Diversification of the vertebrate limb: sequencing the events

Abstract

Naturalists leading up to the early 20th century were captivated by the diversity of limb form and function and described its development in a variety of species. The advent of discoveries in genetics followed by molecular biology led to focused efforts in few 'model' species, namely mouse and chicken, to understand conserved mechanisms of limb axis specification and development of the musculoskeletal system. 'Non-traditional' species largely fell by the wayside until their recent resurgence into the spotlight with advances in next-generation sequencing technologies (NGS). In this review, we focus on how the use of NGS has provided insights into the development, loss, and diversification of amniote limbs. Coupled with advances in chromatin interrogation techniques and functional tests in vivo, NGS is opening possibilities to understand the genetic mechanisms that govern the remarkable radiation of vertebrate limb form and function.

Figure 1.

Development of hindlimb buds and external genitalia in (a) mouse, (b) anole lizard, and (c) snake. HLB, hind limb bud. GT, genital tubercle. HP, hemi-penis. In each species, cis-regulatory control of Tbx4 by the hindlimb element A (HLEA) and dual activity hindlimb element B (HLEB) and control of sonic hedgehog by the ZPA regulatory sequence (ZPA) are depicted. Hindlimb activity of all three elements has degenerated in snakes.

Figure 2.

In the developing hindlimbs (green) of the (a) scaled feet pigeon breeds, hindlimb-specific cis-regulatory regions drive robust Pitx1 expression. Forelimb-specific Tbx5 cis-regulatory regions are inactive (grey circle) in the hindlimbs, resulting in no Tbx5 expression. (b) In feather-footed breeds, a deletion upstream of Pitx1 is associated with reduced hindlimb Pitx1 expression, and cis-regulatory mutations near Tbx5 are associated with its misexpression in the hindlimbs. These cis-regulatory mutations could lead to the reactivation of forelimb-specific Tbx5 enhancers (conceptually represented using a red circle) in the hindlimbs and/or a gain of novel hindlimb-specific enhancers (green star).

Figure 3.

Hindlimbs of (a) mouse and (b) jerboa with femurs (red) and metatarsals (blue) highlighted. Proximally active (red) cis-regulatory regions drive Shox2 expression in mouse and jerboa femurs. These regions are inactive (grey) in the distal mouse limb, and no Shox2 expression is detected in mouse metatarsals. Jerboa metatarsals express Shox2, which may be explained by the activation of cis-regulatory sequences typically restricted to the proximal limb (red).