Figure 1.
String Is Both Necessary and Sufficient for the Initiation of Histoblast Proliferation
(A and B) Wild-type (A)and UAS-String–expressing (B) third instar larva (L3) ventral histoblast nests labeled with GFP. Misexpression of String induced premature proliferation of histoblasts in the larva.
(C and D) UAS-String overexpressing FLP-OUT/FRT clones at 22 h (C) and 28 h (D) after heat shock (aHS) in third instar larvae (His2RFP [red] and GFP [green]). String/GFP-positive cells autonomously enter mitosis (pHis3 staining; blue) and generate multicellular clones.
(E and F) Snapshots from a time-lapse movie of a UAS-Wee1–expressing ventral histoblast nests at 0 h (E) and 4 h APF (F). The misexpression of Wee1 delays the onset of histoblast proliferation, and cell numbers do not change.
Figure 2.
Ecdysone Signaling Is Necessary for string Expression in Histoblasts
(A) String (String-LacZ; red) is not expressed in histoblasts during larval stages. L3, third instar larva.
(B) During the early cell cycles (1–2 h APF), String-LacZ becomes strongly up-regulated in proliferating histoblasts.
(C and D) Esg-Gal4, UAS-GFP; UAS-EcR-RNAi/String-LacZ pupae at 4 h APF (anterior dorsal nest). The expression of EcR-RNAi in histoblasts inhibits the expression of String-LacZ and the onset of histoblast proliferation.
(E and F) In situ hybridization of a string probe on a wild-type (E) or Esg-Gal4, UAS-GFP; UAS-EcR-RNAi/+ (F) ventral histoblast nest at 4 h APF.
Figure 3.
Cyclin E Accumulates in Histoblasts in the Larva and Facilitates the Early Cell Cycles
(A) Histoblasts from larval and early pupal stages were stained with DAPI and Cyclin E and specifically labeled using GFP. Cyclin E levels are high in histoblasts in the third instar larva (L3) and before the onset of proliferation at 0 h APF. During the first two cycles (2 h and 4 h APF), the stored Cyclin E decreases to barely detectable levels.
(B) cycE mutant clones were induced in the embryo and positively marked with GFP using MARCM. Staining in the third instar larva shows that mutant cells (green) fail to accumulate Cyclin E. Such cells arrest after only one division and form two cell clones (C).
(D) cycE mutant clones were generated after the first cell division in pupal stages and were positively marked with GFP using MARCM. At late stages (30 h APF), clones are composed of up to six cells, suggesting that each mutant cycE cell has divided at least two or three times. In contrast, mutant cells in other tissues like the brain (E) and the wing (unpublished data) are arrested and form mostly one- or two-cell clones.
Figure 4.
EGFR/Ras Signaling Is Necessary for the Second Stage of Histoblasts Proliferation
(A) Representative clones induced in the blastoderm by MARCM and analyzed in the interval 24–28 h APF in anterior dorsal nests. Wild-type (WT) clones have more cells than homozygous ras or egfr mutant clones. A mitotic figure labeled with phospho-histone 3 (pHis3; red) could be observed outside of the clone in the WT panel.
(B) Quantification of cell numbers per clone for the three genotypes (WT = 37 [n = 17], ras = 20 [n = 19], egfr = 21 [n = 9]). Error bars represent the standard deviation based on the number of cells per clone.
(C) Twin clonal analysis (ventral nest) of egfr clones induced in the larva shows that at 10 h APF, mutant cells (marked by absence of GFP) have the same proliferation rate as twin wild-type cells (bright green). At 2 2 h (ventral nest) and 30 h APF (dorsal nest), the wild-type twins outnumber egfr mutant cells.
(D) Snapshots from a time-lapse analysis of a dorsal nest expressing a dominant-negative EGFR (UAS-EGFRDN) using the permanent Esg-Gal4 driver (Movie S3). The expression of EGFRDN does not affect histoblast proliferation in early cell cycles, and histoblasts triplicate by 8 h APF. Anterior and posterior dorsal nests expressing EGFRDN have stopped proliferation at 24 h APF (E). The anterior and posterior dorsal nests failed to fuse and contain fewer cells than wild-type nests at this stage of development.
Figure 5.
EGFR/Ras Is Active in Histoblasts and Promotes G2/M Progression
(A and B) spitz (Spitz-LacZ; red) is not expressed at 1 h APF (A) but is strongly up-regulated in both histoblasts and LECs at 24 h APF (B).
(C) DpERK (green) is expressed in histoblasts (arrowhead) and LECs (dorsal nest: 22 h APF).
(D) Expression of EGFRDN using the permanent Esg-Gal4 driver blocks histoblasts at the G2 phase of the cell cycle (FACS analysis: 22 h APF)). In wild-type conditions (left), 12.5% of histoblasts are found in G1 (red), 30% in S (yellow), and 58% in G2 (blue). After EGFRDN overexpression (right), histoblasts become arrested in G2 (89%) with only 2.5% found in G1 and 8.5% in S.
(E) spitz histoblast clones (black; absence of GFP marker) stained with phospho-histone 3 (pHis3; red) to visualize cells in mitosis. Mutant histoblasts do not enter in M except at positions adjacent to wild-type Spitz-expressing cells.
(F) A wild-type dorsal nest stained with PH3. Cells in mitosis are randomly present across the nest.
(G) The overexpression of UAS-Argos in LECs blocks mitosis in adjacent histoblasts at the nest periphery, which are closest to the Argos-expressing LECs. The nest is smaller compared to wild-type controls (F).
Figure 6.
PI3K Signaling Is Required for Histoblasts Growth and Proliferation
(A) A dp110 (PI3K) cell (GFP [green]; arrowhead) induced at blastoderm using MARCM and examined at 0 h APF. The size of the mutant cell (outlined in yellow, right) is approximately 40% of its neighbor wild-type cell (outlined in white), indicating a requirement for PI3K for cell growth during larval stages. Cell membranes were stained with an anti-Dlg antibody (red).
(B) Snapshots from a time-lapse movie (Movie S5) showing a single-cell dp110 clone (GFP; yellow arrowheads) induced at blastoderm using MARCM. All cells are labeled with His2-YFP. The mutant cell enters mitosis at the same time as its wild-type neighbor (white arrowheads) and proceeds to a second division at 200 min in synchrony with the entry in cycle of the two daughter cells from the wild-type neighbor (note the condensed mitotic chromatin; His2-YFP). Overall, the dp110 cell divides three times in 430 min and generates a clone of eight cells, as do its wild-type neighbors, indicating a normal division rate.
(C) Quantification of cell numbers of wild-type (n = 17) and dp110 clones (n = 18) induced in the embryo and examined 26 h APF. On average, dp110 clones are composed of 14 cells, a strong reduction compared to wild-type (w.t.) clones.
(D) A two-cell FLP-OUT/FRT UAS-PTEN clone generated in an early third instar larva marked with GFP. The clone was followed by time-lapse photography (Movie S7) during the early stage of proliferation (anterior dorsal nest: 0 h APF). PTEN-expressing cells undergo the first three rounds of division and generate a clone of 16 cells in 335 min, as do wild-type histoblasts (His2-YFP).
(E) FLP-OUT/FRT UAS-PTEN clones analyzed at 25 h APF (ventral nest) in fixed preparation. These clones are composed of few and scattered cells.
(F) dp110 clones stained with anti-Dlg (red) and DAPI (blue) at 28 h APF. The cells of the clone show reduced size compared to wild-type cells.
Figure 7.
A Model of Histoblasts Cell-Cycle Progression
(A) Four histoblast nests pairs are specified in each hemisegment of the embryo: Anterior dorsal nest composed of around 18 cells and posterior dorsal nest composed of five cells (red), spiracular nest (blue) formed by three cells, and a ventral nest composed of 14 cells (green).
(B) During metamorphosis, histoblast nests develop to form the different structures that compose the abdominal adult epidermis, tergites and intersegmental membranes (red), spiracle (blue), and pleurites and sternites (green). The second abdominal segment (A2) is highlighted.
(C) During larval stages, histoblasts arrested in G2 and grow in response to PI3K activity (Stage 0).
(D) At the onset of metamorphosis, a hormonal input mediated by ecdysone is required for the expression of String, which promotes G2 arrest relief and reentry in the cell cycle. As a consequence, histoblasts undergo several fast synchronous G1-less cell cycles utilizing a stored pool of G1 regulators, including Cyclin E. During these divisions, histoblasts cleave into smaller cells, not undergoing interphase growth (Stage 1).
(E) Subsequent to these cell cycles, division synchrony is lost, the cell cycle slows down with the restoration of a G1 phase, and histoblasts keep their size constant by growing between cycles (Stage 2). These late divisions are coupled to epithelial expansion and replacement of LECs. EGFR signaling triggered by the ligand Spitz is essential for progression of the cell cycle, and in the absence of EGFR signaling, histoblasts arrest in G2. Growth at this stage is mediated by insulin/PI3K signaling.