Gene and cluster-specific expression of the Iroquois family members during mouse development

Mammalian homologues of the Drosophila Iroquois homeobox gene complex, involved in patterning and regionalization of differentiation, have recently been identified (Mech. Dev., 69 (1997) 169; Dev. Biol., 217 (2000) 266; Dev. Dyn., 218 (2000) 160; Mech. Dev., 91 (2000) 317; Dev. Biol., 224 (2000) 263; Genome Res., 10 (2000) 1453; Mech. Dev., 103 (2001) 193). The six members of the murine family were found to be organized in two cognate clusters of three genes each, Irx1, -2, -4 and Irx3, -5, -6, respectively (Peters et al., 2000). As a basis for further study of their regulation and function we performed a comparative analysis of the genomic organization and of the expression patterns of all six Irx genes. The genes are expressed in highly specific and regionalized patterns of ectoderm, mesoderm and endoderm derived tissues. In most tissues the pattern of expression of the clustered genes, especially of Irx1 and -2 and of Irx3 and -5, respectively, closely resembled each other while those of Irx4 and -6 were very divergent. Interestingly, the expression of cognate genes was found to be mutually exclusive in adjacent and interacting tissues of limb, heart and the laryncho-pharyncheal region. The results indicate that the Irx genes are coordinately regulated at the level of the cluster.     

Pancreas development in zebrafish: early dispersed appearance of endocrine hormone expressing cells and their convergence to form the definitive islet

To begin to understand pancreas development and the control of endocrine lineage formation in zebrafish, we have examined the expression pattern of several genes shown to act in vertebrate pancreatic development: pdx-1, insulin (W. M. Milewski et al., 1998, Endocrinology 139, 1440-1449), glucagon, somatostatin (F. Argenton et al., 1999, Mech. Dev. 87, 217-221), islet-1 (Korzh et al., 1993, Development 118, 417-425), nkx2.2 (Barth and Wilson, 1995, Development 121, 1755-1768), and pax6.2 (Nornes et al., 1998, Mech. Dev. 77, 185-196). To determine the spatial relationship between the exocrine and the endocrine compartments, we have cloned the zebrafish trypsin gene, a digestive enzyme expressed in differentiated pancreatic exocrine cells. We found expression of all these genes in the developing pancreas throughout organogenesis. Endocrine cells first appear in a scattered fashion in two bilateral rows close to the midline during mid-somitogenesis and converge during late-somitogenesis to form a single islet dorsal to the nascent duodenum. We have examined development of the endocrine lineage in a number of previously described zebrafish mutations. Deletion of chordamesoderm in floating head (Xnot homolog) mutants reduces islet formation to small remnants, but does not delete the pancreas, indicating that notochord is involved in proper pancreas development, but not required for differentiation of pancreatic cell fates. In the absence of knypek gene function, which is involved in convergence movements, the bilateral endocrine primordia do not merge. Presence of trunk paraxial mesoderm also appears to be instrumental for convergence since the bilateral endocrine primordia do not merge in spadetail mutants. We discuss our findings on zebrafish pancreatogenesis in the light of evolution of the pancreas in chordates.     

Dll3 pudgy mutation differentially disrupts dynamic expression of somite genes

Mutations in the notch ligand delta-like 3 have been identified in both the pudgy mouse (Dll3(pu); Kusumi et al.: Nat Genet 19:274-278, 1998) and the human disorder spondylocostal dysostosis (SCD; Bulman et al.: Nat Genet 24:438-441, 2000), and a targeted mutation has been generated (Dll3(neo); Dunwoodie et al.: Development 129:1795-1806, 2002). Vertebral and rib malformations deriving from defects in somitic patterning are key features of these disorders. In the mouse, notch pathway genes such as Lfng, Hes1, Hes7, and Hey2 display dynamic patterns of expression in paraxial mesoderm, cycling in synchrony with somite formation (Aulehla and Johnson: Dev Biol 207:49-61, 1999; Forsberg et al.: Curr Biol 8:1027-1030, 1998; Jouve et al.: Development 127:1421-1429, 2000; McGrew et al.: Curr Biol 8:979-982, 1998; Nakagawa et al.: Dev Biol 216:72-84, 1999). We report here that the Dll3(pu) mutation has different effects on the expression of cycling (Lfng and Hes7) and stage-specific genes (Hey3 and Mesp2). This suggests a more complex situation than a single oscillatory mechanism in somitogenesis and provides an explanation for the unique radiological features of the human DLL3-type of SCD.     

Expression of the vertebrate Slit gene family and their putative receptors, the Robo genes, in the developing murine kidney

The slit (sli) gene, encoding a secreted glycoprotein, has been demonstrated to play a vital role in axonal guidance in Drosophila melanogaster by acting as a signalling ligand for the robo receptor (Rothberg, J.M., Jacobs, J.R., Goodman, C.S., Artavanis-Tsakonas, S., 1990. slit: an extracellular protein necessary for development of midline glia and commissural axon pathways contains both EGF and LRR domains. Genes Dev. 4, 2169-2187; Kidd, T., Bland, K.S., Goodman, C. S., 1999. Slit is the midline repellent for the robo receptor in Drosophila. Cell 96, 785-794). Multiple homologs of both sli and robo have been identified in vertebrates and are thought to play similar roles to their fly counterparts in neural development (Brose, K., Bland, K.S., Wang, K.H., Arnott, D., Henzel, W., Goodman, C.S., Tessier-Lavigne, M., Kidd, T., 1999. Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96, 795-806). Slit2 has been shown to bind Robo1, mediating both neuronal and axonal guidance in the developing central nervous system (CNS), (Brose et al., 1999; Hu, H., 1999. Chemorepulsion of neuronal migration by Slit2 in the developing mammalian forebrain. Neuron 23, 703-711). Importantly, both gene families display distinct expression patterns outside the CNS (Holmes, G.P., Negus, K., Burridge, L., Raman, S., Algar, E., Yamada, T., Little, M.H., 1998. Distinct but overlapping expression patterns of two vertebrate slit homologs implies functional roles in CNS development and organogenesis. Mech. Dev. 79, 57-72; Yuan, W., Zhou, L., Chen, J.H., Wu, J.Y., Rao, Y., Ornitz, D.M., 1999. The mouse SLIT family: secreted ligands for ROBO expressed in patterns that suggest a role in morphogenesis and axon guidance. Dev. Biol. 212, 290-306). Using in situ hybridization on metanephric explant cultures and urogenital tract sections, the expression patterns of Slit1, 2, 3 and Robo1 and 2 were investigated during murine metanephric development. Slit1 was expressed in the metanephric mesenchyme (MM) surrounding the invading ureteric tree (UT). Slit2 was expressed at the tips of the UT and both Slit2 and Slit3 were expressed at the far proximal end of the comma shaped and S-shaped bodies. Expression of Robo1 was initially diffuse throughout the MM, then upregulated in the pretubular aggregates, and maintained at the distal end of the comma and S-shaped bodies. Robo2 was detected in the induced MM surrounding the arborizing UT tips and later in the proximal end of the S-shaped bodies. Coincident expression of Robo1 with Slit1 in the metanephric mesenchyme and Robo2, Slit2 and Slit3 in the far proximal end of the S-shaped bodies was observed during metanephric development.     

Identification and characterization of roundabout orthologs in zebrafish

The Roundabout (Robo) family of receptors and their extracellular ligands, the Slit protein family, play important roles in repulsive axon guidance. First identified in Drosophila, Robo receptors form an evolutionarily conserved sub-family of the immunoglobulin (Ig) superfamily that are characterized by the presence of five Ig repeats and three fibronectin-type III repeats in the extracellular domain, a transmembrane domain, and a cytoplasmic domain with several conserved motifs that play important roles in Robo-mediated signaling (Cell 92 (1998) 205; Cell 101 (2000) 703). Robo family members have now been identified in C. elegans, Xenopus, rat, mouse, and human (Cell 92 (1998) 205; Cell 92 (1998) 217; Cell 96 (1999) 807; Dev. Biol. 207 (1999) 62). Furthermore, multiple robo genes have been described in Drosophila, rat, mouse and humans, raising the possibility of potential redundancy and diversity in robo gene function. As a first step in elucidating the role of Robo receptors during vertebrate development, we identified and characterized two Robo family members from zebrafish. We named these zebrafish genes robo1 and robo3, reflecting their amino acid sequence similarity to other vertebrate robo genes. Both genes are dynamically expressed in the developing nervous system in distinct patterns. robo3 is expressed during the first day of development in the hindbrain and spinal cord and is later expressed in the tectum and retina. robo1 nervous system expression appears later in development and is more restricted. Moreover, both genes are expressed in non-neuronal tissues consistent with additional roles for these genes during development.     

Role of endothelin in the human craniofacial morphogenesis

Human craniofacial morphogenesis is a complex biological event: it is mediated by several factors and different types tissue interaction. Recent studies on animal models have led to an improved understanding of human craniofacial malformations. In particular, the endothelins, peptides that are involved in various biological functions in many tissues and organs, have been shown to play a crucial role in the development of the first branchial-arch-derived structures in mice [Kurihara et al., Nature 368:703-710, 1994]. We previously reported the identification and localization of endothelin-1 (ET-1) and its receptors in human fetal jaw [Barni et al., Dev Biol 168:373-377, 1995]. In the present study, the gene expression of ET-1 and its receptors were demonstrated in human jaw from 11-12-week-old fetuses. By using in situ hybridization, mRNA for ET-1 was localized in the epithelial cells of the oral mucosa: mRNA for ET receptors (ETA and ETB subtypes) was expressed in the mesenchyme. In situ binding experiments confirmed the presence of ETA and ETB receptors in the cells involved in the osteogenesis of the mandible. Furthermore, ET-1 was able to stimulate thymidine uptake and the expression of the oncoprotein c-fos in the same cell types. Our results indicate that ET-1 may play a putative role in epithelium-mesenchyme interaction during human craniofacial morphogenesis. Our findings are in complete accord with those of the most recent works by Yanagisawa [Yanagisawa H et al., 1998] and Clouthier [Clouthier et al., Development 125:813-824, 1998]. They most probably confirm the primary role of ET-1 in the development of the pharyngeal arches.     

Notch1 and 2 cooperate in limb ectoderm to receive an early Jagged2 signal regulating interdigital apoptosis

Spontaneous and engineered mutations in the Notch ligand Jagged2 produced the Syndactylism phenotype (Jiang, R.L., Lan, Y., Chapman, H.D., Shawber, C., Norton, C.R., Serreze, D.V., Weinmaster, G., Gridley, T., 1998. Defects in limb, craniofacial, and thymic Development in Jagged2 mutant mice. Genes Dev. 12, 1046-1057; Sidow, A., Bulotsky, M.S., Kerrebrock, A.W., Bronson, R.T., Daly, M.J., Reeve, M.P., Hawkins, T.L., Birren, B.W., Jaenisch, R., Lander, E.S., 1997. Serrate2 is disrupted in the mouse limb-development mutant syndactylism. Nature 389, 722-725). Given that additional ligands may be expressed in the developing limb bud, it was possible that loss of Jagged2 disabled only part of Notch function in the limb. In addition, it is not clear from the expression pattern of Jagged2 in the apical ectodermal ridge (AER) whether the ectodermal or mesenchymal compartment of the limb bud receives the Jagged2 signal. To elucidate the requirement for the Notch pathway in limb development, we have analyzed single and compound Notch receptor mutants as well as gamma-secretase-deficient limbs. Floxed alleles were removed either from the developing limb bud ectoderm (using Msx2-Cre) or from the mesenchyme (using Prx1-Cre). Our results confirm that Jagged2 loss describes the contribution of the entire Notch pathway to the mouse limb development and revealed that both Notch1 and 2 are required in the ectoderm to receive the Jagged2 signal. Interestingly, our allelic series allowed us to determine that Notch receives this signal at an early stage in the developmental process and that memory of this event is retained by the mesenchyme, where Notch signaling appears to be dispensable. Thus, Notch signaling plays a non-autonomous role in digit septation.     

Polyploid tissues in the nematode Caenorhabditis elegans

During larval development, the number of somatic nuclei in C. elegans hermaphrodites increases from 558 to 959 (J. E. Sulston and H. R. Horvitz, Dev. Biol. 56, 110-156, 1977; J. E. Sulston et al., Dev. Biol. 100, 64-119, 1983). At the same time, the animals increase about 60-fold in volume. We have measured the DNA contents of several classes of nuclei by quantitating the fluorescence of Hoescht 33258 stained DNA (D. G. Albertson et al., Dev. Biol. 63, 165-178, 1978). Probably all embryonic nuclei, including those of neurons, muscles, hypodermis, and intestine, are diploid at hatching. Neurons, muscles, and nondividing hypodermal nuclei remain diploid throughout larval development. The DNA content of the intestinal nuclei doubles at the end of each larval stage, reaching 32C by the adult stage. New hypodermal cells, generated by division of seam cells in the larval stages, undergo an additional round of DNA replication before fusing with the major syncytium (hyp7, Sulston et al., 1983). Thus the larval hyp7 syncytium comprises a fixed number of diploid embryonic nuclei plus an increasing number of tetraploid postembryonic nuclei. Some of the endoreduplications that occur in the intestinal and hypodermal lineages of C. elegans may correspond to nuclear or cellular divisions in another nematode Panagrellus redivivus (P. W. Sternberg and H. R. Horvitz, Dev. Biol. 93, 181-205, 1982).     

Abdominal B-type Hox gene expression in Xenopus laevis

Previously, we reported a zebrafish iroquois gene, ziro3, and its expression during early embryogenesis (Mech. Dev. 87 (1999) 165). In the present study, we have isolated two novel zebrafish iroquois genes, ziro1 and ziro5, homologs of mouse Irx1 and mouse Irx5, respectively. The expression of both genes is initiated in dorsal neuroectoderm and mesoderm during gastrulation. Later, their expression appears in the central nervous system (CNS), excluding the telencephalon and most of the diencephalon. ziro1 expression is complementary to that of ziro3 in the notochord and later in the gut. In contrast, ziro5 expression mostly overlaps with that of ziro3. Interestingly, all three iroquois zebrafish genes are expressed in the notochord while only Irx3 is active in the mouse notochord. Their expression in later stages of embryogenesis was also compared.     

Coordination of N-glycosylation and protein translocation across the endoplasmic reticulum membrane by Sss1 protein

Secretory proteins are translocated across the endoplasmic reticulum (ER) membrane through a channel formed by three proteins, namely Sec61p, Sbh1p, and Sss1p (Johnson, A. E., and van Waes, M. A. (1999) Annu. Rev. Cell Dev. Biol. 15, 799-842). Sec61p and Sss1p are essential for translocation (Esnault, Y., Blondel, M. O., Deshaies, R. J., Schekman, R., and Kepes, F. (1993) EMBO J. 12, 4083-4093). Sec61p is a polytopic membrane protein that lines the protein translocation channel. The role of Sss1p is unknown. During import into the ER through the Sec61p channel, many proteins are N-glycosylated before translocation is completed. In addition, both the Sec61 channel and oligosaccharyl transferase (OST) copurify with ribosomes from rough ER, suggesting that OST is located in close proximity to the Sec61 channel (Gorlich, D., Prehn, S., Hartmann, E., Kalies, K.-U., and Rapoport, T. A. (1992) Cell 71, 489-503 and Wang, L., and Dobberstein, B. (1999) FEBS Lett. 457, 316-322). Here, we demonstrate a direct interaction between Sss1p and a subunit of OST, Wbp1p, using the split-ubiquitin system and co-immunoprecipitation. We generated mutants in the cytoplasmic domain of Sss1p that disturb the interaction with OST and are viable but display a translocation defect specific for proteins with glycosylation acceptor sites. Our data suggest that Sss1p coordinates translocation across the ER membrane and N-linked glycosylation of secretory proteins.     

Expression of Lrrn1 marks the prospective site of the zona limitans thalami in the early embryonic chicken diencephalon

An unknown chicken gene selected from a published substractive hybridization screen (GenBank Accession No. ; [Christiansen, J.H., Coles, E.G., Robinson, V., Pasini, A., Wilkinson, D.G., 2001. Screening from a subtracted embryonic chick hindbrain cDNA library: identification of genes expressed during hindbrain, midbrain and cranial neural crest development. Mech. Dev. 102, 119-133.]) was deemed of interest because of its dynamic pattern of expression across the forebrain and midbrain regions. A 528bp fragment cloned from early chick embryo cDNA and used for in situ hybridization corresponded to part of the 3' untranslated region of the chicken gene Leucine-rich repeat neuronal protein 1 (Lrrn1). The expression of this gene, mapped in the embryonic chick brain between stages HH10 and HH26, apparently preconfigures the zona limitans thalami site before overt formation of this boundary structure. Apart of colateral expression in the forebrain, midbrain and hindbrain basal plate, the most significant expression of Lrrn1 was found early on across the entire alar plate of midbrain and forebrain (HH10). This unitary domain soon divides at HH14 into a rostral part, across alar secondary prosencephalon and prospective alar prosomere 3 (prethalamus; caudal limit at the prospective zona limitans), and a caudal part in alar prosomere 1 (pretectum) and midbrain. The rostral forebrain domain later downregulates gradually most extratelencephalic signal of Lrrn1, but the rostral shell of zona limitans retains expression longer. Expression in the caudal alar domain also changes by downregulation within its pretectal subdomain. Caudally, the midbrain domain ends at the isthmo-mesencephalic junction throughout the studied period. Embryonic Lrrn1 signal also appears in the somites and in the otic vesicle.