François Van Herp

Molecular biology of neurohormone precursors in the eyestalk of Crustacea

Abstract Our knowledge concerning the primary structures of crustacean neuropeptides has been broadened considerably during the last few years and has greatly contributed to the successful application of molecular biological techniques to crustacean neuroendocrine research. In this review, we compare and discuss the preprohormones of the Red Pigment Concentrating Hormone (RPCH), the Pigment-Dispersing Hormone (PDH) and the different members of the Crustacean Hyperglycemic Hormone, Molt-Inhibiting and Gonad-Inhibiting Hormone family (CHH/MIH/ GIH peptide family), recently elucidated by cloning and sequencing of the respective cDNAs. Expression studies, using in situ hybridization, Northern blots and RNase protection assays, have demonstrated that the mRNAs encoding some of the aforementioned preprohormones (for example, preproPDH and preproCHH) are not only expressed in the eyestalk but also in other parts of the central nervous system. The combination of molecular biological techniques with (bio)chemical and immunochemical methods provides elegant tools to study neuropeptides at the level of mRNA and peptide in individual animals during different physiological conditions. The fundamental knowledge obtained by such a combined approach will give detailed insight into how neuropeptides are involved in the adaptation of Crustacea to a broad spectrum of natural and aquacultural conditions.

Cloning and expression of two mRNAs encoding structurally different crustacean hyperglycemic hormone precursors in the lobster Homarus americanus

The crustacean hyperglycemic hormone (CHH) of the X-organ sinus gland complex is a multifunctional neurohormone primarily involved in the regulation of blood sugar levels. HPLC analysis of lobster sinus glands revealed two CHH-immunoreactive groups, each consisting of two isoforms with identical amino acid sequences and molecular weights. In order to obtain more information concerning the number and sequences of preproCHHs, and to study their expression, we isolated two full-length cDNAs encoding two different CHH preprohormones. Both preprohormone structures consist of a signal peptide, a CHH-precursor-related peptide and a highly-conserved CHH peptide. Expression studies revealed that the X-organ is not the only source of CHH mRNA because the ventral nerve system also expresses this mRNA. Based on these findings and earlier studies on the effect of eyestalk ablation, implantation of thoracic/abdominal ganglia as well as the multifunctionality of CHH, we postulate that CHH, present in the ventral nerve system is a good candidate for a supplementary role in the control of reproduction and molting.

Cloning and expression of mRNA encoding prepro-gonad-inhibiting hormone (GIH) in the lobster Homarus americanus

The gonad‐inhibiting hormone (GIH) is produced in the eyestalk X‐organ sinus gland complex of male and female lobsters, and plays a prominent role in the regulation of reproduction, e.g. inhibition of vitellogenesis in female animals. To study this neurohormone at the mRNA level, we cloned and sequenced a cDNA which encodes GIH in the lobster Homarus americanus. The structure of preproGIH consists of a signal peptide and the GIH peptide itself. A comparative analysis revealed that lobster GIH, together with crab molt‐inhibiting hormone, belongs to a separate group of the crustacean hyperglycemic hormone (CHH) peptide family which seems to be unique for crustaceans. Expression studies showed that GIH mRNA is expressed in the eyestalk, indicating that the neuroendocrine center in this optic structure is the only source of GIH. As this center modulates the other (neuro)endocrine organs in crustaceans, it is postulated that GIH regulates production and release of hormones involved in reproduction/molting processes.

Involvement of the hyperglycemic neurohormone family in the control of reproduction in decapod crustaceans

Summary In the last few years, (bio)chemical and molecular biological studies have shown that several members of the hyperglycemic hormone family are present in different molecular forms. In vivo and in vitro bioassays revealed that some of these isoforms also play a role in the control of reproduction in decapod crustaceans. This communication gives a review of the cytological aspects of the eyestalk X-organ sinus gland complex, responsible for the synthesis, storage and release of these neuropeptides, and the molecular and functional aspects of those members involved in the control of reproduction. Finally, the role of the hyperglycemic hormone family in the regulation of reproduction in the female lobster is described as an example of the (possible) interactions of the members of the hyperglycemic hormone family with other (neuro)endocrine factors in the reproductive process of crustaceans.

Localization of crustacean hyperglycemic hormone (CHH) and gonad-inhibiting hormone (GIH) in the eyestalk ofHomarus gammarus larvae by immunocytochemistry and in situ hybridization

This study deals with the localization of crustacean hyperglycemic hormone (CHH) and gonad-inhibiting hormone (GIH) in the eyestalk of larvae and postlarvae ofHomarus gammarus, by immunocytochemistry and in situ hybridization. The CHH and GIH neuropeptides are located in the perikarya of neuroendocrine cells belonging to the X-organ of the medulla terminalis, in their tract joining the sinus gland, and in the neurohemal organ itself, at larval stages I, II and III and at the first postlarval stage (stage IV). In all the investigated stages, the mRNA encoding the aforementioned neuropeptides could only be detected in the perikarya of these neuroendocrine cells. In stage I, approximately 19 CHH-immunopositive and 20 GIH-immunopositive cells are present, both with a mean diameter of 7±1 μm. GIH cells are preferably localized at the periphery of the X-organ surrounding the CHH cells that are centrally situated. Colocalization of CHH and GIH immunoreactions can be observed in some cells. The cell system producing CHH and GIH in the larval and postlarval eyestalk is thus functional and is morphologically comparable to the corresponding neuroendocrine center in the adult lobster.

Transgene-driven protein expression specific to the intermediate pituitary melanotrope cells of Xenopus laevis.

In the present study, we examined the amphibian Xenopus laevis as a model for stable transgenesis and in particular targeted transgene protein expression to the melanotrope cells in the intermediate pituitary. For this purpose, we have fused a Xenopus proopiomelanocortin (POMC) gene promoter fragment to the gene encoding the reporter green fluorescent protein (GFP). The transgene was integrated into the Xenopus genome as short concatemers at one to six different integration sites and at a total of one to ∼20 copies. During early development the POMC gene promoter fragment gave rise to GFP expression in the total prosencephalon, whereas during further development expression became more restricted. In free‐swimming stage 40 embryos, GFP was found to be primarily expressed in the melanotrope cells of the intermediate pituitary. Immunohistochemical analysis of cryosections of brains/pituitaries from juvenile transgenic frogs revealed the nearly exclusive expression of GFP in the intermediate pituitary. Metabolic labelling of intermediate and anterior pituitaries showed newly synthesized GFP protein to be indeed primarily expressed in the intermediate pituitary cells. Hence, stable Xenopus transgenesis with the POMC gene promoter is a powerful tool to study the physiological role of proteins in a well‐defined neuroendocrine system and close to the in vivo situation.

Structure and localization of mRNA encoding a pigment dispersing hormone (PDH) in the eyestalk of the crayfish Orconectes limosus

The pigment‐dispersing hormone (PDH) is produced in the eyestalks of Crustacea where it induces light‐adapting movements of pigment in the compound eye and regulates the pigment dispersion in the chromatophores. To study this hormone at the mRNA level, we cloned and sequenced cDNA encoding PDH in the crayfish Orconectes limosus. The structure of the PDH preprohormone consists of a signal peptide, a PDH precursor‐related peptide (PPRP) and the highly conserved PDH peptide at the carboxy‐terminal end. In situ hybridization in combination with immunocytochemistry revealed four cell clusters expressing PDH in the optic ganglia of the eyestalk. Three clusters stained both with the PDH cRNA probe and the PDH antiserum, however, the perikarya in the lamina ganglionaris (LG) only stained with the PDH antiserum, suggesting the presence of a PDH‐like peptide in the LG.

Detection of mRNA encoding Crustacean Hyperglycemic Hormone (CHH) in the eyestalk of the crayfish Orconectes limosus using non-radioactive in situ hybridization

A non-radioactive in situ hybridization procedure for the localization of the mRNA encoding the crustacean hyperglycemic hormone (CHH) in the eyestalk of the crayfish Orconectes limosus has been developed. Based on the partial amino acid sequence of CHH, polymerase chain reactions were performed to generate complementary DNA (cDNA) clones encoding CHH. Non-radioactively labelled probes derived from the cDNA sequence were used to establish suitable conditions in terms of tissue fixation and pretreatment for detection of the CHH-encoding mRNA in combination with an immunocytochemical staining using a polyclonal antibody for CHH. Localization of the mRNA in the CHH perikarya was obtained with a complementary RNA probe in combination with pepsin/HCl treated Bouin-fixed eyestalks. The immunocytochemical staining confirmed that this cRNA probe specifically hybridized with mRNA of cell somata belonging to the CHH-producing cell system in the eyestalk of Orconectes limosus.

Differential onset of expression of mRNAs encoding proopiomelanocortin, prohormone convertases 1 and 2, and granin family members during Xenopus laevis development

The production of peptide hormones through proteolytic cleavage of prohormones, e.g., proopiomelanocortin (POMC), involves a number of regulated secretory proteins, such as prohormone convertase PC1, PC2 and granin family members, that are co-expressed with the prohormone. Although the expression of these proteins has been well-studied in adult animals, data on their expression during development are limited. We used whole-mount in situ hybridization to visualize POMC mRNA expression in the intermediate and anterior pituitary of Xenopus tadpoles. A more sensitive analysis, namely semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) on total RNA isolated from Xenopus developmental stages, revealed that the expression of POMC, PC1 and PC2 mRNA commenced at stages 13 (neural plate stage), 15 (neural fold stage) and 19 (neural tube stage), respectively, with a gradual increase in their expression levels during further development. Surprisingly, and in contrast to what holds for POMC and the convertases, mRNAs for secretogranin II and III (SgII, SgIII) and 7B2 were not only expressed during neural development, but could already be detected in unfertilized mature oocytes, the first cleavage stages and in blastula-stage embryos. These granins are thus maternally present in Xenopus embryos suggesting that they may have a role during oogenesis and/or early embryonic development.

A proteome map of the pituitary melanotrope cell activated by black‐background adaptation of Xenopus laevis

Upon transfer of Xenopus laevis from a white to a black background, the melanotrope cells in the pituitary pars intermedia secrete α‐melanocyte‐stimulating hormone, which stimulates dispersion of melanin pigment in skin melanophores. This adaptive behavior is under the control of neurotransmitters and neuropeptides of hypothalamic origin. The α‐melanocyte‐stimulating hormone‐producing cells and their hypothalamic control system provide an interesting model to study proteins required for biosynthetic and secretory processes involved in peptide hormone production and for brain–pituitary signaling. We present a 2‐D PAGE‐based proteome map of melanotrope cells from black‐adapted animals, identifying 204 different proteins by MS analysis.