In Arthropods, development of the central nervous system relies heavily on the gene single-minded ( sim ; FlyBaseID: FBgn0004666) (Linne et al., 2012) . sim encodes a bHLH-PAS transcription factor that acts as a key regulator in the development of the ventral midline (Crews et al., 1988; Nambu et al., 1991) . In the fruit fly Drosophila melanogaster , sim is active throughout the whole of embryogenesis, with transcript first being detected at the cellular blastoderm stage and persisting until the end of embryo development (Thomas et al., 1988; Wheeler et al., 2006) . Most of the noncoding DNA of the sim locus has been assayed for regulatory activity, and enhancers have been defined for early embryonic, larval, and adult stages of development (Freer et al., 2011; Hong et al., 2013; Jenett et al., 2012; Kvon et al., 2014; Markstein et al., 2004; Pearson et al., 2016; Sandmann et al., 2007) . However, sim regulation is still not completely defined. For example, enhancers governing midline expression later in embryonic development have yet to be identified. Although much of the locus has already been characterized, we reasoned that a finer-scale mapping of specific regions might better resolve discrete regulatory activities and possibly discover unknown enhancers, improving our understanding of sim regulation.

In this study, we focus on the 3' end of D. melanogaster sim , where sequence comparison across 27 insects shows distinct blocks of high conservation ( Fig. 1A ). Using this conservation as a guide, we isolated three fragments— sim_3pA , sim_3pB , and sim_3pC —from the genome and characterized them by reporter gene assay in transgenic embryos. sim_3pA is negative in all embryonic tissues at all stages ( Fig. 1B ). sim_3pC shows limited activity in the central nervous system (CNS) of stage 16 embryos, but this expression is outside of the midline in regions ectopic to sim ( Fig. 1C ). Therefore, this element is likely not a true sim enhancer. Neither sim_3pC nor sim_3pA were explored further. sim_3pB , on the other hand, is active in the midline from the end of germband retraction (late stage 12) through at least stage 16, with the strongest signal at stages 13 and 14 ( Fig. 1D -F, arrowheads). sim_3pB also displays extensive ectopic activity throughout the medial portion of the CNS, starting earlier at approximately stage 10 and continuing into stage 16 ( Fig. 1E, arrows).

All known sim midline enhancers in D. melanogaster possess autoregulatory capability mediated by binding of Sim:Tango heterodimers to CNS Midline Elements (CMEs) (Pearson and Crews, 2014; Sonnenfeld et al., 1997; Wharton et al., 1994) . Three matches to the CME consensus sequence, RCGTG (Hong et al., 2013; Sonnenfeld et al., 1997) , are present in sim_3pB . However, mutation of these sites leads to only a modest reduction in reporter expression. Midline activity is slightly reduced, whereas background expression is mostly unchanged ( Fig. 1G, arrowheads). Careful quantitative analysis will be required to confirm whether this difference in activity is significant. Surprisingly, when introduced into a sim null environment, sim_3pB activity is strongly reduced in the entire CNS ( Fig. 1H ). These contradictory results suggest that sim_3pB may be both directly and indirectly dependent on Sim for activity, although we cannot rule out additional direct Sim autoregulatory binding through non-canonical sites.

During early stage 11 in D. melanogaster , the embryonic midline consists of the anterior and posterior midline glia (AMG and PMG, respectively), the median neuroblast (MNB), and midline precursors (MP1, MP3, MP4, MP5, and MP6) (Wheeler et al., 2006) . Toward the end of stage 11, the MP3 precursors divide into H-cell and H-cell sib while MP4-6 each give rise to one Ventral Unpaired Median motoneuron and one Ventral Unpaired Median interneuron (mVUM and iVUM, respectively). Each cell type expresses a specific set of genes that allows it to be uniquely identified (Wheeler et al., 2006) . By co-staining sim_3pB embryos for the reporter gene and one of four known midline markers—Slit, Mab 22C10, Engrailed, and Nubbin—we were able to identify the midline cells in which sim_3pB is active. In stage 13/14 embryos, sim_3pB activity did not coincide with either Slit or Nubbin, which rules out midline glia (AMG/PMG), MP1 neurons, and H-cell/H-cell sib ( Fig. 1I -K). Co-localization with 22C10 in stage 16 embryos suggests activity in the mVUMs ( Fig. 1L ), which is confirmed by sim_3pB staining relative to Engrailed-positive cells at stage 13 ( Fig. 1M -O, S). Additionally, sporadic activity is observed in the iVUMs ( Fig. 1P -R, S). sim itself is not expressed in the mVUMs at stage 13 (Wheeler et al., 2006) , but is it possible that activity begins weakly prior to stage 13 (in the MP4-6 precursor cells) and is already fading by the time they have divided. This could also explain the inconsistent activity seen in the iVUMs.

sim_3pB shares several characteristics with a recently discovered sim midline enhancer in the mosquito Aedes aegypti , sim_5P3 (Schember et al., 2024) . It is also dissimilar in key ways. Although both elements begin activity later than other known sim midline primordium enhancers, sim_5P3 becomes active at the end of stage 9 and sim_3pB not until the end of stage 12 (with respect to its midline activity). Neither are active in the full set of midline cells and are instead restricted to the MP4 lineage, but whereas sim_5P3 expression includes all the descendants of the initial MP4 equivalence group (PMG, MNB, the MP4-6 precursors and their VUM progeny), sim_3pB appears limited to the VUMs. Additionally, while neither element is directly autoregulated, they differ in their dependance on Sim protein. The sim_5P3 sequence contains no CME sites and is fully functional in a sim null background. On the other hand, sim_3pB remains weakly active with the loss of three CME sites yet is not functional in a sim null background. Taken together, despite interesting similarities between these two regulatory elements, it cannot necessarily be concluded that sim_3pB is a functional counterpart to A. aegypti sim_5P3 .

sim_3pB activity, though it includes sim positive cells, also extends to regions where sim is not expressed in the embryo. The sequence we isolated could be lacking repressors necessary for midline-only activity. Alternatively, sim_3pB may not act as an enhancer in its native context. Kalay et al. (2019) introduced the concept of “cryptic enhancers”—sequences that have the ability to drive gene expression in isolation but are repressed in their endogenous chromosomal environment. A cryptic enhancer might thus be located within a larger region that is negative for regulatory activity (Kalay et al., 2019) . Such may be the case with sim_3pB . In two separate experiments, regions containing and extending beyond the sim_3pB sequence failed to drive reporter expression: sim_I2.6 , which overlaps all but the first ~70bp of sim_3pB (Freer et al., 2011) , and VT40848 , which contains the entirety of the sim_3pB sequence (Kvon et al., 2014) , were both reported to be inactive ( Fig. 1A ). Taking this into consideration, it is possible that sim_3pB is a cryptic enhancer for sim . An intriguing alternative possibility is that sim_3pB acts in its native location as a silencer element to prevent sim transcription in the mVUMs and cells outside of the midline, but manifests as an enhancer in an out-of-context reporter assay. This could explain the extensive activity observed in sim- negative cells. Further investigation will be necessary to test these various possibilities. Nonetheless, currently sim_3pB stands as the only identified embryonic D. melanogaster sim regulatory element with midline activity beginning at or after the late midline primordium stage. Continuing to explore the sim locus may reveal more enhancers governing midline activity at these later stages, providing a clearer picture of how sim is regulated throughout embryonic development in D. melanogaster and in Arthropods as a whole.

Reporter Constructs

sim_3pA and sim_3pC were isolated via PCR on genomic DNA from D. melanogaster strain OregonR (OrgR) and cloned into the pattBnucGFP vector (Kazemian et al., 2011) . sim_3pB was synthesized as a gBlock (IDT, Coralville IA) and cloned into the placZattB vector (Bischof et al., 2013) using the NEBuilder HiFi DNA Assembly kit (New England Biolabs, Beverley, MA). To generate the sim_3pBmut line, MacVector (Apex, NC) was used to scan for Sim:Tgo binding sequences ACGTG (Sonnenfeld et al., 1997) and DDRCGTG (Hong et al., 2013) . Three matches were found, and each site was mutated to GGATCC, as described by Wharton et al. (1994). The sim_3pBmut fragment was synthesized as a gBlock (IDT, Coralville IA) and inserted into placZattB using the NEBuilder HiFi DNA Assembly kit. Transgenic flies were generated by Rainbow Transgenic Flies (Camarillo, CA) by phiC31 recombination using line attP2 8622.

Sequence conservation

Sequence conservation was obtained from the UCSC Genome Browser (Raney et al., 2024) using the “phyloP 27 species” conservation track. Data in this track are obtained using phyloP (Pollard et al., 2010) and the following species: Drosophila melanogaster, D. simulans, D. sechellia, D. yakuba, D. erecta, D. biarmipes, D. suzukii, D. ananassae, D. bipectinata, D. eugracilis, D. elegans, D. kikkawai, D. takahashii, D. rhopaloa, D. ficusphila, D. pseudoobscura, D. persimilis, D. miranda, D. willistoni, D. virilis, D. mojavensis, D. albomicans, D. grimshawi, Musca domestica, Anopheles gambiae, Apis mellifera, Tribolium castaneum.

Immunohistochemistry

Immunohistochemistry on D. melanogaster embryos was conducted using standard methods. Staining was done with primary antibodies mouse anti-β-galactosidase (1:500, Promega Z378) and rabbit anti-GFP (1:10000, Ab-cam ab290) then visualized using the Vectastain® Standard ABC kit (Vector Laboratories). Imaging was performed using a Zeiss Axioskop equipped with a Jenoptix Arctur camera. For fluorescent staining, primary antibodies were mouse anti-Slit C555.6D (1:10; Developmental Studies Hybridoma Bank), mouse anti-Nubbin 2D4 (1:10, DSHB), mouse anti-Engrailed 4D9 (DSHB), and Mab 22C10 (1:20, DSHB). Secondary antibodies were anti-mouse Alexa Fluor 488 (1:250, Molecular Probes, A11029) and anti-rabbit Alexa Fluor 647 (1:500, Molecular Probes, A21245). Fluorescent staining was visualized using a Leica SP8 confocal microscope.

Test for autoregulation

The sim_3pB construct was recombined onto a sim null chromosome ( sim ² , Bloomington Stock Center stock #2055) and balanced with a TM3 balancer containing ftz-lacZ . Loss of sim function was confirmed by complementation testing with a sim- null deficiency ( Df(3R)Excel6167/TM6B, Bloomington Stock Center stock #7646).