The proprotein convertase BLI-4 promotes collagen secretion prior to assembly of the Caenorhabditis elegans cuticle.

Some types of collagens, including transmembrane MACIT collagens and C. elegans cuticle collagens, are N-terminally cleaved at a dibasic site that resembles the consensus for furin or other proprotein convertases of the subtilisin/kexin (PCSK) family. Such cleavage may release transmembrane collagens from the plasma membrane and affect extracellular matrix assembly or structure. However, the functional consequences of such cleavage are unclear and evidence for the role of specific PCSKs is lacking. Here, we used endogenous collagen fusions to fluorescent proteins to visualize the secretion and assembly of the first collagen-based cuticle in C. elegans and then tested the role of the PCSK BLI-4 in these processes. Unexpectedly, we found that cuticle collagens SQT-3 and DPY-17 are secreted into the extraembryonic space several hours before cuticle matrix assembly. Furthermore, this early secretion depends on BLI-4/PCSK; in bli-4 and cleavage-site mutants, SQT-3 and DPY-17 are not efficiently secreted and instead form large intracellular puncta. Their later assembly into cuticle matrix is reduced but not entirely blocked. These data reveal a role for collagen N-terminal processing in intracellular trafficking and the control of matrix assembly in vivo. Our observations also prompt a revision of the classic model for C. elegans cuticle matrix assembly and the pre-cuticle-to-cuticle transition, suggesting that cuticle layer assembly proceeds via a series of regulated steps and not simply by sequential secretion and deposition.

The proprotein convertase BLI-4 promotes collagen secretion during assembly of the Caenorhabditis elegans cuticle.

Some types of collagens, including transmembrane MACIT collagens and C. elegans cuticle collagens, are N-terminally cleaved at a dibasic site that resembles the consensus for furin or other proprotein convertases of the subtilisin/kexin (PCSK) family. Such cleavage may release transmembrane collagens from the plasma membrane and affect extracellular matrix assembly or structure. However, the functional consequences of such cleavage are unclear and evidence for the role of specific PCSKs is lacking. Here, we used endogenous collagen fusions to fluorescent proteins to visualize the secretion and assembly of the first collagen-based cuticle in C. elegans and then tested the role of the PCSK BLI-4 in these processes. Unexpectedly, we found that cuticle collagens SQT-3 and DPY-17 are secreted into the extraembryonic space several hours before cuticle matrix assembly. Furthermore, this early secretion depends on BLI-4/PCSK; in bli-4 and cleavage-site mutants, SQT-3 and DPY-17 are not efficiently secreted and instead form large intracellular aggregates. Their later assembly into cuticle matrix is reduced but not entirely blocked. These data reveal a role for collagen N-terminal processing in intracellular trafficking and in the spatial and temporal restriction of matrix assembly in vivo . Our observations also prompt a revision of the classic model for C. elegans cuticle matrix assembly and the pre-cuticle-to-cuticle transition, suggesting that cuticle layer assembly proceeds via a series of regulated steps and not simply by sequential secretion and deposition.

Insulin signaling and osmotic stress response regulate arousal and developmental progression of C. elegans at hatching.

The progression of animal development from embryonic to juvenile life depends on the coordination of organism-wide responses with environmental conditions. We found that two transcription factors that function in interneuron differentiation in Caenorhabditis elegans, fax-1, and unc-42, are required for arousal and progression from embryogenesis to larval life by potentiating insulin signaling. The combination of mutations in either transcription factor and a mutation in daf-2 insulin receptor results in a novel perihatching arrest phenotype; embryos are fully developed but inactive, often remaining trapped within the eggshell, and fail to initiate pharyngeal pumping. This pathway is opposed by an osmotic sensory response pathway that promotes developmental arrest and a sleep state at the end of embryogenesis in response to elevated salt concentration. The quiescent state induced by loss of insulin signaling or by osmotic stress can be reversed by mutations in genes that are required for sleep. Therefore, countervailing signals regulate late embryonic arousal and developmental progression to larval life, mechanistically linking the two responses. Our findings demonstrate a role for insulin signaling in an arousal circuit, consistent with evidence that insulin-related regulation may function in control of sleep states in many animals. The opposing quiescent arrest state may serve as an adaptive response to the osmotic threat from high salinity environments.

A tissue-specific enhancer of the C. elegans nhr-67/tailless gene drives coordinated expression in uterine stem cells and the differentiated anchor cell.

The nhr-67 nuclear receptor gene of Caenorhabditis elegans encodes the ortholog of the Drosophila tailless and vertebrate Tlx genes. In C. elegans, nhr-67 plays multiple roles in the development of the uterus during L2 and L3 larval stages. Four pre-VU cells are born in the L2 stage and form the precursor complement for the ventral surface of the mature uterus. One of the four pre-VU cells becomes the anchor cell (AC), which exits the cell cycle and differentiates, while the remaining three VU cells serve as stem cells that populate the ventral uterus. The nhr-67 gene functions in the development of both VU cell lineages and AC differentiation. Hypomorphic mutations in nhr-67 identify a 276bp region of the distal promoter that is sufficient to activate nhr-67 expression in pre-VU cells and the AC. The 276bp region includes 8 conserved potential cis-acting sites, including two E boxes and a nuclear receptor binding site. Mutational analysis demonstrates that the two E boxes are required for expression of nhr-67 in uterine precursor cells. The E/daughterless ortholog HLH-2 binds these sites as a homodimer, thus playing a central role in activating nhr-67 expression in the uterine precursors. At least two other binding activities, one of which may be the nhr-25/Ftz-F1 nuclear receptor transcription factor, also contribute to uterine precursor cell expression. The organization of the nhr-67 uterine precursor enhancer is compared to similar conserved enhancers in the egl-43, lag-2, and lin-3 genes, which contain the same HLH-2-binding E boxes and are similarly expressed in both pre-VU cells and the AC. This basic regulatory module allows the coordinated expression of at least four genes. Expression of genes in different cells that must coordinate to form a mature organ is driven by a shared set of promoter elements, which integrate multiple transcription factor inputs.