Factors affecting proliferation and differentiation of Lepidopteran midgut stem cells.

Midgut stem cells of last instar larvae and pupae of Heliothis virescens, Lymantria dispar and several other Lepidopteran species have been cultured in vitro and have been induced to proliferate using low titers of ecdysteroids and the 77-Kda peptide fragment, alpha-arylphorin, isolated and identified from pupal fat body tissue. The insulin-related hormone, Bombyxin, also induced mitosis in cultured midgut stem cells; it appeared to be fast-acting and quickly inactivated, while alpha-arylphorin was slower to act and had a longer lasting effect in vitro, indicating different functions for these proliferation agents. Changes in Calcium ion concentration within or outside the cells discretely affected stem cell differentiation, indicating a role for second messenger participation in peptide regulation of this process.

Role of integrin beta1-like protein in proliferation and differentiation of cultured stem cells from midgut of Heliothis virescens.

Cultured midgut cells from Heliothis virescens larvae were incubated with anti-human integrin beta1 made in rabbit and then passed over a column of magnetic beads bound to anti-rabbit IgG (MACS, Miltenyi Bergisch Gladbach, Germany). Cells bound to integrin beta1 antibody also bound to the anti-rabbit IgG on the magnetic beads (MACS) and were retained in the column while it remained in the magnetic field. Non-bound cells were eluted at this time.

Altering the fate of stem cells from midgut of Heliothis virescens:the effect of calcium ions.

Cultured stem cells from larval midgut tissue of the lepidopteran Heliothis virescens respond to alterations in external calcium ion concentration (Ca(2+) (out)) by changing the rate of stem cell proliferation and by differentiating to larval or non-larval phenotypes. Decreasing the external concentration of Ca(2+) with the Ca(2+) chelating agent EGTA increased proliferation of stem cells in culture, and doubled the proportion of cells differentiating to columnar and goblet cells typical of larval midgut compared to controls. In contrast, increasing inward transport of Ca(2+) into the cells by increasing the concentration of external calcium ion concentration, or by incubation with the Ca(2+) ionophore A23187 (which tends to open inward plasma membrane Ca(2+) channels), induced dose-dependent differentiation to non-midgut cell types such as squamous and scale-like cells.

Bombyxin stimulates proliferation of cultured stem cells derived from heliothis virescens and mamestra brassicae larvae1.

Bombyxin stimulated proliferation of cultured midgut stem cells that were derived from two noctuiid moth larvae, Heliothis virescens and Mamestra brassicae. Bombyxin exhibited the highest activity at 10(-12) M. The number of cells increased for 3 d after the addition of bombyxin.

Stimulation of midgut stem cell proliferation and differentiation by insect hormones and peptides.

Stem cells derived from midguts of the caterpillar, Spodoptera littoralis, can be induced to multiply and differentiate in vitro. Ecdysone (E) and 20-hydroxyecdysone (20E) had a concentration-dependent effect: E was more active in cell proliferation and 20E in differentiation. Ecdysteroid receptors in midgut stem cell nuclei were stained with the antibody 9B9.

Implications for the functions of the four known midgut differentiation factors: An immunohistologic study of Heliothis virescens midgut.

Antibodies to the peptides that induce differentiation of midgut larval stem cells, the midgut differentiating factors MDF-2, MDF-3, and MDF-4, bind to columnar cells in midgut cultures and in intact midgut of Heliothis virescens, in manners similar to the binding of anti- MDF-1 to those tissues. Antibodies to MDF-2 and MDF-3 also stained droplets in the midgut lumen, suggesting that columnar cells may also release MDF-2- and MDF-3-like cytokines to the lumen. Antibody to MDF-4 exhibited similar staining patterns but also recognized stem and differentiating cells, the presumed targets of peptides that regulate stem cell differentiation.

Stimulation of midgut stem cell proliferation by Manduca sexta alpha-arylphorin.

Extracts of the green-colored perivisceral fat body of newly ecdysed Manduca sexta pupae stimulate mitosis in midgut stem cells of Heliothis virescens cultured in vitro. Using a combination of cation- and anion-exchange chromatography, we have isolated a protein from these fat body extracts that accounts for the observed stem cell proliferation. SDS-PAGE analysis of the protein results in a single band of 77 kDa.

Effects of a fat body extract on larval midgut cells and growth of lepidoptera.

Treatment with fat body extract (FBX) from pupae of the tobacco hornworm, Manduca sexta, caused mortality in larvae of two pest lepidopterans, the gypsy moth, Lymantria dispar, and the cotton leafworm, Spodoptera littoralis. In FBX-treated larvae, the feeding rate was depressed, causing reduced weight gain and then larval death. Their midgut showed formation of multicellular layers of midgut epidermis, indicating stem-cell hyperplasia.

Stem cells from midguts of Lepidopteran larvae: clues to the regulation of stem cell fate.

Previously, we showed that isolated stem cells from midguts of Heliothis virescens can be induced to multiply in response to a multiplication protein (MP) isolated from pupal fat body, or to differentiate to larval types of mature midgut cells in response to either of 4 differentiation factors (MDFs) isolated from larval midgut cell-conditioned medium or pupal hemolymph. In this work, we show that the responses to MDF-2 and MP in H. virescens stem cells decayed at different time intervals, implying that the receptors or response cascades for stem cell differentiation and multiplication may be different.

Peptides that elicit midgut stem cell differentiation isolated from chymotryptic digests of hemolymph from Lymantria dispar pupae.

Isolated stem cells of Heliothis virescens, cultured in vitro, were induced to differentiate by Midgut Differentiation Factors 3 and 4. These were peptides identified from a chymotrypsin digest of hemolymph taken from newly pupated Lymantria dispar. Partial purification was obtained by filtration through size exclusion filters.