Automated profiling of gene function during embryonic development.

Systematic functional profiling of the gene set that directs embryonic development is an important challenge. To tackle this challenge, we used 4D imaging of C. elegans embryogenesis to capture the effects of 500 gene knockdowns and developed an automated approach to compare developmental phenotypes. The automated approach quantifies features-including germ layer cell numbers, tissue position, and tissue shape-to generate temporal curves whose parameterization yields numerical phenotypic signatures. In conjunction with a new similarity metric that operates across phenotypic space, these signatures enabled the generation of ranked lists of genes predicted to have similar functions, accessible in the PhenoBank web portal, for ∼25% of essential development genes. The approach identified new gene and pathway relationships in cell fate specification and morphogenesis and highlighted the utilization of specialized energy generation pathways during embryogenesis. Collectively, the effort establishes the foundation for comprehensive analysis of the gene set that builds a multicellular organism.

HP1α is a chromatin crosslinker that controls nuclear and mitotic chromosome mechanics.

Chromatin, which consists of DNA and associated proteins, contains genetic information and is a mechanical component of the nucleus. Heterochromatic histone methylation controls nucleus and chromosome stiffness, but the contribution of heterochromatin protein HP1α (CBX5) is unknown. We used a novel HP1α auxin-inducible degron human cell line to rapidly degrade HP1α. Degradation did not alter transcription, local chromatin compaction, or histone methylation, but did decrease chromatin stiffness. Single-nucleus micromanipulation reveals that HP1α is essential to chromatin-based mechanics and maintains nuclear morphology, separate from histone methylation. Further experiments with dimerization-deficient HP1αI165E indicate that chromatin crosslinking via HP1α dimerization is critical, while polymer simulations demonstrate the importance of chromatin-chromatin crosslinkers in mechanics. In mitotic chromosomes, HP1α similarly bolsters stiffness while aiding in mitotic alignment and faithful segregation. HP1α is therefore a critical chromatin-crosslinking protein that provides mechanical strength to chromosomes and the nucleus throughout the cell cycle and supports cellular functions.

Micromanipulation of prophase I chromosomes from mouse spermatocytes reveals high stiffness and gel-like chromatin organization.

Meiosis produces four haploid cells after two successive divisions in sexually reproducing organisms. A critical event during meiosis is construction of the synaptonemal complex (SC), a large, protein-based bridge that physically links homologous chromosomes. The SC facilitates meiotic recombination, chromosome compaction, and the eventual separation of homologous chromosomes at metaphase I. We present experiments directly measuring physical properties of captured mammalian meiotic prophase I chromosomes. Mouse meiotic chromosomes are about ten-fold stiffer than somatic mitotic chromosomes, even for genetic mutants lacking SYCP1, the central element of the SC. Meiotic chromosomes dissolve when treated with nucleases, but only weaken when treated with proteases, suggesting that the SC is not rigidly connected, and that meiotic prophase I chromosomes are a gel meshwork of chromatin, similar to mitotic chromosomes. These results are consistent with a liquid- or liquid-crystal SC, but with SC-chromatin stiff enough to mechanically drive crossover interference.