Chronic activation of the D2 dopamine autoreceptor inhibits synaptogenesis in mesencephalic dopaminergic neurons in vitro.

Chronic blockade or activation of dopamine receptors is critical for the pharmacological treatment of diseases like schizophrenia, Parkinson's or attention deficit and hyperactivity disorder. However, the long-term impact of such treatments on dopamine neurons is unclear. Chronic blockade of the dopamine D2 receptor in vivo triggers an increase in the axonal arborization of dopamine neurons [European Journal of Neuroscience, 2002, 16, 787-794]. However, the specific involvement of presynaptic (autoreceptors) vs. postsynaptic D2 receptors as well as the molecular mechanisms involved have not been determined. Here, we examined the role of D2 autoreceptors in regulating the ability of mouse dopamine neurons to establish axon terminals. Chronic activation of this receptor with quinpirole, a specific agonist, decreased the number of axon terminals established by isolated dopamine neurons. This effect was accompanied by a decrease in dopamine release and was mediated through inhibition of protein kinase A. The decrease in axon terminal number induced by D2 receptor activation was also occluded when the mammalian Target of Rapamycin pathway of mRNA translation was blocked. Our results suggest that chronic activation of the D2 autoreceptor inhibits synaptogenesis by mesencephalic dopamine neurons through translational regulation of the synthesis of proteins required for synapse formation. This study provides a better understanding of the impact of long-term pharmacological interventions acting through the D2 receptor.

Molecular cloning, tissue distribution, and chromosomal localization of MMEL2, a gene coding for a novel human member of the neutral endopeptidase-24.11 family.

Members of the neutral endopeptidase (NEP, also known as MME for membrane metallo-endopeptidase in the Human Gene Nomenclature database) family play significant roles in pain perception, arterial pressure regulation, phosphate metabolism, and homeostasis. In this paper, we report the cloning of a new human member of the NEP family that we named MMEL2 for membrane metallo-endopeptidase-like 2. The MMEL2 protein has the structural characteristics of type II transmembrane proteins, although the presence of a furin-like cleavage site in the ectodomain suggests that it may be released into the medium following proteolytic cleavage. The MMEL2 protein contains the zinc-binding consensus sequence HEXXH and all the residues known to be essential for the enzymatic activity of other members of the family. The MMEL2 mRNA was detected predominantly in testis, but weak expression also was observed in brain, kidney, and heart. The human MMEL2 gene was mapped to 1p36 by fluorescence in situ hybridization. It will be important to test whether MMEL2 defects are associated with diseases such as hereditary motor sensory neuropathy 2A, Schwartz-Jampel-Aberfeld syndrome, or neuroblastoma, which all map to this locus.

Disease-causing missense mutations in the PHEX gene interfere with membrane targeting of the recombinant protein.

PHEX is homologous to the M13 zinc metallopeptidases, a class of type II membrane glycoproteins. Although more than 140 mutations in the PHEX gene have been identified in patients with X-linked hypophosphatemia (XLH), the most prevalent form of inherited rickets, the molecular consequences of disease-causing PHEX mutations have not yet been investigated. We examined the effect of PHEX missense mutations on cellular trafficking of the recombinant protein. Four mutant PHEX cDNAs were generated by PCR mutagenesis: C85R, G579R and S711R, identified in XLH patients, and E581V, previously engineered in neutral endopeptidase 24.11, where it abolished catalytic activity but not plasma membrane targeting. Wild-type and mutant PHEX cDNAs were transfected in HEK(293) cells and PHEX protein expression was characterized. In contrast to the wild-type and E581V PHEX proteins, the C85R, G579R and S711R mutants were completely sensitive to endoglycosidase H digestion, indicating that they were not fully glycosylated. Sequestration of the disease-causing mutant proteins in the endoplasmic reticulum (ER) and plasma membrane localization of wild-type and E581V PHEX proteins was demonstrated by immunofluorescence and cell surface biotinylation. Of the three mutant PHEX proteins, the S711R was the least stable and the only one that could be rescued from the ER to the plasma membrane in cells grown at 26 degrees C. The chemical chaperone glycerol failed to correct defective targeting of all three mutant proteins. Our data provide a mechanism for loss of PHEX function in XLH patients expressing the C85R, G579R and S711R mutations.

Neutral endopeptidase is expressed on the follicular granulosa cells of rabbit ovaries.

Neutral endopeptidase (NEP) is a zinc metallopeptidase ubiquitously distributed in various tissues in mammals. This peptidase is involved in the post-secretory metabolism of various neuropeptides and peptide hormones in vivo, such as enkephalins, bradykinin, atrial natriuretic peptide, substance P and endothelins. In this paper we show that NEP is expressed in ovaries as a 110-kDa glycosylated integral membrane protein with enzymatic properties similar to those of the kidney protein. Using immunohistochemistry, we localize the peptidase in the granulosa cells of follicles at all stages of maturation, with the exception of atretic follicles. We also observe immunoreactive staining in the epithelia that lines the blood vessels in the medulla and the surface of the ovary. The co-localization of NEP and bioactive peptides known to be physiological substrates of NEP in other tissues suggests an important role for this protein in processes such as follicle maturation, ovulation, and/or regulation of ovarian blood flow, by modulating the physiological function of these peptides.

Characterization of PHEX endopeptidase catalytic activity: identification of parathyroid-hormone-related peptide107-139 as a substrate and osteocalcin, PPi and phosphate as inhibitors.

Mutations in the PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) are responsible for X-linked hypophosphataemia, and studies in the Hyp mouse model of the human disease implicate the gene product in the regulation of renal phosphate (P(i)) reabsorption and bone mineralization. Although the mechanism for PHEX action is unknown, structural homologies with members of the M13 family of endopeptidases suggest a function for PHEX protein in the activation or degradation of peptide factors involved in the control of renal P(i) transport and matrix mineralization. To determine whether PHEX has endopeptidase activity, we generated a recombinant soluble, secreted form of human PHEX (secPHEX) and tested the activity of the purified protein with several peptide substrates, including a variety of bone-related peptides. We found that parathyroid-hormone-related peptide(107-139) is a substrate for secPHEX and that the enzyme cleaves at three positions within the peptide, all located at the N-terminus of aspartate residues. Furthermore, we show that osteocalcin, PP(i) and P(i), all of which are abundant in bone, are inhibitors of secPHEX activity. Inhibition of secPHEX activity by osteocalcin was abolished in the presence of Ca(2+). We suggest that PHEX activity and mineralization may be controlled in vivo by PP(i)/P(i) and Ca(2+) and, in the latter case, the regulation requires the participation of osteocalcin.

Two rat brain staufen isoforms differentially bind RNA.

In neurones, a limited number of mRNAs is found in dendrites, including transcripts encoding the microtubule-associated protein 2 (MAP2). Recently, we identified a cis-acting dendritic targeting element (DTE) in MAP2 mRNAs. Here we used the yeast tri-hybrid system to identify potential trans-acting RNA-binding factors of the DTE. A cDNA clone was isolated that encodes a member of a mammalian protein family that is highly homologous to the Drosophila RNA-binding protein Staufen. Mammalian Staufen appears to be expressed in most tissues and brain areas. Two distinct rat brain Staufen isoforms, rStau+I6 and rStau-I6, are encoded by alternatively spliced mRNAs. Both isoforms contain four double-stranded RNA-binding domains (dsRBD). In the larger rStau+I6 isoform, six additional amino acids are inserted in the second dsRBD. Although both isoforms interacted with the MAP2-DTE and various additional RNA fragments in an in vitro north-western assay, rStau-I6 exhibited a stronger signal of bound radioactively labelled RNAs as compared with rStau+I6. Using an antibody directed against mammalian Staufen, the protein was detected in somata and dendrites of neurones of the adult rat hippocampus and cerebral cortex. Ultrastructural studies revealed that in dendrites, rat Staufen accumulates along microtubules. Thus in neurones, rat Staufen may serve to link RNAs to the dendritic microtubular cytoskeleton and may thereby regulate their subcellular localization.

Gene products from LUQ neurons in the abdominal ganglion are present at the renal pore of Aplysia californica.

The L2-4,6 and L5 cells located in the left upper quadrant of the abdominal ganglion of Aplysia californica express the L5-67 and LUQ-1 genes, respectively, in a nonoverlapping manner. These cells send major neurites to the kidney and at least some of them were shown to innervate the renal pore closer muscle, and thereby control its function. By using in-situ hybridization and immunofluorescence, the presence of L5-67 and LUQ-1 mRNAs and peptides was studied in the kidney, with emphasis on the region of the renal pore. We detected immunoreactive materials in many small varicose nerve fibers running along the central epithelium in the inner parts of the kidney, and in neurites located within a large nerve associated with muscles inside the renal pore. Our observations represent the first direct evidence of the presence of gene products from LUQ cells at the renal pore, suggesting that they may be responsible for mediating LUQ cell signals. Furthermore, mRNAs coding for the L5-67 and LUQ-1 peptides were also found in the nerve structure inside the renal pore. Our report documents a striking example of neuropeptide mRNA targeting nerve terminals that are very distant from their cell bodies.

Developmental expression and tissue distribution of Phex protein: effect of the Hyp mutation and relationship to bone markers.

Mutations in PHEX, a phosphate-regulating gene with homology to endopeptidases on the X chromosome, are responsible for X-linked hypophosphatemia (XLH). The murine Hyp homologue has the phenotypic features of XLH and harbors a large deletion in the 3' region of the Phex gene. We characterized the developmental expression and tissue distribution of Phex protein, using a monoclonal antibody against human PHEX, examined the effect of the Hyp mutation on Phex expression, and compared neprilysin (NEP), osteocalcin, and parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) receptor gene expression in bone of normal and Hyp mice. Phex encodes a 100- to 105-kDa glycoprotein, which is present in bones and teeth of normal mice but not Hyp animals. These results were confirmed by in situ hybridization (ISH) and ribonuclease protection assay. Phex protein expression in femur and calvaria decreases with age, suggesting a correlation between Phex expression and bone formation. Immunohistochemical studies detected Phex protein in osteoblasts, osteocytes, and odontoblasts, but not in osteoblast precursors. In contrast to Phex, the abundance of NEP messenger RNA (mRNA) and protein is not significantly altered in Hyp bone. Similarly, osteocalcin and PTH/PTHrP receptor gene expression are not compromised in bone of Hyp mice. Our results are consistent with the hypothesis that loss of Phex function affects the mineralizing activity of osteoblasts rather than their differentiation.

Genomic organization of the human and mouse stau genes.

Mammalian Staufen is a double-stranded RNA-binding protein potentially involved in mRNA transport and localization. Recently, we reported that the human gene is located on chromosome 20, region q13.1. We now report the genomic organization of both the human and mouse stau genes. Amplification of genomic DNA and sequencing of the resulting PCR products indicated that the human and mouse genes are fragmented into 15 and 12 exons, distributed over at least 65 and 17 kb of genomic DNA, respectively. The three additional exons found in the human gene are subjected to differential splicing, generating four different transcripts. Corresponding exons have not been found in mouse transcripts. Apart from those three exons, the overall organization of the stau gene is similar in the two species, and the positions of the exon-intron junctions are perfectly conserved. Even an alternative choice between two splicing acceptor sites, which causes an insertion of 18 nucleotides in exon 5, is conserved in both humans and mice. An extremely G+C-rich region lacking canonical TATA and CAAT boxes was found upstream of the most 5' RACE sequence, suggesting that a housekeeping-like promoter drives the broad expression of Staufen in mammalian cells. This work represents the first step toward production of knockout mice and the elucidation of putative Staufen-linked hereditary diseases in humans.

A novel murine Staufen isoform modulates the RNA content of Staufen complexes.

Mouse Staufen (mStau) is a double-stranded RNA-binding protein associated with polysomes and the rough endoplasmic reticulum (RER). We describe a novel endogenous isoform of mStau (termed mStau(i)) which has an insertion of six amino acids within dsRBD3, the major double-stranded RNA (dsRNA)-binding domain. With a structural change of the RNA-binding domain, this conserved and widely distributed isoform showed strongly impaired dsRNA-binding ability. In transfected cells, mStau(i) exhibited the same tubulovesicular distribution (RER) as mStau when weakly expressed; however, when overexpressed, mStau(i) was found in large cytoplasmic granules. Markers of the RER colocalized with mStau(i)-containing granules, showing that overexpressed mStau(i) could still be associated with the RER. Cotransfection of mStau(i) with mStau relocalized overexpressed mStau(i) to the reticular RER, suggesting that they can form a complex on the RER and that a balance between these isoforms is important to achieve proper localization. Coimmunoprecipitation demonstrated that the two mStau isoforms are components of the same complex in vivo. Analysis of the immunoprecipitates showed that mStau is a component of an RNA-protein complex and that the association with mStau(i) drastically reduces the RNA content of the complex. We propose that this new isoform, by forming a multiple-isoform complex, regulates the amount of RNA in mStau complexes in mammalian cells.

Expression of autocrine motility factor/phosphohexose isomerase in Cos7 cells.

Autocrine motility factor (AMF) is identical to the glycolytic enzyme phosphohexose isomerase (PHI) and overexpression of AMF/PHI is associated with tumor malignancy. In order to study the overexpression of AMF/PHI, an HA-tagged AMF construct was transiently transfected into Cos7 cells. Expression of a tagged AMF-HA allowed us to determine that over a period of 16 hours only a small amount (0.1-1%) of total cellular AMF-HA was secreted into the cell medium. Cell-associated AMF-HA was exclusively cytosolic as it could be completely extracted with Triton X-100 and concentrated within actin rich pseudopodial domains. Treatment of the cells with the glycolysis inhibitor oxamate disrupted the association of AMF-HA with actin concentrations demonstrating that glycolysis regulates the formation of these AMF/PHI-associated actin-rich protrusions. AMF/PHI is a well-characterized tumor cell secreted cytokine and we identify here an alternate intracellular function for this glycolytic enzyme/cytokine in cell motility.

The double-stranded RNA-binding protein Staufen is incorporated in human immunodeficiency virus type 1: evidence for a role in genomic RNA encapsidation.

Human Staufen (hStau), a double-stranded RNA (dsRNA)-binding protein that is involved in mRNA transport, is incorporated in human immunodeficiency virus type 1 (HIV-1) and in other retroviruses, including HIV-2 and Moloney murine leukemia virus. Sucrose and Optiprep gradient analyses reveal cosedimentation of hStau with purified HIV-1, while subtilisin assays demonstrate that it is internalized. hStau incorporation in HIV-1 is selective, is dependent on an intact functional dsRNA-binding domain, and quantitatively correlates with levels of encapsidated HIV-1 genomic RNA. By coimmunoprecipitation and reverse transcription-PCR analyses, we demonstrate that hStau is associated with HIV-1 genomic RNA in HIV-1-expressing cells and purified virus. Overexpression of hStau enhances virion incorporation levels, and a corresponding, threefold increase in HIV-1 genomic RNA encapsidation levels. This coordinated increase in hStau and genomic RNA packaging had a significant negative effect on viral infectivity. This study is the first to describe hStau within HIV-1 particles and provides evidence that hStau binds HIV-1 genomic RNA, indicating that it may be implicated in retroviral genome selection and packaging into assembling virions.

Molecular cloning and biochemical characterization of a new mouse testis soluble-zinc-metallopeptidase of the neprilysin family.

Because of their roles in controlling the activity of several bio-active peptides, members of the neprilysin family of zinc metallopeptidases have been identified as putative targets for the design of therapeutic agents. Presently, six members have been reported, these are: neprilysin, endothelin-converting enzyme (ECE)-1 and ECE-2, the Kell blood group protein, PHEX (product of the phosphate-regulating gene with homologies to endopeptidase on the X chromosome) and X-converting enzyme (XCE). In order to identify new members of this important family of peptidases, we designed a reverse transcriptase-PCR strategy based on conserved amino acid sequences of neprilysin, ECE-1 and PHEX. We now report the cloning from mouse testis of a novel neprilysin-like peptidase that we called NL1. NL1 is a glycoprotein that, among the members of the family, shows the strongest sequence identity with neprilysin. However, in contrast with neprilysin and other members of the family which are type II integral membrane proteins, NL1 was secreted when expressed in cultured mammalian cells, likely due to cleavage by a subtilisin-like convertase at a furin-like site located 22 amino acid residues in the C-terminus of the transmembrane domain. The recombinant enzyme exhibited neprilysin-like peptidase activity and was efficiently inhibited by phosphoramidon and thiorphan, two inhibitors of neprilysin. Northern blot analysis and in situ hybridization showed that NL1 mRNA was found predominantly in testis, specifically in round and elongated spermatids. This distribution of NL1 mRNA suggests that it could be involved in sperm formation or other processes related to fertility.

Microtubule-dependent recruitment of Staufen-green fluorescent protein into large RNA-containing granules and subsequent dendritic transport in living hippocampal neurons.

Dendritic mRNA transport and local translation at individual potentiated synapses may represent an elegant way to form synaptic memory. Recently, we characterized Staufen, a double-stranded RNA-binding protein, in rat hippocampal neurons and showed its presence in large RNA-containing granules, which colocalize with microtubules in dendrites. In this paper, we transiently transfect hippocampal neurons with human Staufen-green fluorescent protein (GFP) and find fluorescent granules in the somatodendritic domain of these cells. Human Stau-GFP granules show the same cellular distribution and size and also contain RNA, as already shown for the endogenous Stau particles. In time-lapse videomicroscopy, we show the bidirectional movement of these Staufen-GFP-labeled granules from the cell body into dendrites and vice versa. The average speed of these particles was 6.4 microm/min with a maximum velocity of 24. 3 microm/min. Moreover, we demonstrate that the observed assembly into granules and their subsequent dendritic movement is microtubule dependent. Taken together, we have characterized a novel, nonvesicular, microtubule-dependent transport pathway involving RNA-containing granules with Staufen as a core component. This is the first demonstration in living neurons of movement of an essential protein constituent of the mRNA transport machinery.

Cloning and characterization of Aplysia neutral endopeptidase, a metallo-endopeptidase involved in the extracellular metabolism of neuropeptides in Aplysia californica.

Cell surface metallo-endopeptidases play important roles in cell communication by controlling the levels of bioactive peptides around peptide receptors. To understand the relative relevance of these enzymes in the CNS, we characterized a metallo-endopeptidase in the CNS of Aplysia californica, whose peptidergic pathways are well described at the molecular, cellular, and physiological levels. The membrane-bound activity cleaved Leu-enkephalin at the Gly3-Phe4 bond with an inhibitor profile similar to that of the mammalian neutral endopeptidase (NEP). This functional homology was supported by the molecular cloning of cDNAs from the CNS, which demonstrated that the Aplysia and mammalian NEPs share all the same amino acids that are essential for the enzymatic activity. The protein is recognized both by specific anti-Aplysia NEP (apNEP) antibodies and by the [125I]-labeled NEP-specific inhibitor RB104, demonstrating that the apNEP gene codes for the RB104-binding protein. In situ hybridization experiments on sections of the ganglia of the CNS revealed that apNEP is expressed in neurons and that the mRNA is present both in the cell bodies and in neurites that travel along the neuropil and peripheral nerves. When incubated in the presence of a specific NEP inhibitor, many neurons of the buccal ganglion showed a greatly prolonged physiological response to stimulation, suggesting that NEP-like metallo-endopeptidases may play a critical role in the regulation of the feeding behavior in Aplysia. One of the putative targets of apNEP in this behavior is the small cardioactive peptide, as suggested by RP-HPLC experiments. More generally, the presence of apNEP in the CNS and periphery may indicate that it could play a major role in the modulation of synaptic transmission in Aplysia and in the metabolism of neuropeptides close to their point of release.

Mammalian staufen is a double-stranded-RNA- and tubulin-binding protein which localizes to the rough endoplasmic reticulum.

Staufen (Stau) is a double-stranded RNA (dsRNA)-binding protein involved in mRNA transport and localization in Drosophila. To understand the molecular mechanisms of mRNA transport in mammals, we cloned human (hStau) and mouse (mStau) staufen cDNAs. In humans, four transcripts arise by differential splicing of the Stau gene and code for two proteins with different N-terminal extremities. In vitro, hStau and mStau bind dsRNA via each of two full-length dsRNA-binding domains and tubulin via a region similar to the microtubule-binding domain of MAP-1B, suggesting that Stau cross-links cytoskeletal and RNA components. Immunofluorescent double labeling of transfected mammalian cells revealed that Stau is localized to the rough endoplasmic reticulum (RER), implicating this RNA-binding protein in mRNA targeting to the RER, perhaps via a multistep process involving microtubules. These results are the first demonstration of the association of an RNA-binding protein in addition to ribosomal proteins, with the RER, implicating this class of proteins in the transport of RNA to its site of translation.

Molecular cloning, sequence analysis and expression distribution of an aminopeptidase in Aplysia california.

We are investigating the role of membrane-bound peptidases in the inactivation of neuropeptides in Aplysia californica. Recently, we reported the biochemical characterization of a membrane-bound neuropeptide-degrading enzyme which has enzymatic characteristics similar to those of the mammalian aminopeptidase N (Bawab W, Querido E, Crine P, DesGroseillers L. Identification and characterization of aminopeptidases from Aplysia californica, Biochem J 1992;286:967-975). We now report the cloning and sequencing of a cDNA encoding an aminopeptidase enzyme (apAP) and the localization of the apAP transcript in Aplysia. The apAP cDNA encodes a putative protein of 1007 amino acids, which shows around 34% sequence identity to mammalian aminopeptidases A and N sequences. The deduced amino acid sequence suggests that apAP is a type II membrane-bound protein, with a long extracellular domain in which the consensus sequence of zinc-binding metallopeptidases (His-Glu-Xxx-Xxx-His) is found. RT-PCR and Northern blot experiments showed that the apAP gene is expressed as a single 6.8-kb transcript in the central nervous system, gill, heart, kidney and ovotestis.

Cloning and functional expression of an Aplysia 5-HT receptor negatively coupled to adenylate cyclase.

Serotonin (5-HT) is involved in the control of various behaviors in Aplysia californica, including reproduction, feeding, locomotion, circadian rhythm, synaptic plasticity, and synaptic growth. The large variety of functions of 5-HT is mediated by different receptor subtypes that are coupled to different second-messenger systems. Here, we report the cloning of a cDNA coding for an Aplysia G-protein-coupled 5-HT receptor (5-HTap1). Its deduced amino acid sequence resembles those of the 5-HT1 receptor subfamily. When expressed in stable cell lines, 5-HTap1 exhibits high-affinity binding for the serotonergic radioligand [N-methyl-3H]lysergic acid diethylamide. This binding is competed by several 5-HT agonists and antagonists, and the pharmacological profile of inhibition has some similarities with those of 5-HT1 and 5-HT7 receptors. Application of 5-HT or its agonists 5-carboxamidotryptamine maleate and (+/-)-8-hydroxy-2-(di-n-propyl-amino) tetralin hydrobromide on cells transformed with 5-HTap1 produced a dose-dependent inhibition of forskolin-stimulated cAMP accumulation. 5-HTap1 is thus negatively coupled to adenylate cyclase. The production of antiserum against the 5-HTap1 receptor allowed us to examine its expression in animal tissues. The receptor protein is detected in every tissue examined, although it seems only weakly expressed in some samples. The receptor is also found in every ganglia of the nervous system, both in the sheath and in the neurons. 5-HTap1 mRNA is absent from the sheath, indicating that the protein observed there is probably located on the nerve terminals.

Pex mRNA is localized in developing mouse osteoblasts and odontoblasts.

Mutations in PEX, a phosphate-regulating gene with homology to endopeptidase on the X chromosome, were recently identified in patients with X-linked hypophosphatemia (XLH), an inherited disorder of phosphate homeostasis characterized by growth retardation and rachitic and osteomalacic bone disease. To understand the mechanism by which loss of PEX function elicits the mutant phenotype, a study of its mRNA localization and ontogenesis was undertaken. Using the reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) with polyA+ RNA purified from mouse testis, a 337-bp Pex cDNA fragment was generated and cloned in the pCRII plasmid. The cDNA was used to generate sense and anti-sense Pex riboprobes for in situ hybridization (ISH) and Northern analysis. To survey a large number of different tissues, sagittal sections of embryos and newborn mice were examined. ISH showed the presence of Pex mRNA in osteoblasts and odontoblasts. Pex gene expression was detectable on Day 15 of embryonic development, which coincides with the beginning of intercellular matrix deposition in bones. Finally, Northern analysis of total RNA from calvariae and teeth of 3-day-old and adult mice showed that the abundance of the 7-kb Pex transcript is decreased in adult bones and in nongrowing teeth. The present study demonstrates that Pex mRNA is expressed in bones and teeth and suggests that this putative endopeptidase plays an important role in the development of these tissues.