Molecular evolution of HoxA13 and the multiple origins of limbless morphologies in amphibians and reptiles

Developmental processes and their results, morphological characters, are inherited through transmission of genes regulating development. While there is ample evidence that cis-regulatory elements tend to be modular, with sequence segments dedicated to different roles, the situation for proteins is less clear, being particularly complex for transcription factors with multiple functions. Some motifs mediating protein-protein interactions may be exclusive to particular developmental roles, but it is also possible that motifs are mostly shared among different processes. Here we focus on HoxA13, a protein essential for limb development. We asked whether the HoxA13 amino acid sequence evolved similarly in three limbless clades: Gymnophiona, Amphisbaenia and Serpentes. We explored variation in ω (dN/dS) using a maximum-likelihood framework and HoxA13sequences from 47 species. Comparisons of evolutionary models provided low ω global values and no evidence that HoxA13 experienced relaxed selection in limbless clades. Branch-site models failed to detect evidence for positive selection acting on any site along branches of Amphisbaena and Gymnophiona, while three sites were identified in Serpentes. Examination of alignments did not reveal consistent sequence differences between limbed and limbless species. We conclude that HoxA13 has no modules exclusive to limb development, which may be explained by its involvement in multiple developmental processes

Insights into Hox Protein Function from a Large Scale Combinatorial Analysis of Protein Domains

Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA), we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences

A zinc-finger fusion protein refines Gal4-defined neural circuits.

The analysis of behavior requires that the underlying neuronal circuits are identified and genetically isolated. In several major model species-most notably Drosophila-neurogeneticists identify and isolate neural circuits with a binary heterologous expression-control system: Gal4-UASG. One limitation of Gal4-UASG is that expression patterns are often too broad to map circuits precisely. To help refine the range of Gal4 lines, we developed an intersectional genetic AND operator. Interoperable with Gal4, the new system's key component is a fusion protein in which the DNA-binding domain of Gal4 has been replaced with a zinc finger domain with a different DNA-binding specificity. In combination with its cognate binding site (UASZ) the zinc-finger-replaced Gal4 ('Zal1') was functional as a standalone transcription factor. Zal1 transgenes also refined Gal4 expression ranges when combined with UASGZ, a hybrid upstream activation sequence. In this way, combining Gal4 and Zal1 drivers captured restricted cell sets compared with single drivers and improved genetic fidelity. This intersectional genetic AND operation presumably derives from the action of a heterodimeric transcription factor: Gal4-Zal1. Configurations of Zal1-UASZ and Zal1-Gal4-UASGZ are versatile tools for defining, refining, and manipulating targeted neural expression patterns with precision