A molecular ruler, FliK, controls the length of the flagellar hook. FliK measures hook length and catalyzes the secretion-substrate specificity switch from rod-hook substrate specificity to late substrate secretion, which includes the filament subunits. Here, we show normal hook-length control and filament assembly in the complete absence of the C-ring thus refuting the previous “cup” model for hook-length control. Mutants of C-ring components, which are reported to produce short hooks, show a reduced rate of hook-basal body assembly thereby allowing for a premature secretion-substrate specificity switch. Unlike fliK null mutants, hook-length control in an autocleavage-defective mutant of flhB, the protein responsible for the switch to late-substrate secretion, is completely abolished. FliK deletion variants that retain the ability to measure hook length are secreted thus demonstrating that FliK directly measures rod-hook length during the secretion process. Finally, we present a unifying model accounting for all published data on hook-length control in which FliK acts as a molecular ruler that takes measurements of rod-hook length while being intermittently secreted during the assembly process of the hook-basal body complex.

237 words Main text: 3834 words Number of figures: 5 Number of supplementary figures: 5 Number of supplementary videos: 6 Number of references: 71 Key Points: 1) Syndecan 2 regulates the formation of Hematopoietic Stem/Progenitor Cells, differentiation into hematopoietic populations and the CHT architecture 2) Syndecan 2 mutants are significantly more susceptible to bacterial infection 2 Abstract Syndecan 2 (Sdc2) is a transmembrane cell-surface heparan sulfate proteoglycan (HSPG) that has been implicated in the regulation of cell-cell signaling pathways and cell-matrix interactions.Surprisingly, homozygous recessive maternal zygotic (MZ) sdc2 null mutants in zebrafish appear to have normal development, normal morphology and are viable and fertile in adulthood. Whole transcriptome RNA sequencing, FACS analyses, and imaging of transgenic reporter lines that distinguish specific hematopoietic lineages revealed that sdc2 mutants have defects in the specification and proportions of red blood cells and neutrophils that initiate during embryonic hematopoiesis and likely persist through adulthood. During bacterial infections, MZsdc2 mutants have markedly reduced neutrophil recruitment and significantly higher death rates.Hematopoietic stem/progenitor cell (HSPC) numbers are also significantly reduced in MZsdc2 mutants. In zebrafish, cells that bud off of the ventral region of somites are thought to give rise to the reticular stromal cells of the caudal hematopoietic tissue (CHT) stem cell niche. In MZsdc2 mutants, these budding cells have abberant blebbing morphology associated with widespread apoptosis during induction of HSPCs and with changes in the vascularization and stromal cell structure of the CHT stem cell niche. This suggests that loss of sdc2 disrupts the earliest events of definitive hematopoiesis. Our findings of hematopoietic defects, nascent immune system alterations and inability to resolve infection in sdc2 mutants sets the stage for examining the roles of HSPG genes in a wide range of hematopoietic and immune defects in humans. Fn Fn DAPI DAPI J

Zebrafish Gdf3 (Dvr1) is a member of the TGFb superfamily of cell signaling ligands that includes Xenopus Vg1 and mammalian Gdf1/3. Surprisingly, engineered homozygous mutants in zebrafish have no apparent phenotype. Elimination of Gdf3 in oocytes of maternal-zygotic mutants results in embryonic lethality that can be fully rescued with gdf3 RNA, demonstrating that Gdf3 is required only early in development, beyond which mutants are viable and fertile. Gdf3 mutants are refractory to Nodal ligands and Nodal repressor Lefty1. Signaling driven by TGFb ligand Activin and constitutively active receptors Alk4 and Alk2 remain intact in gdf3 mutants, indicating that Gdf3 functions at the same pathway step as Nodal. Targeting gdf3 and ndr2 RNA to specific lineages indicates that exogenous gdf3 is able to fully rescue mutants only when coexpressed with endogenous Nodal. Together, these findings demonstrate that Gdf3 is an essential cofactor of Nodal signaling during establishment of the embryonic axis.