Highlights 32 33 • Tbx20 plays a role in the development of non-image-forming pathways. 34 35 • Loss of Tbx20-expressing RGCs alters specific aspects of the pupil reflex arc. 36 37 • Previously unknown role for a specific subset of Tbx20-expressing RGCs in 38 modulating pupil size. 39 40 eTOC 41 Dhande et al. identify a novel genetic program that marks and is required for the 42 development of non-image-forming parallel visual pathways. Moreover, chemogenetic 43 activation of a specific retina-to-OPN pathway reveals a novel circuit element controlling 44 pupil size. These findings provide new insight into the mechanisms that establish defining 45 features of functionally specialized sensory pathways. 46 47space. We discovered that Tbx20 expression is mostly restricted to three RGC-types: M1 56 intrinsically photosensitive RGCs (ipRGCs), M2 ipRGCs and 'diving' RGCs. The axonal 57 projections of Tbx20-RGCs innervate non-image-forming brain centers including the 58 suprachiasmatic nucleus, the medial division of the posterior pretectal nucleus, and the 59 olivary pretectal nucleus (OPN), a principal station in the neural pathway for generating 60 the pupillary light reflex (PLR). Conditional deletion of Tbx20 resulted in reduced Tbx20-61 RGC axonal innervation of these targets and revealed a key role of these neurons in driving 62 specific phases of the PLR. Furthermore, chemogenetic activation uncovered a novel role 63 for a specific subset of Tbx20-RGCs in controlling pupil dilation. These data offer a new 64 understanding of the genetic and molecular mechanisms that establish specific, 65 behaviorally-relevant visual circuits. 66 67