Patterning of the third pharyngeal pouch into thymus/parathyroid by Six and Eya1

Previous studies have suggested a role of the homeodomain Six family proteins in patterning the developing vertebrate head that involves appropriate segmentation of three tissue layers, the endoderm, the paraxial mesoderm and the neural crest cells; however, the developmental programs and mechanisms by which the Six genes act in the pharyngeal endoderm remain largely unknown. Here, we examined their roles in pharyngeal pouch development. Six1-/- mice lack thymus and parathyroid and analysis of Six1-/- third pouch endoderm demonstrated that the patterning of the third pouch into thymus/parathyroid primordia is initiated. However, the endodermal cells of the thymus/parathyroid rudiments fail to maintain the expression of the parathyroid-specific gene Gcm2 and the thymus-specific gene Foxn1 and subsequently undergo abnormal apoptosis, leading to a complete disappearance of organ primordia by E12.5. This thus defines the thymus/parathyroid defects present in the Six1 mutant. Analyses of the thymus/parathyroid development in Six1-/-;Six4-/- double mutant show that both Six1 and Six4 act synergistically to control morphogenetic movements of early thymus/parathyroid tissues, and the threshold of Six1/Six4 appears to be crucial for the regulation of the organ primordia-specific gene expression. Previous studies in flies and mice suggested that Eya and Six genes may function downstream of Pax genes. Our data clearly show that Eya1 and Six1 expression in the pouches does not require Pax1/Pax9 function, suggesting that they may function independently from Pax1/Pax9. In contrast, Pax1 expression in all pharyngeal pouches requires both Eya1 and Six1 function. Moreover, we show that the expression of Tbx1, Fgf8 and Wnt5b in the pouch endoderm was normal in Six1-/- embryos and slightly reduced in Six1-/-;Six4-/- double mutant, but was largely reduced in Eya1-/- embryos. These results indicate that Eya1 appears to be upstream of very early events in the initiation of thymus/parathyroid organogenesis, while Six genes appear to act in an early differentiation step during thymus/parathyroid morphogenesis. Together, these analyses establish an essential role for Eya1 and Six genes in patterning the third pouch into organ-specific primordia.     

Eya 1 acts as a critical regulator for specifying the metanephric mesenchyme

Although it is well established that the Gdnf-Ret signal transduction pathway initiates metanephric induction, no single regulator has yet been identified to specify the metanephric mesenchyme or blastema within the intermediate mesoderm, the earliest step of metanephric kidney development and the molecular mechanisms controlling Gdnf expression are essentially unknown. Previous studies have shown that a loss of Eya 1 function leads to renal agenesis that is a likely result of failure of metanephric induction. The studies presented here demonstrate that Eya 1 specifies the metanephric blastema within the intermediate mesoderm at the caudal end of the nephrogenic cord. In contrast to its specific roles in metanephric development, Eya 1 appears dispensable for the formation of nephric duct and mesonephric tubules. Using a combination of null and hypomorphic Eya 1 mutants, we now demonstrated that approximately 20% of normal Eya 1 protein level is sufficient for establishing the metanephric blastema and inducing the ureteric bud formation but not for its normal branching. Using Eya 1, Gdnf, Six 1 and Pax 2 mutant mice, we show that Eya 1 probably functions at the top of the genetic hierarchy controlling kidney organogenesis and it acts in combination with Six 1 and Pax 2 to regulate Gdnf expression during UB outgrowth and branching. These findings uncover an essential function for Eya 1 as a critical determination factor in acquiring metanephric fate within the intermediate mesoderm and as a key regulator of Gdnf expression during ureteric induction and branching morphogenesis.     

Eya1 is required for the morphogenesis of mammalian thymus,parathyroid and thyroid

Eyes absent (Eya) genes regulate organogenesis in both vertebrates and invertebrates. Mutations in human EYA1 cause congenital Branchio-Oto-Renal (BOR) syndrome, while targeted inactivation of murine Eya1 impairs early developmental processes in multiple organs, including ear, kidney and skeletal system. We have now examined the role of Eya1 during the morphogenesis of organs derived from the pharyngeal region, including thymus, parathyroid and thyroid. The thymus and parathyroid are derived from 3rd pharyngeal pouches and their development is initiated via inductive interactions between neural crest-derived arch mesenchyme, pouch endoderm, and possibly the surface ectoderm of 3rd pharyngeal clefts. Eya1 is expressed in all three cell types during thymus and parathyroid development from E9.5 and the organ primordia for both of these structures failed to form in Eya1(-/-) embryos. These results indicate that Eya1 is required for the initiation of thymus and parathyroid gland formation. Eya1 is also expressed in the 4th pharyngeal region and ultimobranchial bodies. Eya1(-/-) mice show thyroid hypoplasia, with severe reduction in the number of parafollicular cells and the size of the thyroid lobes and lack of fusion between the ultimobranchial bodies and the thyroid lobe. These data indicate that Eya1 also regulates mature thyroid gland formation. Furthermore, we show that Six1 expression is markedly reduced in the arch mesenchyme, pouch endoderm and surface ectoderm in the pharyngeal region of Eya1(-/-) embryos, indicating that Six1 expression in those structures is Eya1 dependent. In addition, we show that in Eya1(-/-) embryos, the expression of Gcm2 in the 3rd pouch endoderm is undetectable at E10.5, however, the expression of Hox and Pax genes in the pouch endoderm is preserved at E9.5-10.5. Finally, we found that the surface ectoderm of the 3rd and 4th pharyngeal region show increased cell death at E10.5 in Eya1(-/-) embryos. Our results indicate that Eya1 controls critical early inductive events involved in the morphogenesis of thymus, parathyroid and thyroid.     

ARI-1, an RBR family ubiquitin-ligase, functions with UBC-18 to regulate pharyngeal development in C. elegans

The LIN-35 retinoblastoma protein homolog and the ubiquitin-conjugating enzyme UBC-18 function redundantly to control an early step of pharyngeal morphogenesis in C. elegans. In order to identify ubiquitin-ligases acting downstream of UBC-18, we carried out a two-hybrid screen using UBC-18 as the bait molecule. Our screen identified three putative ubiquitin-ligases, one of which, ARI-1, showed genetic interactions leading to defective pharyngeal development that were identical to that previously observed for UBC-18. ARI-1 is a member of the RBR family of ubiquitin-ligases and contains a C-terminal motif that places it within the highly conserved Ariadne subfamily of RBR ligases. Our analyses indicate that ARI-1 is the principal Ariadne family member in C. elegans that is involved in the control of pharyngeal development with UBC-18. Using GFP reporters, we find that ARI-1 is expressed dynamically in a wide range of tissues including muscles and neurons during embryonic and postembryonic development. We also provide evidence that dsRNA species containing 14 or fewer base pairs of contiguous identity with closely related mRNAs are sufficient to mediate off-target silencing in C. elegans.     

C. elegans pharyngeal morphogenesis requires both de novo synthesis of pyrimidines and synthesis of heparan sulfate proteoglycans

The C. elegans pharynx undergoes elongation and morphogenesis to its characteristic bi-lobed shape between the 2- and 3-fold stages of embryogenesis. During this period, the pharyngeal muscles and marginal cells forming the isthmus between the anterior and posterior pharyngeal bulbs elongate and narrow. We have identified the spontaneous mutant pyr-1(cu8) exhibiting defective pharyngeal isthmus elongation, cytoskeletal organization defects, and maternal effect lethality. pyr-1 encodes CAD, a trifunctional enzyme required for de novo pyrimidine synthesis, and pyr-1(cu8) mutants are rescued by supplying exogenous pyrimidines. Similar pharyngeal defects and maternal effect lethality were found in sqv-1, sqv-8, rib-1 and rib-2 mutants, which affect enzymes involved in heparan sulfate proteoglycan (HSPG) synthesis. rib-1 mutant lethality was enhanced in a pyr-1 mutant background, indicating that HSPG synthesis is very sensitive to decreased pyrimidine pools, and HS disaccharides are moderately decreased in both rib-1 and pyr-1 mutants. We hypothesize that HSPGs are necessary for pharyngeal isthmus elongation, and pyr-1 functions upstream of proteoglycan synthesizing enzymes by providing precursors of UDP-sugars essential for HSPG synthesis.     

The coordinate regulation of pharyngeal development in C. elegans by lin-35/Rb, pha-1, and ubc-18

Organ development is a complex process involving the coordination of cell proliferation, differentiation, and morphogenetic events. Using a screen to identify genes that function coordinately with lin-35/Rb during animal development, we have isolated a weak loss-of-function (LOF) mutation in pha-1. lin-35; pha-1 double mutants are defective at an early step in pharyngeal morphogenesis leading to an abnormal pharyngeal architecture. pha-1 is also synthetically lethal with other class B synthetic multivulval (SynMuv) genes including the C. elegans E2F homolog, efl-1. Reporter analyses indicate that pha-1 is broadly expressed during embryonic development and that its functions reside in the cytoplasm. We also provide genetic and phenotypic evidence to support the model that PHA-1, a novel protein, and UBC-18, a ubiquitin-conjugating enzyme that we have previously shown to function with lin-35 during pharyngeal development, act in parallel pathways to regulate the activity of a common cellular target.     

The chitin synthase genes chs-1 and chs-2 are essential for C. elegans development and responsible for chitin deposition in the eggshell and pharynx, respectively

It is widely accepted that chitin is present in nematodes. However, its precise role in embryogenesis is unclear and it is unknown if chitin is necessary in other nematode tissues. Here, we determined the roles of chitin and the two predicted chitin synthase genes in Caenorhabditis elegans by chitin localization and gene disruption. Using a novel probe, we detected chitin in the eggshell and discovered elaborate chitin localization patterns in the pharyngeal lumen walls. Chitin deposition in these two sites is likely regulated by the activities of chs-1 (T25G3.2) and chs-2 (F48A11.1), respectively. Reducing chs-1 gene activity by RNAi led to eggs that were fragile and permeable to small molecules, and in the most severe case, absence of embryonic cell division. Complete loss of function in a chs-1 deletion resulted in embryos that lacked chitin in their eggshells and failed to divide. These results showed that eggshell chitin provides both mechanical support and chemical impermeability essential to developing embryos. Knocking down chs-2 by RNAi caused a defect in the pharynx and led to L1 larval arrest, indicating that chitin is involved in the development and function of the pharynx.     

Analysis of twin of eyeless regulation during early embryogenesis in Drosophila melanogaster

The Drosophila Pax6 homolog twin of eyeless (toy) is so far the first zygotically expressed gene involved in eye morphogenesis in Drosophila. The study of its expression during embryogenesis is therefore informative of the initial events of eye development in Drosophila. We have analyzed how the initial expression domain of toy at cellular blastoderm is regulated. We show that the three maternal patterning systems active in the cephalic region (the anterior, terminal and dorsal-ventral systems) cooperate with zygotically activated gap genes to shape the initial expression domain of toy. Whereas Bicoid, Dorsal and Torso signaling synergistically act as activators, Hunchback, Knirps and Decapentaplegic act as repressors.     

Expression of 'segmentation' genes during larval and juvenile development in the polychaetes Capitella sp. I and H. elegans

Polychaete annelids and arthropods are both segmented protostome invertebrates. To investigate whether the segmented body plan of these two phyla share a common molecular ground pattern, we report the developmental expression of orthologues of the arthropod segment polarity genes engrailed (en), hedgehog (hh), and wingless (wg/Wnt1) in larval and juvenile stages of the polychaete annelid Capitella sp. I and en in a second polychaete, Hydroides elegans. Temporally, neither Wnt1 nor hh are detected in the segmented region of the larval body until after morphological segmentation is apparent. Expression of CapI-Wnt1 is limited to a ring of ectoderm marking the future anus during larval segmentation. CapI-hh is expressed in a ring of the hindgut internal to that of CapI-Wnt1, as well as in a subset of ventral nerve cord neurons, anterior gut tissue, and mesoderm. In both H. elegans and Capitella sp. I, en is expressed in a spatially and temporally dynamic manner in segmentally iterated structures as well as a population of cells that migrate internally from ectoderm to mesoderm, possibly representing a population of ecto-mesodermal precursors. Significantly, the expression patterns we report for wg, en, and hh orthologues in Capitella sp. I and for en in larval development of H. elegans are not comparable to the highly conserved ectodermal segment polarity pattern observed in arthropods at any life history stage, consistent with distinct origins of segmentation between annelids and arthropods.     

The hedgehog-related gene wrt-5 is essential for hypodermal development in Caenorhabditis elegans

The Caenorhabditis elegans genome encodes a series of hedgehog-related genes, which are thought to have evolved and diverged from an ancestral Hh gene. They are classified into several families based on their N-terminal domains. Here, we analyze the expression and function of a member of the warthog gene family, wrt-5, that lacks the Hint/Hog domain. wrt-5 is expressed in seam cells, the pharynx, pharyngeal-intestinal valve cells, neurons, neuronal support cells, the excretory cell, and the reproductive system. WRT-5 protein is secreted into the extracellular space during embryogenesis. Furthermore, during larval development, WRT-5 protein is secreted into the pharyngeal lumen and the pharyngeal expression changes in a cyclical manner in phase with the molting cycle. Deletion mutations in wrt-5 cause embryonic lethality, which are temperature sensitive and more severe at 15 degrees C than at 25 degrees C. Animals that hatch exhibit variable abnormal morphology, for example, bagging worms, blistering, molting defects, or Roller phenotypes. We examined hypodermal cell junctions using the AJM-1Colon, two colonsGFP marker in the wrt-5 mutant background and observed cell boundary abnormalities in the arrested embryos. AJM-1Colon, two colonsGFP protein is also misplaced in pharyngeal muscle cells in the absence of WRT-5. In conclusion, we show that wrt-5 is an essential gene that - despite its lack of a Hint domain - has multiple functions in C. elegans and is implicated in cell shape integrity.     

The zebrafish dog-eared mutation disrupts eya1, a gene required for cell survival and differentiation in the inner ear and lateral line

To understand the molecular basis of sensory organ development and disease, we have cloned and characterized the zebrafish mutation dog-eared (dog) that is defective in formation of the inner ear and lateral line sensory systems. The dog locus encodes the eyes absent-1 (eya1) gene and single point mutations were found in three independent dog alleles, each prematurely truncating the expressed protein within the Eya domain. Moreover, morpholino-mediated knockdown of eya1 gene function phenocopies the dog-eared mutation. In zebrafish, the eya1 gene is widely expressed in placode-derived sensory organs during embryogenesis but Eya1 function appears to be primarily required for survival of sensory hair cells in the developing ear and lateral line neuromasts. Increased levels of apoptosis occur in the migrating primordia of the posterior lateral line in dog embryos and as well as in regions of the developing otocyst that are mainly fated to give rise to sensory cells of the cristae. Importantly, mutation of the EYA1 or EYA4 gene causes hereditary syndromic deafness in humans. Determination of eya gene function during zebrafish organogenesis will facilitate understanding the molecular etiology of human vestibular and hearing disorders.