The Drosophila homologue of CTIP1 (Bcl11a) and CTIP2 (Bcl11b) regulates neural stem cell temporal patterning.

In the developing nervous system, neural stem cells (NSCs) use temporal patterning to generate a wide variety of different neuronal subtypes. In Drosophila, the temporal transcription factors, Hunchback, Kruppel, Pdm and Castor, are sequentially expressed by NSCs to regulate temporal identity during neurogenesis. Here, we identify a new temporal transcription factor that regulates the transition from the Pdm to Castor temporal windows. This factor, which we call Chronophage (or 'time-eater'), is homologous to mammalian CTIP1 (Bcl11a) and CTIP2 (Bcl11b). We show that Chronophage binds upstream of the castor gene and regulates its expression. Consistent with Chronophage promoting a temporal switch, chronophage mutants generate an excess of Pdm-specified neurons and are delayed in generating neurons associated with the Castor temporal window. In addition to promoting the Pdm to Castor transition, Chronophage also represses the production of neurons generated during the earlier Hunchback and Kruppel temporal windows. Genetic interactions with Hunchback and Kruppel indicate that Chronophage regulates NSC competence to generate Hunchback- and Kruppel-specified neurons. Taken together, our results suggest that Chronophage has a conserved role in temporal patterning and neuronal subtype specification.

The developmental origin of heart size and shape differences in Astyanax mexicanus populations.

Regulation of heart size and shape is one of the least understood processes in developmental biology. We have for the first time analysed the hearts of Astyanax mexicanus and identified several differences in heart morphology between the surface (epigean morph) and cave-dwelling (troglomorph) morphs. Examination of the adult revealed that the troglomorph possesses a smaller heart with a rounder ventricle in comparison to the epigean morph. The size differences identified appear to arise early in development, as early as 24 h post-fertilisation (hpf), while shape differences begin to appear at 2 days post-fertilisation. The heart of the first-generation cross between the cave-dwelling and river-dwelling morph shows uncoupling of different phenotypes observed in the parental populations and indicates that the cardiac differences have become embedded in the genome during evolution. The differences in heart morphology are accompanied by functional changes between the two morphs, with the cave-dwelling morph exhibiting a slower heart rate than the river-dwelling morph. The identification of morphological and functional differences in the A. mexicanus heart could allow us to gain more insight into how such parameters are regulated during cardiac development, with potential relevance to cardiac pathologies in humans.

NanoDam identifies Homeobrain (ARX) and Scarecrow (NKX2.1) as conserved temporal factors in the Drosophila central brain and visual system.

Temporal patterning of neural progenitors is an evolutionarily conserved strategy for generating neuronal diversity. Type II neural stem cells in the Drosophila central brain produce transit-amplifying intermediate neural progenitors (INPs) that exhibit temporal patterning. However, the known temporal factors cannot account for the neuronal diversity in the adult brain. To search for missing factors, we developed NanoDam, which enables rapid genome-wide profiling of endogenously tagged proteins in vivo with a single genetic cross. Mapping the targets of known temporal transcription factors with NanoDam revealed that Homeobrain and Scarecrow (ARX and NKX2.1 orthologs) are also temporal factors. We show that Homeobrain and Scarecrow define middle-aged and late INP temporal windows and play a role in cellular longevity. Strikingly, Homeobrain and Scarecrow have conserved functions as temporal factors in the developing visual system. NanoDam enables rapid cell-type-specific genome-wide profiling with temporal resolution and is easily adapted for use in higher organisms.

In vivo, genome-wide profiling of endogenously tagged chromatin-binding proteins with spatial and temporal resolution using NanoDam in Drosophila.

NanoDam is a technique for genome-wide profiling of the binding targets of any endogenously tagged chromatin-binding protein in vivo, without the need for antibodies, crosslinking, or immunoprecipitation. Here, we explain the procedure for NanoDam experiments in Drosophila, starting from a genetic cross, to the generation of sequencing libraries and, finally, bioinformatic analysis. This protocol can be readily adapted for use in other model systems after simple modifications. For complete details on the use and execution of this protocol, please refer to Tang et al. (2022).