Identifying Roles of Dlx Paralogs in Zebrafish Development

Abstract

The Dlx homeobox genes are conserved among vertebrates and encode homeodomain transcription factors involved in development of multiple tissues. These genes are organized as bigene clusters that are partially regulated by conserved cis-regulatory elements. These regulatory elements contain binding sequences for Dlx proteins suggesting they are sites of cross-regulation. The Dlx genes are important in the differentiation and migration of GABAergic neurons in the mouse brain. Additionally, Dlx are also important in the development of craniofacial tissues by providing patterning information to neural crest cells. However, deletion of single and compound Dlx mutants in the mouse lead to early lethality, precluding detailed analysis on the role of individual Dlx during development. In zebrafish, these genes are also involved in the development of craniofacial tissues and are co-expressed in brain areas containing GABAergic neurons. Therefore, the role of four paralogs dlx1a, dlx2a, dlx5a, and dlx6a in the nervous system and craniofacial development were investigated in this project. Two additional mutants, inter56 where the enhancers of dlx5a/dlx6a are deleted, and dlx5i6 where the entire dlx5a/dlx6a locus is deleted were created for further analysis. These mutants were generated by CRISPR-Cas9 genome editing and all homozygotes were found to be viable and fertile as adults. They were also capable of normal feeding indicating no abnormalities in jaw function. Whole mount in situ hybridization for dlx genes in dlx mutants demonstrated dlx can cross-regulate in zebrafish. Relationships observed in the mouse were identified in zebrafish however some differences were also elucidated, such as the presence of dlx5a expression in dlx2a mutants until 3dpf. During craniofacial development, loss of dlx genes affected the size of ventral cartilage structures. In particular, the Meckel’s cartilage (MC) of dlx5a-/- and dlx5i6-/- larvae had consistent morphological defects. The expression of chondrogenic markers and proliferation was also altered in dlx5a-/- and dlx5i6-/- mutants at 2- 3dpf. Importantly, the size and shape of the MC may be due to abnormal expression of the noncanonical Wnt wnt5b during morphogenesis. These mutants exhibited more random distribution of microtubule organizing center in MC chondrocytes indicative of abnormal non-canonical Wnt signaling. To address consequences of dlx loss in the nervous system of zebrafish, the distribution and number of calretinin (CR) neurons, a type of GABAergic neuron were investigated. There was a trend towards fewer CR+ neurons in the optic tectum of dlx5a-/- and dlx5i6-/- mutant adults suggesting possible defects in integrating visual, auditory and vestibular information. These mutants also have abnormal swimming behaviour at around 2 months old characterized by frequent turning and spinning during a swim bout without changes in inactivity duration or total distance travelled. The number of neuromasts, a type of sensory organ that provides positional information, was reduced in dlx5a mutants and have reduced regeneration. The maintenance and regeneration of neuromasts depend on Schwann cells, a type of neural crest derivative. Defects in craniofacial development and neuromast biology suggest dlx5a may have a general role in neural crest cell biology beyond craniofacial development.