In this dataset we provide the original z-stacks of anti-serotonin stainings used in the article by Coll-Tan? and colleagues entitled ?The CHD8/CHD7/Kismet family links blood-brain barrier glia and serotonin to ASD-associated sleep defects? (doi: 10.1126/sciadv.abe2626).In this paper we show that Kismet-related sleep disturbances are caused by high serotonin during development, paralleling a well-established but genetically unsolved autism endophenotype.In order to investigate serotonin levels during development, we performed anti-serotonin fluorescence immunohistochemistry and quantitative imaging (via photon counting, see Materials and Methods), in pan-glial and SPG-specific kismet knockdown wandering third instar larvae. We focused on the projections of serotonergic neurons of the subesophagal ganglion that innervate the proventriculus and anterior midgut of the larval digestive tract. In controls, serotonin was visible in the proventricular nerve and at the level of proventricular innervations, but absent or hard to discern in the distal projections that innervate the anterior midgut. In contrast, loss of glial Kismet led to elevated serotonin signal, with serotonergic midgut innervations strongly labeled. As the innervated tissues appeared to show slightly higher levels of staining too, we quantified total signal intensities of ?-serotonin signal across the proventriculus and in the anterior midgut, containing both the serotonergic terminals and the innervated tissues. We observed significantly increased serotonin at the midgut region of pan-glial kismet knockdown larvae. Similarly, serotonin was significantly increased at the level of the proventriculus and midgut of SPG-specific kismet knockdown larvae. Together, these results demonstrate that Kismet loss in glia leads to increased serotonin during development.