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

Abstract

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. The most active preparation was subsequently subjected to a series of 3 Reverse Phase-HPLC procedures. Partial sequences of the peptides were identified via automated Edman degradation as the nanomers EEVVKNAIA-OH (MDF 3) and ITPTSSLAT-OH (MDF 4). These sequences were commercially synthesized. The synthetic compounds proved active in a dose-dependent manner. Stem cells responded to synthetic MDF 3 and MDF 4 as they did to previously identified peptides MDF 1 and 2, which have quite different amino acid sequences. All of the 4 MDFs administered singly induced statistically similar differentiation responses at 2 x 10(-8), 2 x 10(-9), and 2 x 10(-10) M. However, pairs of the 4 MDFs produced even more differentiation, the same response as one alone, no response, or were inhibitory, dependent on the MDF pair and its concentration. The data suggests complicated receptor interactions.

Factors affecting proliferation and differentiation of lepidopteran midgut stem cells

“…Each was very active in inducing 4th instar-like differentiation of H. virescens midgut stem cells. Peak activity was detected at 10 À6 M (EEVVKNAIA) AND 10 À8 M (ITPTSSLAT), respectively, well within the physiological range for bioactivity (Loeb and Jaffe, 2002).…”

“…Specific stem cell fates can be elicited by cellfree media in which a particular developmental stage of the midgut epithelium had grown, indicating that different growth factors were released into the media by the different mature cells in the cultures. We have described and characterized a few growth factors, the MDFs (Loeb et al, 1999(Loeb et al, , 2003Loeb and Jaffe, 2002), and have elicited differentiation to other cell forms by commercially available vertebrate growth factors (Loeb et al, 2003), emphasizing the complexity of regulation of midgut stem cell differentiation and the plasticity of lepidopteran midgut stem cells. Vertebrate stem cells multiply and function under the control of a bevy of locally interacting peptidic growth factors (Watt and Hogan, 2000), and may also occur as developmental fates of lepidopteran stem cells are regulated.…”

“…Alteration of the calcium titer of the culture medium surrounding midgut stem cells of H. virescens had a profound effect on stem cell fate (Loeb, 2005). Decreasing Ca 21 out by use of the Ca 21 chelating agent ethylene glycol-bis (2-amino ethyl ether)-N, N, N 0 N 0 -tetraacetic acid (EGTA) promoted dose-dependent stem cell multiplication up to 1.5-fold and differentiation to larval forms of midgut epithelium in vitro, similar to that induced by the MDFs (Loeb et al, 1999;Loeb and Jaffe, 2002). In contrast, increasing the concentration of Ca 21 out by adding Ca 21 to the culture medium to a concentration equal to or more than 55 mM, or increasing Ca 21 entry into stem cells by use of the calcium ionophore A23187 (Rosenberger and Triggle, 1978), stimulated cultured larval midgut stem cells to produce scale-like, squamous-like, and vacuolated cells in a dose-dependent fashion, similar to effects of mammalian growth factors PDGF, RA, and EGF.…”

“…Over 60% of freshly isolated larval midgut stem cells from H. virescens (approximately 10 mm in diameter) exposed to MDF1 differentiated to larval forms of columnar or goblet cells approximately 30 mm long within 6-8 days (Loeb et al, 1999;Loeb and Jaffe, 2002). In contrast, after 15 days of exposure to platelet-derived growth factor (PDGF), approximately 11% of the cells differentiated to oval cells, approximately 60 mm long, each with a prominent vacuole and no visible microvilli.…”

“…Several types of endocrine cells in the midgut can be recognized by differences in morphology and their immunologic responses to a large number of peptides, including allatostatins, leukokinin (a diuretic hormone), myoinhibitory peptide, tachykinins (Veenstra et al, 2008;Veenstra, 2009), cholecystekinin, substance P, vasoactive intestinal peptide, somatoliberin, gonadoliberin (Andries and Tramu, 2004), FMRFamide, allatotropin, neuropeptide F, myosuppressins, and sulfakinins, among others (Zitnan et al, 2005) as well as to antibody raised against a differentiation factor that we isolated, sequenced, and named MDF-4 (Loeb and Jaffe, 2002).…”