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Rhythmic and sequential segmentation of the growing vertebrate body relies on the segmentation clock, a multi-cellular oscillating genetic network. The clock is visible as tissue-level kinematic waves of gene expression that travel through the presomitic mesoderm (PSM) and arrest at the position of each forming segment. Here, we test how this hallmark wave pattern is driven by culturing single maturing PSM cells. We compare their cell-autonomous oscillatory and arrest dynamics to those we observe in the embryo at cellular resolution, finding similarity in the relative slowing of oscillations and arrest in concert with differentiation. This shows that cell-extrinsic signals are not required by the cells to instruct the developmental program underlying the wave pattern. We show that a cell-autonomous timing activity initiates during cell exit from the tailbud, then runs down in the anterior-ward cell flow in the PSM, thereby using elapsed time to provide positional information to the clock. Exogenous FGF lengthens the duration of the cell-intrinsic timer, indicating extrinsic factors in the embryo may regulate the segmentation clock via the timer. In sum, our work suggests that a noisy cell-autonomous, intrinsic timer drives the slowing and arrest of oscillations underlying the wave pattern, while extrinsic factors in the embryo tune this timer's duration and precision. This is a new insight into the balance of cell-intrinsic and -extrinsic mechanisms driving tissue patterning in development., Competing Interests: LR, AB, GV, SN, RD, DS, AO No competing interests declared, PS Co-founder of Viventis Microscopy, (© 2024, Rohde, Bercowsky-Rama et al.)

Gastrulation is a morphogenetic process that results in the formation of the embryonic germ layers. Here we detail the major cell movements that occur during zebrafish gastrulation: epiboly, internalization, and convergent extension. Although gastrulation is known to be regulated by signaling pathways such as the Wnt/planar cell polarity pathway, many questions remain about the underlying molecular and cellular mechanisms. Key factors that may play a role in gastrulation cell movements are cell adhesion and cytoskeletal rearrangement. In addition, some of the driving force for gastrulation may derive from tissue interactions such as those described between the enveloping layer and the yolk syncytial layer. Future exploration of gastrulation mechanisms relies on the development of sensitive and quantitative techniques to characterize embryonic germ-layer properties.

The first morphological sign of vertebrate postcranial body segmentation is the sequential production from posterior paraxial mesoderm of blocks of cells termed somites. Each of these embryonic structures is polarized along the anterior/posterior axis, a subdivision first distinguished by marker gene expression restricted to rostral or caudal territories of forming somites. To better understand the generation of segment polarity in vertebrates, we have studied the zebrafish mutant fused somites (fss), because its paraxial mesoderm lacks segment polarity. Previously examined markers of caudal half-segment identity are widely expressed, whereas markers of rostral identity are either missing or dramatically down-regulated, suggesting that the paraxial mesoderm of the fss mutant embryo is profoundly caudalized. These findings gave rise to a model for the formation of segment polarity in the zebrafish in which caudal is the default identity for paraxial mesoderm, upon which is patterned rostral identity in an fss-dependent manner. In contrast to this scheme, the caudal marker gene ephrinA1 was recently shown to be down-regulated in fss embryos. We now show that notch5, another caudal identity marker and a component of the Delta/Notch signaling system, is not expressed in the paraxial mesoderm of early segmentation stage fss embryos. We use cell transplantation to create genetic mosaics between fss and wild-type embryos in order to assay the requirement for fss function in notch5 expression. In contrast to the expression of rostral markers, which have a cell-autonomous requirement for fss, expression of notch5 is induced in fss cells at short range by nearby wild-type cells, indicating a cell-non-autonomous requirement for fss function in this process. These new data suggest that segment polarity is created in a three-step process in which cells that have assumed a rostral identity must subsequently communicate with their partially caudalized neighbors in order to induce the fully caudalized state.

Zebrafish embryonic red blood cells (RBCs) develop in trunk intermediate mesoderm (IM), and early macrophages develop in the head, suggesting that local microenvironmental cues regulate differentiation of these two blood lineages. spadetail (spt) mutant embryos, which lack trunk paraxial mesoderm (PM) due to a cell-autonomous defect in tbx16, fail to produce embryonic RBCs but retain head macrophage development. In spt mutants, initial hematopoietic gene expression is absent in trunk IM, although endothelial and pronephric expression is retained, suggesting that early blood progenitor development is specifically disrupted. Using cell transplantation, we reveal that spt is required cell autonomously for early hematopoietic gene expression in trunk IM. Further, we uncover an interaction between embryonic trunk PM and blood progenitors that is essential for RBC development. Importantly, our data identify a hematopoietic microenvironment that allows embryonic RBC production in the zebrafish.

The T-box genes Tbx4 and Tbx5 have been shown to have key functions in the specification of the identity of the vertebrate forelimb (Tbx5) and hindlimb (Tbx4). Here we show that in zebrafish, Tbx5 has an additional early function that precedes the formation of the limb bud itself. Functional knockdown of zebrafish tbx5 through the use of an antisense oligonucleotide resulted in a failure to initiate fin bud formation, leading to the complete loss of pectoral fins. The function of the tbx5 gene in the development of zebrafish forelimbs seems to involve the directed migration of individual lateral-plate mesodermal cells into the future limb-bud-producing region. The primary defect seen in the tbx5-knockdown phenotype is similar to the primary defects described in known T-box-gene mutants such as the spadetail mutant of zebrafish and the Brachyury mutant of the mouse, which both similarly exhibit an altered migration of mesodermal cells. A common function for many of the T-box genes might therefore be in mediating the proper migration and/or changes in adhesive properties of early embryonic cells.