formation and causes loss of germline cells.

<p>Images compare the germline phenotype of <i>tj</i>^<i>eo2</i><i>/tj</i>^<i>eo2</i> mutants (<i>tj</i>^null) with a control (wt) at different stages of gonad development. Vasa (blue) marks germline cells. LamC (A,B,E,F), Engrailed (G,H), or <i>bab-lacZ</i> (I,J) highlight TFs in green. Hts (green in C,D) marks spectrosomes and fusomes and outlines all cells. (<b>A-D'</b>) In a wild-type ovary at the prepupal stage, GSCs (arrowheads) occupy the anterior-most row in the germ cell population as indicated by the presence of pMad (red) (A) and the absence of <i>bam</i>-GFP (red in C and white in C'). Of the anterior-most row of germline cells in a <i>tj</i>^null ovary, only a few are positive for pMad (arrowheads) (B), and some express <i>bam</i>-GFF (D,D'). The anterior-most row of the germ cell population is marked by arrows and/or a stippled line in C-D'. (<b>E-H'</b>) At the mid-pupal stage, wild-type ovarioles are already filled with developing germ cells and contain pMad-positive GSCs next to TFs (E), whereas few germline cells remain in a <i>tj</i>^null ovary and pMad is not detected (F). (G,G') In wild-type ovarioles, <i>bam</i>-GFP is not detected in GSCs (asterisk), barely visible in adjacent cystoblasts (CB) and strongly expressed in cystocytes (CC). (H,H') The few persisting germline clusters in a <i>tj</i>^null ovary that are associated with TFs show different levels of <i>bam</i>-GFP expression (arrowheads). (<b>I-J'</b>) Remaining germ cells in adult <i>tj</i> mutant ovaries are organized in small clusters and are usually associated with TFs (I,J), and contain a spectrosome (arrows in J') that is marked by Hts (red in J and white in J'). Genotypic markers: <i>bam-GFP</i> (C-D',G-H'), <i>bab-lacZ/+</i> (I-J'). Anterior is up in all panels. Scale bars: 20 μm in A-D,G,H,J; 50 μm in E,F,I.</p