Kidney function depends on the nephron, which comprises a blood filter, a tubule that is subdivided into functionally distinct segments, and a collecting duct. How these regions arise during development is poorly understood. The zebrafish pronephros consists of two linear nephrons that develop from the intermediate mesoderm along the length of the trunk. Here we show that, contrary to current dogma, these nephrons possess multiple proximal and distal tubule domains that resemble the organization of the mammalian nephron. We examined whether pronephric segmentation is mediated by retinoic acid (RA) and the caudal (cdx) transcription factors, which are known regulators of segmental identity during development. Inhibition of RA signaling resulted in a loss of the proximal segments and an expansion of the distal segments, while exogenous RA treatment induced proximal segment fates at the expense of distal fates. Loss of cdx function caused abrogation of distal segments, a posterior shift in the position of the pronephros, and alterations in the expression boundaries of raldh2 and cyp26a1, which encode enzymes that synthesize and degrade RA, respectively. These results suggest that the cdx genes act to localize the activity of RA along the axis, thereby determining where the pronephros forms. Consistent with this, the pronephric-positioning defect and the loss of distal tubule fate were rescued in embryos doubly-deficient for cdx and RA. These findings reveal a novel link between the RA and cdx pathways and provide a model for how pronephric nephrons are segmented and positioned along the embryonic axis.

The subtilisin-like prohormone convertase PC2 and the polypeptide 7B2 (an intracellularly cleaved protein of unknown function) are both selectively present in the regulated secretory pathway of neurons and endocrine cells.Here we demonstrate that intact recombinant 7B2 is a potent inhibitor of PC2 and prevents proPC2 cleavage in vitro, whereas the 7B2 cleavage product is virtually inactive. The PC2-related proteinase PC1/PC3 is not inhibited by 7B2.Furthermore, the carboxyl-terminal half of the 7B2 protein sequence is distantly related to the so-called potato inhibitor I family (which includes subtilisin inhibitors Synthetic compounds (e.g., peptidyl chloromethanes containing basic amino acid residues) have been shown to inhibit the Kex2 enzyme and furin-mediated cleavage of the human immunodeficiency viral coat protein gpl60 (5, 6). In addition, the furin enzyme is inhibited by a1-antitrypsin genetically engineered to contain the consensus cleavage site of furin at its reactive site (7) and furin is moderately inhibited by the similarly mutated turkey ovomucoid third domain (8). However, as yet, potent naturally occurring inhibitors of the proprotein cleavage enzymes have not been identified.The neuroendocrine-specific polypeptide 7B2 was initially isolated from porcine anterior pituitary glands as a protein of ;21 kDa (9). Biosynthesis ofthe %21-kDa 7B2 protein occurs through carboxyl-terminal processing of a --27-kDa precursor protein (10,11) and only the cleaved form of 7B2 is released (10). Secretion of the 7B2 cleavage product could be regulated (10,12,13), establishing that, like PC1/PC3 and PC2, the polypeptide 7B2 is in the regulated secretory pathway. The 7B2 protein is highly conserved and widely distributed in the central nervous system and endocrine tissues (14)(15)(16) and was generated by PCR using specific primers; the 5' primer corresponded to nucleotides 107-129 with a BamHI site introduced at the 5' end and the 3' primer consisted of nucleotides 538-562 with an introduced 5' stop codon and 5' HindIII site. The recombinant 21-kDa 7B2 protein represents amino acids 1-151 of the human 7B2 protein and corresponds to the 7B2 cleavage product isolated from anterior pituitaries (9,11). The expression plasmid for the intact 27-kDa 7B2 precursor protein was constructed by replacing the =0.2-kb Kpn I-HindIII fiagment of the 21-kDa 7B2 construct by the -0.8-kb Kpn 1-HindIII fragment (encoding the carboxylterminal half of the precursor protein) of a full-length human 7B2 cDNA clone (18). Recombinant 7B2 was purified by Ni2+-NTA agarose affinity chromatography according to the instructions of the manufacturer (Qiagen, Chatsworth, CA).Preparation of PC1/PC3 and PC2 Enzymes. Active 87-kDa PC1/PC3 was purified from medium of overexpressing CHO cells as described (19). PC2 was obtained from the conditioned medium of ,BTC3 cells through immunopurification (20). One hundred milliliters of 16-h conditioned fffC3 cell culture medium (containing aprotinin at 100 jug/ml) was collected, centrifuged, and conce...

Early embryonic development in many organisms relies upon maternal molecules deposited into the egg prior to fertilization. We have cloned and characterized a maternal T-box gene in the zebrafish, eomesodermin(eomes). During oogenesis, the eomes transcript becomes localized to the cortex of the oocyte. After fertilization during early cleavage stages, eomes is expressed in a vegetal to animal gradient in the embryo, whereas Eomesodermin protein (Eom) is distributed cytoplasmically throughout the blastoderm. Strikingly, following midblastula transition, nuclear-localized Eomesodermin is detected on the dorsal side of the embryo only. Overexpression of eomes results in Nodal-dependent and nieuwkoid/dharma (nwk/dhm) independent ectopic expression of the organizer markers goosecoid (gsc), chordin (chd) and floating head (flh) and in the formation of secondary axes. The same phenotypes are observed when a VP16-activator construct is injected into early embryos, indicating that eomes acts as a transcriptional activator. In addition, a dominant-negative construct and antisense morpholino oligonucleotides led to a reduction in gsc and flh expression. Together these data indicate that eomes plays a role in specifying the organizer.

SUMMARYPrimordial germ cells (PGCs) in Xenopus are specified through the inheritance of germ plasm. During gastrulation, PGCs remain totipotent while surrounding cells in the vegetal mass become committed to endoderm through the action of the vegetal localized maternal transcription factor VegT. We find that although PGCs contain maternal VegT RNA, they do not express its downstream targets at the mid-blastula transition (MBT). Transcriptional repression in PGCs correlates with the failure to phosphorylate serine 2 in the carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAPII). As serine 5 is phosphorylated, these results are consistent with a block after the initiation step but before the elongation step of RNAPII-based transcription. Repression of PGC gene expression occurs despite an apparently permissive chromatin environment. Phosphorylation of CTD-serine 2 and expression of zygotic mRNAs in PGCs are first detected at neurula, some 10 hours after MBT, indicating that transcription is significantly delayed in the germ cell lineage. Significantly, Oct-91, a POU subclass V transcription factor related to mammalian Oct3/4, is among the earliest zygotic transcripts detected in PGCs and is a likely mediator of pluripotency. Our findings suggest that PGCs are unable to respond to maternally inherited endoderm determinants because RNAPII activity is transiently blocked while these determinants are present. Our results in a vertebrate system further support the concept that one strategy used repeatedly during evolution for preserving the germline is RNAPII repression.