EvoDevo: when four became two plus two

James C-G Hombría and Sol Sotillos CABD, (CSIC/JA/UPO) 41013 Seville Spain

Wings and halteres are homologous flight appendages whose shape differences are controlled by the Ubx . Recent research shows how Ubx regulates apical and basal extracellular matrix proteases and their inhibitors to achieve this morphological divergence.

The dorsal thoracic appendages of flies differ in size, shape and cellular morphology. Previous research analyzed how the (Ubx) Hox protein controls patterning, cell proliferation and cell differentiation to regulate the differential development of wing vs. haltere [1-4]. A recent paper in Development now completes the study by analyzing how Ubx modulation of the apical and basal extra cellular matrix (ECM) affects their morphogenesis during metamorphosis [5].

Hox proteins are transcription factors with an important function as developmental regulators [6]. Except in sponges, these transcription factors have been found in all from jellyfish to or mammals [7]. Hox proteins regulate the formation of specialized organs and structures at different positions of the anterior posterior body axis. The of Hox or their forced expression in areas where they are not normally expressed can transform one part of the body into the likeness of another. Such transformations are especially evident when comparing homologous structures with different morphologies. In mice, for example, ribs associated to abdominal vertebrae appear when Hox6, normally expressed in the thorax, is activated in the abdominal region during development [8]. However, the most famous transformation caused by Hox expression alteration was obtained by Edward B. Lewis by combining in cis-regulatory regions that affected the expression of the Ubx transforming the two winged fly into a perfect four winged fly [9, 10]. Halteres, required to maintain balance during flight, evolved by the reduction of the posterior pair of wings of the four-winged fly ancestors. Four winged insects, like butterflies, express Ubx in the hind wings where it controls the differential pigmentation patterns between the anterior and posterior pair of wings [11, 12]. Thus, the extreme shape transformation into halteres must have occurred because Ubx acquired new target genes during dipteran evolution. Understanding how Ubx controls the formation of halteres, preventing the formation of wings, offers a unique opportunity to understand how differentially expressed developmental genes lead to the divergent evolution of homologous structures creating diversity.

The wings and the halteres derive after metamorphosis from the dorsal thoracic imaginal discs. The wing and the haltere primordia are specified at embryogenesis and proliferate inside the maggot during larval stages forming large bags of epithelial cells organized as a monolayer. Although the wing and haltere imaginal discs already have a different size in the embryo, both discs have a similar morphology until at metamorphosis major differences become apparent that result from modifications of the developmental wing gene-network induced by Ubx in the haltere imaginal disc [13]. In the mature wing, each cell produces a single hair and is 8 times wider than the haltere cells which secrete 2-3 hairs each. Both dorsal and ventral wing surfaces become tightly apposed with the final wing formed by the fused thin transparent cuticles secreted by the adult cells before they die. The anterior edge of the wing is covered with sensory organs, and longitudinal veins form in the wing strengthening the blade. In contrast, the haltere forms a short, hollow club-shaped structure with a thick, pigmented cuticle secreted by epithelial cells that survive for a longer period. The position of veins and sensory elements becomes specified in the wing disc at larval stages, so the fly faces a morphogenetic challenge during metamorphosis to fold the wing into a perfect functional structure. At the beginning of metamorphosis, the imaginal discs are extruded, substituting the larval ectoderm to form the adult appendages. The precise adhesion of the dorsal and ventral epithelial wing layers requires the complete ECM remodeling, which occurs in two phases (Fig. 1A) [14]. An initial wing apposition occurs during the first few hours after puparium formation (APF). A critical step from 4-7h APF enables a first expansion of the wing. At this early phase the larval apical ECM composed by the ZP protein Dumpy is degraded due to the expression of the apical Stubble (Sb) and Notopleural (Np) proteases. Simultaneously, the basal ECM composed by Collagen IV, Perlecan and Laminin is degraded by the basal Matrix metalloproteases 1 and 2 (Mmp1 and Mmp2) allowing the direct apposition of the dorsal and ventral epithelial layers. The release from the ECM results in the cell´s isodiametric expansion. This is soon followed by polarized proximo-distal cell rearrangements that elongate the wing blade while simultaneously narrowing its antero-posterior axis (Fig. 1B). This convergent-extension movement is driven from 4-5h APF by the planar polarization of the motor protein Myosin-II and Rok (the kinase activating the Myosin-II regulatory subunit) on the apical cell membranes perpendicular to the proximo-distal wing axis. At 6-7h APF Myosin-II re-localizes laterally, which in conjunction with the cells´ release from the ECM allows the epithelium to expand further with the columnar cells becoming cuboidal, transforming what was the pseudostratified wing disc epithelium of the larva into the monolayered epithelium of the wing [15]. When this first phase ends, the wing cells form a pupal cuticle and secrete Dumpy restoring the apical ECM that attaches the epithelium to the cuticle. On the other hand, the re-establishment of the basal ECM leads at 11h APF to the transient separation of the two epithelial wing layers, although the dorsal and ventral cells maintain long thin protrusions across the ECM that keep them in contact [14].

As development proceeds, the second phase of ECM remodeling occurs. Basal ECM degradation at about 18h APF allows the dorsal and ventral layers to rejoin. Degradation of the apical ECM due to a second burst of Sb and Np protease expression allows the epithelial wing cells to separate from the pupal cuticle they secreted [5]. However, the gigantic apical ECM protein Dumpy is not uniformly degraded, remaining at the distal wing margin where it maintains the wing blade attached to the rigid pupal cuticle (Fig. 1A). This attachment will be the source of a global tension when the proximal cells of the wing contract to form the hinge that articulates the blade with the thoracic body. Hinge contraction creates a proximo- distal tension along the wing blade due to its distal anchorage to the rigid cuticle, causing a further stretch (Fig. 1B). This global force influences the final wing blade shape as shown by the shortening of the wing blade caused by either laser cuts in the distal blade or by Dumpy mutations that release this tension by eliminating the blade´s distal attachment to the cuticle [16, 17]. These results show that the final wing shape is the result of both endogenous cell rearrangements and forces imposed by the apical and basal ECM.

The complex wing ECM dynamics described above do not occur in the haltere. Recent studies show how Ubx function affects the differential degradation of the apical and basal ECM in the haltere, explaining how the wing and haltere shapes diverge during metamorphosis (Fig. 1A). It has been observed that while the wing blade, expresses high levels of Mmp1 protease and low levels of the metalloprotease inhibitor Timp, the opposite occurs in the haltere, allowing the persistence of a basal ECM that blocks epithelial apposition [5, 18]. Similarly, Ubx prevents the early 4h APF expression of the apical proteases Sb and Np in the halteres, preventing the degradation of the apical ECM protein Dumpy that maintains its epithelium in a contracted pseudostratified organization. Also, while the second burst of Sb and Np expression at about 40h in the wing cannot degrade the apical Dumpy ECM protein from the wing’s edge, the separation of the haltere epithelium from the pupal cuticle is complete, resulting in the absence of distal elongation observed in the wing blade when hinge contraction occurs. The wing and haltere expansion caused by altering Ubx expression [18] and the observation that Ubx binds to the chromatin regulatory regions of apical and basal proteases as well as to the protease inhibitor Timp implies that Ubx may be controlling the wing and haltere morphological differences arising at metamorphosis through their direct regulation [5]. These results not only resolve how the most famous Hox transformation arises but also add to the growing evidence that, beyond homeosis, the main function performed by Hox proteins is the regulation of morphogenesis and organogenesis [19, 20].

Figure 1 Legend

Schematic wing and haltere transversal and dorsal sections at different hours after puparium formation (APF). (A) At 3h APF apical (blue), and basal (green) ECM surround the columnar epithelial cells. At 5h APF proteases degrade the wing ECM allowing epithelial apposition and expansion, which is prevented in the haltere through Ubx regulation of protease expression and function. At 13h APF ECM secretion reconstitutes the apical and basal ECM transiently separating the epithelia. A pupal cuticle (brown) is secreted to which the edge of the wing blade attaches through Dumpy. A second burst of protease expression degrades the ECM allowing final wing adhesion at 40h APF.

(B) Wing shape changes during metamorphosis. Release from the apical (light blue) EMC present in the larval wing disc, and polarised Myosin contraction allows wing expansion. Distal wing margin restriction of Dumpy at 18h anchors the wing blade cells to the pupal cuticle. Hinge contraction (purple arrows) creates tension elongating the proximo-distal axis of the wing blade (orange arrow). Wing blade (orange), hinge (purple), Dumpy (light blue). Black arrows at 4h APF mark the convergent extension movements. Purple and orange arrows mark respectively the hinge contraction and the wing extension. Cross-sections in the anterior-posterior axis are represented below.

References:

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A Wing Haltere

3h APF

Stubble/Notopleural No apposition of D/V layers Mmps expression Columnar cell shape Apposition of D/V layers Timp expression Columnar cell shape Ubx aECM and bECM degradation 5-6h APF

Myo II/Rok apical P/D polarization Convergent-extension expansion No apposition of D/V layers Cuboidal cell shape Columnar cell shape

13h APF

a/b ECM secretion. D/V layers separation No apposition of D/V layers Dumpy at the wing blade binds Columnar cell shape to the pupal cuticle

Stubble/Notopleural 40h APF expression Ubx

Wing elongation Adhesion II Dumpy degradation No apposition of D/V layers Columnar cell shape apical ECM: Dumpy basal ECM: Col IV, Perlecan

B

LIII 4-5h APF 18-30h APF Imaginal Disc Convergent extension Hinge contraction Tension=>Wing elongation