In contrast to the DP, where distinction between trunk and appendage occurs later in imaginal development (see below), the VP is organized in gene expression domains that subdivide the primordia in different territories (
Figure 1C) [
16,
56,
57,
58]. The development of the VP requires the function of
Dll and of the two paralogous genes
btd and
Sp1. Btd and Sp1 are members of the highly conserved Sp family of transcription factors required for appendage formation in vertebrates and invertebrates [
30,
59,
60,
61,
62,
63].
btd/Sp1 are expressed in the embryonic progenitors of the leg imaginal discs starting around stage 10-11 [
29,
30,
54,
64]. As it is the case with
Dll,
btd is activated by Wg and repressed dorsally and in the abdominal segments by Dpp and Ubx, respectively [
30]. At stage 11,
btd,
Sp1 and
Dll (
Dll-304) are activated in parallel in the TP and are genetically independent of each other. Importantly, as mentioned before, these TP cells could contribute to the DP and to the entire VP. Some hours later, at stage 14, VP and DP are already separated and the activity of the
Dll-304 CRM decays. At this time point,
Dll expression is controlled by two
Dll CRMs with mutually exclusive patterns at the VP, the leg trigger (
Dll-LT) and the Keilin organ (
Dll-KO) elements (
Figure 1C) [
16]. The progressive refinement of the appendage primordium developmental potential is reflected by the
cis-architecture of the
Dll gene [
14,
16,
65]. At the end of embryogenesis, the VP is genetically subdivided in domains with different cell fates that correspond to the P-D subdivision of the arthropod leg proposed by Robert E. Snodgrass (see below) [
66,
67] (
Figure 1C). VP cells that express
esg but do not activate
Dll will form the coxopodite, the most proximal and unsegmented domain of the appendage that forms as an outgrowth of the bodywall, including the coxa. VP cells co-expressing
esg and
Dll (through the
Dll-LT CRM) will form the telopodite or the leg proper, which includes all the distal leg segments that are articulated. Additionally, cells at the center of the VP that are
esg negative and Dll positive (
Dll-KO) are fated to form a larval mechanosensory structure that shares a common lineage with the leg disc called the Keilin’s organ (KO) [
16,
67]. While
Dll-KO cells do not contribute to the leg imaginal disc, the progeny of
Dll-LT activating cells form the entire telopodite. Consistently, in
Dll mutants the telopodite is lost while the coxopodite is present [
8,
16,
68,
69].
Dll-LT is positively regulated by Wg and Dpp and repressed in the center of the VP by members of the
achaete–scute complex (ASC) [
14,
16,
67]. At the same time, Dll and ASC positively regulate the
Dll-KO CRM restricting its activity to the VP [
16]. At this stage
btd and
Sp1 act upstream of
Dll and only the elimination of both genes suppresses
Dll expression and the activity of the
Dll-LT and
Dll-KO enhancers [
14,
16,
54]. Initially, some studies proposed that the two related homeobox genes Homothorax (Hth) and nuclear Extradenticle (Exd) were markers, along with
esg, of coxopodite fate [
33,
40,
41,
70,
71]. However, a reevaluation of the expression of
hth,
esg and
Dll and their specific cell fate helped define a high-resolution map of the VP fates formed by three molecularly different domains with distinct developmental potential [
16,
67].