In preprints: of genitalia and six-legged mice

It may seem unlikely that limbs would share common developmental mechanisms with external genitalia. Yet closer examination suggests that they may share homology. The genitalia and hindlimbs develop in proximity in most amniotes (Tschopp et al., 2014), and both organs begin as buds that require common signaling pathways for growth and patterning (Cohn, 2011). It is therefore not surprising that evolutionary biologists might suggest a common origin for these two organs. But what distinguishes their different outcomes? A recent preprint by Lozovska and colleagues (2023 preprint), provides intriguing data that suggest limb and genitalia development are linked, but different outcomes require transforming growth factor β (TGFβ) receptor 1 signaling, providing a mechanistic adaptation for the evolution and development of external genitalia.

Over 300 million years ago, the first tetrapods were emerging from their shallow aquatic habitats. This feat prompted evolutionary changes that morphed the fins of sarcopterygian/lobe-finned fish into the limbs of tetrapods. As such, fossils from the Devonian revealed various fossils with appendages that were simultaneously fin- and limb-like (Shubin et al., 2006). However, it was not until the Carboniferous that tetrapods would further evolve the amniotic and cleidoic (shelled) egg, and release themselves from an obligate watery environment for reproduction (Benton and Donoghue, 2007). The earliest tetrapods may have had limbs that allowed them to walk on land, but they had to return to their shallow seas and inland ponds to reproduce. Much like today's amphibians, their embryos would have been susceptible to desiccation and would fail to develop without a moist environment. Furthermore, the aqueous environment permits broadcast sperm to swim to the egg. Conversely, amniotes form a water-tight membrane that encases the embryo, allowing for development on dry land. But although this broadened the potential environments that tetrapods could claim, it also required anatomical changes to deliver motile sperm directly to the ovulated egg. Reproduction in many amniote lineages solved this challenge by evolving external genitalia to enable the internal delivery of sperm cells, thereby negating the need for an aquatic habitat. The molecular mechanisms that facilitated the development of external genitalia in the amniote lineage remain unidentified in evolution and developmental biology.

As mentioned above, alterations to the lobe-fin developmental program were elaborated to generate articulated tetrapod limbs. Accordingly, recent work has revealed molecular and genetic mechanisms coopting this program to induce and pattern genitalia. Interactions between posterior Hox proteins, sonic hedgehog (Shh) and fibroblast growth factor (Fgf) signaling first produce paired buds that subsequently grow and fuse into the genital tubercle (Cohn, 2011). This mechanism of using Hox genes, Shh and Fgfs to generate an appendage is common in limb development. However, key differences occur between limb and genital development. The limb bud is composed solely of mesoderm wrapped in ectoderm. Reciprocal Fgf signaling between the ectoderm and the mesoderm induces proliferation and outgrowth of the bud, whereas Shh produced by the posterior mesenchyme serves to pattern the limb along the anterior-posterior axis (Zeller et al., 2009). Hox proteins both initiate limb formation and subsequently expand the segments of the limb (Zakany and Duboule, 2007). In contrast, the development of external genitalia begins as paired cellular outgrowths that flank the cloaca. These then fuse into the genital tubercle, which includes cells from all three germ layers. The cloacal endoderm, which forms the urethral plate epithelium of the genital tubercle, is the source of Shh (Perriton et al., 2002); however, Fgf8, which is required for initiation (Harada et al., 2015), has not been shown to be required for genital bud outgrowth (Seifert et al., 2009). Nevertheless, the similarities between hindlimb and genital bud formation are striking. Several species of snakes and lizards possess hemipenes, paired penises that develop from paired limb bud-like structures in the embryo (Cope, 1896). Furthermore, Tschopp and colleagues (2014) used four different species of amniotes to show that a shift in the positioning of the cloaca allows for a common primordium to generate both the limbs and the genitalia. Yet if the limb and the genitalia share a common precursor, how are the two appendages distinguished from one another?

A major advance in answering this question is provided by Lozovska and colleagues, who analyzed the consequences of knocking out Tgfbr1 in the prospective genitalia and hindlimbs of mouse embryos. Using a conditional approach, the authors found that loss of Tgfbr1 after the formation of the hindlimb and genital tubercle primordia caused the genitalia to be transformed into a second set of hindlimbs with fully patterned and articulated skeletal elements. Subsequent analyses confirmed that the genital tubercles of the knockouts were indeed limb-like. Consistent with the formation of limbs, the expression domain of Fgf8 extended from the hindlimbs to the cloaca region, the genital tubercle of the mutants expressed Lin28a and the hindlimb-specific Pitx1, and then subsequently expressed the dorsal-ventral limb markers Lmx1b and En1. Together, these data strongly support their conclusion that loss of Tgfbr1 results in a transformation of the external genitalia into limbs. Perhaps more importantly, this work shows the plasticity of the genital tubercle primordia and lends support to the hypothesis that external genitalia and hindlimbs share a common ontology. This conclusion was supported by ATAC-seq results that show changes in the accessibility of the regulatory genomic regions that normally coordinate the expression of specific limb genes, such as Fgf10, or genitalia factors, such as Isl1. In a surprising finding, the zone of polarizing activity (ZPA) regulatory sequence, which directs posterior limb bud expression of Shh, was found to be inaccessible in both the hindlimb and genital tubercle of Tgfbr1 mutants, suggesting a dual role for Tgfbr1 in hindlimb and genital development. Overall, it's likely that signaling through Tgfbr1 is coordinating both limb and genitalia development via different downstream effectors that act as pioneer factors in transcriptional regulation.

Taken together, the results presented in this preprint go a long way in linking the developmental genetics between limb formation and external genitalia. Furthermore, this work strongly supports the logical links between the hindlimbs of tetrapods and the genitalia of amniotes proposed by evolutionary biologists. However, many questions remain. For example, which ligand, if any, is signaling through Tgfbr1? How is this signaling regulated in time and space during appendage development? What are the different downstream effectors that regulate limb formation distinctly from genital development? Answers to these questions will be needed to clarify different developmental outcomes in mammals as well as other amniotes. For example, how do some lizard species form hemipenes and hindlimbs, while snakes generate only hemipenes? These evolutionary questions aside, the work by Lozovska and colleagues provides new and deep insight into the developmental and evolutionary biology of tetrapods and amniotes.