Thyroid hormone regulates proximodistal patterning in fin rays.

Processes that regulate size and patterning along an axis must be highly integrated to generate robust shapes; relative changes in these processes underlie both congenital disease and evolutionary change. Fin length mutants in zebrafish have provided considerable insight into the pathways regulating fin size, yet signals underlying patterning have remained less clear. The bony rays of the fins possess distinct patterning along the proximodistal axis, reflected in the location of ray bifurcations and the lengths of ray segments, which show progressive shortening along the axis. Here, we show that thyroid hormone (TH) regulates aspects of proximodistal patterning of the caudal fin rays, regardless of fin size. TH promotes distal gene expression patterns, coordinating ray bifurcations and segment shortening with skeletal outgrowth along the proximodistal axis. This distalizing role for TH is conserved between development and regeneration, in all fins (paired and medial), and between Danio species as well as distantly related medaka. During regenerative outgrowth, TH acutely induces Shh-mediated skeletal bifurcation. Zebrafish have multiple nuclear TH receptors, and we found that unliganded Thrab-but not Thraa or Thrb-inhibits the formation of distal features. Broadly, these results demonstrate that proximodistal morphology is regulated independently from size-instructive signals. Modulating proximodistal patterning relative to size-either through changes to TH metabolism or other hormone-independent pathways-can shift skeletal patterning in ways that recapitulate aspects of fin ray diversity found in nature.

Growth patterns of caudal fin rays are informed by both external signals from the regenerating organ and remembered identity autonomous to the local tissue.

Regenerating tissues must remember or interpret their spatial position, using this information to restore original size and patterning. The external skeleton of the zebrafish caudal fin is composed of 18 rays; after any portion of the fin is amputated, position-dependent regenerative growth restores each ray to its original length. We tested for transcriptional differences during regeneration of proximal versus distal tissues and identified 489 genes that differed in proximodistal expression. Thyroid hormone directs multiple aspects of ray patterning along the proximodistal axis, and we identified 364 transcripts showing a proximodistal expression pattern that was dependent on thyroid hormone context. To test what aspects of ray positional identity are directed by extrinsic environental cues versus remembered identity autonomous to the tissue, we transplanted distal portions of rays to proximal environments and evaluated regeneration within the new location. Native regenerating proximal tissue showed robust expression of scpp7, a transcript with thyroid-regulated proximal enrichment; in contrast, regenerating rays originating from transplanted distal tissue showed reduced (distal-like) expression during outgrowth. These distal-to-proximal transplants regenerated far beyond the length of the graft itself, indicating that cues from the proximal environment promoted additional growth. Nonetheless, these transplants initiated regeneration at a much slower rate compared to controls, suggesting memory of distal identity was retained by the transplanted tissue. This early growth retardation caused rays that originated from transplants to grow noticeably shorter than neighboring native rays. While several aspects of fin ray morphology (bifurcation, segment length) were found to be determined by the environment, we found that both regeneration speed and ray length are remembered autonomously by tissues, and that persist through multiple rounds of amputation and regeneration.

Developmental effector gene regulation: Multiplexed strategies for functional analysis.

The staggering complexity of the genome controls for developmental processes is revealed through massively parallel cis-regulatory analysis using new methods of perturbation and readout. The choice of combinations of these new methods is tailored to the system, question and resources at hand. Our focus is on issues that include the necessity or sufficiency of given cis-regulatory modules, cis-regulatory function in the normal spatial genomic context, and easily accessible high throughput and multiplexed analysis methods. In the sea urchin embryonic model, recombineered BACs offer new opportunities for consecutive modes of cis-regulatory analyses that answer these requirements, as we here demonstrate on a diverse suite of previously unstudied sea urchin effector genes expressed in skeletogenic cells. Positively active cis-regulatory modules were located in single Nanostring experiments per BAC containing the gene of interest, by application of our previously reported "barcode" tag vectors of which> 100 can be analyzed at one time. Computational analysis of DNA sequences that drive expression, based on the known skeletogenic regulatory state, then permitted effective identification of functional target site clusters. Deletion of these sub-regions from the parent BACs revealed module necessity, as simultaneous tests of the same regions in short constructs revealed sufficiency. Predicted functional inputs were then confirmed by site mutations, all generated and tested in multiplex formats. There emerged the simple conclusion that each effector gene utilizes a small subset of inputs from the skeletogenic GRN. These inputs may function to only adjust expression levels or in some cases necessary for expression. Since we know the GRN architecture upstream of the effector genes, we could then conceptually isolate and compare the wiring of the effector gene driver sub-circuits and identify the inputs whose removal abolish expression.

Growth patterns of caudal fin rays are informed by both external signals from the regenerating organ and remembered identity autonomous to the local tissue.

Regenerating tissues must remember or interpret their spatial position, using this information to restore original size and patterning. The external skeleton of the zebrafish caudal fin is composed of 18 rays; after any portion of the fin is amputated, position-dependent regenerative growth restores each ray to its original length. We tested for transcriptional differences during regeneration of proximal versus distal tissues and identified 489 genes that differed in proximodistal expression. Thyroid hormone directs multiple aspects of ray patterning along the proximodistal axis, and we identified 364 transcripts showing a proximodistal expression pattern that was dependent on thyroid hormone context. To test what aspects of ray positional identity are directed by extrinsic cues versus remembered identity autonomous to the tissue itself, we transplanted distal portions of rays to proximal environments and evaluated regeneration within the new location. While neighboring proximal tissue showed robust expression of scpp7, a transcript with thyroid-regulated proximal enrichment, regenerating rays originating from transplanted distal tissue showed reduced (distal-like) expression during outgrowth. These distal-to-proximal transplants regenerated far beyond the length of the graft itself, indicating that cues from the proximal environment promoted additional growth. Nonetheless, these transplants initially regenerated at a much slower rate compared to controls, suggesting memory of distal identity was retained by the transplanted tissue. This early growth retardation caused rays that originated from transplants to become noticeably shorter than their native neighboring rays. While several aspects of fin ray morphology (bifurcation, segment length) were found to be determined by the environment, regeneration speed and ray length are remembered autonomously by tissues, persisting across multiple rounds of amputation and regeneration.

Discovery of Isoxazole Amides as Potent and Selective SMYD3 Inhibitors.

We report herein the discovery of isoxazole amides as potent and selective SET and MYND Domain-Containing Protein 3 (SMYD3) inhibitors. Elucidation of the structure-activity relationship of the high-throughput screening (HTS) lead compound 1 provided potent and selective SMYD3 inhibitors. The SAR optimization, cocrystal structures of small molecules with SMYD3, and mode of inhibition (MOI) characterization of compounds are described. The synthesis and biological and pharmacokinetic profiles of compounds are also presented.