Drosophila melanogaster head development represents a valuable process to study the developmental control of various organs, such as the antennae, the dorsal ocelli and the compound eyes from a common precursor, the eye-antennal imaginal disc. While the gene regulatory network underlying compound eye development has been extensively studied, the key transcription factors regulating the formation of other head structures from the same imaginal disc are largely unknown. We obtained the developmental transcriptome of the eye-antennal discs covering late patterning processes at the late 2nd larval instar stage to the onset and progression of differentiation at the end of larval development. We revealed the expression profiles of all genes expressed during eye-antennal disc development and we determined temporally co-expressed genes by hierarchical clustering. Since co-expressed genes may be regulated by common transcriptional regulators, we combined our transcriptome dataset with publicly available ChIP-seq data to identify central transcription factors that co-regulate genes during head development. Besides the identification of already known and well-described transcription factors, we show that the transcription factor Hunchback (Hb) regulates a significant number of genes that are expressed during late differentiation stages. We confirm that hb is expressed in two polyploid subperineurial glia cells (carpet cells) and a thorough functional analysis shows that loss of Hb function results in a loss of carpet cells in the eye-antennal disc. Additionally, we provide for the first time functional data indicating that carpet cells are an integral part of the blood-brain barrier. Eventually, we combined our expression data with a de novo Hb motif search to reveal stage specific putative target genes of which we find a significant number indeed expressed in carpet cells.

The size and shape of organs is tightly controlled to achieve optimal function. Natural morphological variations often represent functional adaptations to an ever-changing environment. For instance, variation in head morphology is pervasive in insects and the underlying molecular basis is starting to be revealed in the Drosophila genus for species of the melanogaster group. However, it remains unclear whether similar diversifications are governed by similar or different molecular mechanisms over longer timescales. To address this issue, we used species of the virilis phylad because they have been diverging from D. melanogaster for at least 40 million years. Our comprehensive morphological survey revealed remarkable differences in eye size and head shape among these species with D. novamexicana having the smallest eyes and southern D. americana populations having the largest eyes. We show that the genetic architecture underlying eye size variation is complex with multiple associated genetic variants located on most chromosomes. Our genome wide association study (GWAS) strongly suggests that some of the putative causative variants are associated with the presence of inversions. Indeed, northern populations of D. americana share derived inversions with D. novamexicana and they show smaller eyes compared to southern ones . Intriguingly, we observed a significant enrichment of genes involved in eye development on the 4th chromosome after intersecting chromosomal regions associated with phenotypic differences with those showing high differentiation among D. americana populations. We propose that variants associated with chromosomal inversions contribute to both intra- and interspecific variation in eye size among species of the virilis phylad.

35The size and shape of organs is tightly controlled to achieve optimal function. Natural 36 morphological variations often represent functional adaptations to an ever-changing 37 environment. For instance, variation in head morphology is pervasive in insects and the 38 underlying molecular basis is starting to be revealed in the Drosophila genus for species of the 39 melanogaster group. However, it remains unclear whether similar diversifications are governed 40 by similar or different molecular mechanisms over longer timescales. To address this issue, we 41 used species of the virilis phylad because they have been diverging from D. melanogaster for 42 at least 40 million years. Our comprehensive morphological survey revealed remarkable 43 differences in eye size and head shape among these species with D. novamexicana having the 44 smallest eyes and southern D. americana populations having the largest eyes. We show that 45 the genetic architecture underlying eye size variation is complex with multiple associated 46 genetic variants located on most chromosomes. Our genome wide association study (GWAS) 47 strongly suggests that some of the putative causative variants are associated with the presence 48 of inversions. Indeed, northern populations of D. americana share derived inversions with D. 49 novamexicana and they show smaller eyes compared to southern ones. Intriguingly, we 50 observed a significant enrichment of genes involved in eye development on the 4 th chromosome 51 after intersecting chromosomal regions associated with phenotypic differences with those 52 showing high differentiation among D. americana populations. We propose that variants 53 associated with chromosomal inversions contribute to both intra-and inter-specific variation 54 in eye size among species of the virilis phylad.55 56 Quantitative genetics approaches have revealed multiple loci associated with variation 83 in eye size between D. simulans and D. mauritiana supporting the complex genetic architecture 84 of this trait (Arif et al. 2013). Similar observations were made for intra-specific variation in D. 85 melanogaster (Norry and Gomez 2017; Ramaekers et al. 2019) and D. simulans (Gaspar et al. 86 2020). However, Ramaekers et al. (2019) have shown that a single mutation affecting the 87 regulation of the eyeless/Pax6 gene can explain up to 50% of variation in eye size between two 88 D. melanogaster strains. Although, the genetic architecture underlying eye size variation is 89 starting to be revealed for species of the melanogaster group, it remains unclear whether similar 90 independent morphological diversifications identified in Drosophila (Norry et al. 2000; Keesey 91 et al. 2019) share the same molecular basis over longer timescales. 92 Chromosomal inversions are an interesting genetic variant because suppression of 93 recombination is thought to maintain linkage of favorable alleles which are protected from 94 immigrant alleles carrying variants which decrease fitness (Kirkpatrick and Barton 2006; 95 Kirkpatrick 2010). Therefore...

Background Recent technological advances opened the opportunity to simultaneously study gene expression for thousands of individual cells on a genome-wide scale. The experimental accessibility of such single-cell RNA sequencing (scRNAseq) approaches allowed gaining insights into the cell type composition of heterogeneous tissue samples of animal model systems and emerging models alike. A major prerequisite for a successful application of the method is the dissociation of complex tissues into individual cells, which often requires large amounts of input material and harsh mechanical, chemical and temperature conditions. However, the availability of tissue material may be limited for small animals, specific organs, certain developmental stages or if samples need to be acquired from collected specimens. Therefore, we evaluated different dissociation protocols to obtain single cells from small tissue samples of Drosophila melanogaster eye-antennal imaginal discs. Results We show that a combination of mechanical and chemical dissociation resulted in sufficient high-quality cells. As an alternative, we tested protocols for the isolation of single nuclei, which turned out to be highly efficient for fresh and frozen tissue samples. Eventually, we performed scRNAseq and single-nuclei RNA sequencing (snRNAseq) to show that the best protocols for both methods successfully identified relevant cell types. At the same time, snRNAseq resulted in less artificial gene expression that is caused by rather harsh dissociation conditions needed to obtain single cells for scRNAseq. A direct comparison of scRNAseq and snRNAseq data revealed that both datasets share biologically relevant genes among the most variable genes, and we showed differences in the relative contribution of the two approaches to identified cell types. Conclusion We present two dissociation protocols that allow isolating single cells and single nuclei, respectively, from low input material. Both protocols resulted in extraction of high-quality RNA for subsequent scRNAseq or snRNAseq applications. If tissue availability is limited, we recommend the snRNAseq procedure of fresh or frozen tissue samples as it is perfectly suited to obtain thorough insights into cellular diversity of complex tissue.

The development of different cell types must be tightly coordinated in different organs. The developing head of Drosophila melanogaster represents an excellent model to study the molecular mechanisms underlying this coordination because the eye-antennal imaginal discs contain the organ anlagen of nearly all adult head structures, such as the compound eyes or the antennae. We studied the genome wide gene expression dynamics during eye-antennal disc development in D. melanogaster to identify new central regulators of the underlying gene regulatory network. Expression based gene clustering and transcription factor motif enrichment analyses revealed a central regulatory role of the transcription factor Hunchback (Hb). We confirmed that hb is expressed in two polyploid retinal subperineurial glia cells (carpet cells). Our functional analysis shows that Hb is necessary for carpet cell development and loss of Hb function results in abnormal glia cell migration and photoreceptor axon guidance patterns. Additionally, we show for the first time that the carpet cells are an integral part of the blood-brain barrier.

Background Recent technological advances opened the opportunity to simultaneously study gene expression for thousands of individual cells on a genome-wide scale. The experimental accessibility of such single-cell RNA sequencing (scRNAseq) approaches already allowed gaining insights into the cell type composition of heterogeneous tissue samples of animal model systems and emerging models alike. A major prerequisite for a successful application of the method is the dissociation of complex tissue into individual cells, which often requires large amounts of input material and harsh mechanical, chemical and temperature conditions. However, the availability of tissue material may be limited for small animals, specific organs, certain developmental stages or if samples need to be acquired from collected specimens. Therefore, we evaluated different dissociation protocols to obtain single-cells from small tissue samples of Drosophila melanogaster eye-antennal imaginal discs. ResultsWe show that a combination of mechanical and chemical dissociation resulted in sufficient high-quality cells. As an alternative, we tested protocols for the isolation of single nuclei, which turned out to be highly efficient for fresh and for frozen tissue samples. Eventually, we performed scRNAseq and single-nuclei RNA sequencing (snRNAseq) to show that the best protocols for both methods successfully identified relevant cell types. However, snRNAseq resulted in less artificial gene expression that is caused by rather harsh dissociation conditions needed to obtain single cells for scRNAseq. Conclusion We present two dissociation protocols that allow isolating single cells and single nuclei, respectively, from low input material. Both protocols resulted in extraction of high-quality RNA for subsequent scRNAseq or snRNAseq applications. If tissue availability is limited, we recommend the snRNAseq procedure of fresh or frozen tissue samples as it is perfectly suited to obtain thorough insights into cellular diversity of complex tissue.