Patterning with clocks and genetic cascades: Segmentation and regionalization of vertebrate versus insect body plans

Abstract

Oscillatory and sequential processes have been implicated in the spatial patterning of many embryonic tissues. For example, molecular clocks delimit segmental boundaries in vertebrates and insects and mediate lateral root formation in plants, whereas sequential gene activities are involved in the specification of regional identities of insect neuroblasts, vertebrate neural tube, vertebrate limb, and insect and vertebrate body axes. These processes take place in various tissues and organisms, and, hence, raise the question of what common themes and strategies they share. In this article, we review 2 processes that rely on the spatial regulation of periodic and sequential gene activities: segmentation and regionalization of the anterior-posterior (AP) axis of animal body plans. We study these processes in species that belong to 2 different phyla: vertebrates and insects. By contrasting 2 different processes (segmentation and regionalization) in species that belong to 2 distantly related phyla (arthropods and vertebrates), we elucidate the deep logic of patterning by oscillatory and sequential gene activities. Furthermore, in some of these organisms (e.g., the fruit fly Drosophila), a mode of AP patterning has evolved that seems not to overtly rely on oscillations or sequential gene activities, providing an opportunity to study the evolution of pattern formation mechanisms.

Fig 1. Gene expression patterns in vertebrates.

(A) Morphogenesis in the chicken embryo. Left: Epiblast stage. The primitive streak is shown as a vertical line with the node on the top. Axial progenitors (blue) are located in the anterior region of the primitive streak. Middle panel: 3-somite stage. Paraxial mesoderm is segmented into somites (pink) anteriorly, but unsegmented posteriorly (PSM; green). Axial progenitors (blue) are located in the regressing primitive streak at the posterior end of the embryo, where axial elongation takes place. Right panel: 8-somite stage. Somites (pink) continue to from sequentially from the PSM (green), as the embryo elongates posteriorly. (B) Signaling gradients in somitogenesis. FGF and Wnt signaling (yellow) activity is highest in the posterior progenitor domain and forms a posterior-to-anterior gradient along the PSM. The determination front (dotted line) is positioned by these signaling gradients. (C) The segmentation clock. Waves of gene expression (orange) are initiated in the posterior domain and travel anteriorly along the PSM. When the segmentation clock reaches the determination front (dotted line), a new pair of somites is specified (green). (D) Hox gene spatial and temporal colinearity. The schematic depicts expression of a hypothetical Hox cluster in chicken embryos. Hox genes are first expressed in the progenitor domain and spread anteriorly through cell ingression, thus leading to the formation of nested expression domains (colored regions) through the sequential activation of more posterior Hox genes. Throughout the figure, for all embryo schematics: anterior to the top and posterior to the bottom. PSM, presomitic mesoderm.

Fig 2. Key gene expression patterns during segmentation and regionalization in Tribolium (as a representative of short-germ insects) and Drosophila (as a representative of long-germ insects).

(A) Expression patterns of segmentation clock genes in Tribolium (namely primary pair-rule genes: Tc-eve (shown in blue), Tc-run (red), and Tc-odd (green)) across time (at 24°C) during both the blastoderm and germ-band stages of embryogenesis. Tribolium clock genes are expressed periodically as consecutive waves that propagate from posterior (right) to anterior (left) (frequency doubling of pair-rule gene expressions is not depicted). (B) Key regionalization gene expression patterns in Tribolium (namely gap genes: Tc-hb (blue), Tc-Kr (red), Tc-mlpt (green), and Tc-gt (gold)) across time (at 24°C) during both blastoderm and germ-band stages of embryogenesis (head expressions of gap genes are not depicted for simplicity), in addition to Tc-cad (shown in gray), a master regulator of both segmentation and regionalization in Tribolium. Tribolium gap genes are expressed as consecutive nonperiodic waves that propagate from posterior (right) to anterior (left) within the expression domain of Tc-cad. (C) Expression patterns of selected Drosophila segmentation genes (namely the pair-rule gene Dm-eve (shown in brown)) and regionalization genes (namely the gap genes: Dm-Hb (blue), Dm-Kr (red), Dm-kni (green), and Dm-gt (gold)) across time during the blastoderm stage, in addition to master regulator gradients: Dm-Bcd (orange) and Dm-Cad (gray). Drosophila gap gene expression bands arise more or less simultaneously before nuclear cycle 14 (NC14). Later during NC14, pair-rule gene expressions arise, also more or less simultaneously. Eventually, expression domains of both gap and pair-rule genes undergo posterior-to-anterior shifts, reminiscent of the posterior-to-anterior propagation of gap and pair-rule gene expression waves in Tribolium. Finally, and concomitantly with the degradation of Dm-Bcd and Dm-Cad gradients, gap and pair-rule gene expression domains stabilize, pair-rule gene expressions undergo frequency doubling (not depicted), then both gap and pair-rule gene expressions eventually fade. Throughout the figure, for all embryo schematics: anterior to the left and posterior to the right.

Fig 3. The FF model (see also Box 1).

(A) Top panel: In the FF model, different concentrations of a morphogen gradient (shown in gray) activate different cellular states (shown in different colors). The FF model can pattern nonelongating tissues (bottom left) via a nonregressing gradient (shown in gray), as well as elongating tissues (bottom right) if a retracting short-range gradient (shown in black) activate a slowly decaying morphogen (gray). (B) In a similar fashion, the FF model can generate periodic patterns if a complex regulatory logic of periodically expressed genes (shown in brown) is employed or if an intermediate step of nonperiodic patterning is introduced. FF, French Flag.

Fig 4. Segmentation and regionalization mechanisms in insects.

(A) Maternal Dm-Hb gradient acts as a master regulator of gap genes in Drosophila. Progressive reduction of maternal Dm-Hb gradient (in various mutant backgrounds) results in progressive shifts of gap gene domains toward anterior. (B) Pair-rule stripes (shown for Dm-eve) are specified in a stripe-specific fashion in Drosophila. Left: Each one or pair of Dm-eve stripes are specified by a specific enhancer that receives inputs from upstream gap genes (shown repressive gap inputs for the 3+7 and 4+6 enhancers; strong repression shown in sold lines; weak repression in dashed lines). The full 7-stripe pattern is then stabilized by a 7-stripe (or zebra) enhancer. Right: Shown how the regulatory logics of 3+7 and 4+6 Dm-eve enhancers translates upstream gap gene expressions (shown are those of Dm-Kr and Dm-kni) into stripe pairs [140]. (C) A sketch of the genetic wiring of the Tribolium segmentation clock, composed of the 3 primary pair-rule genes: Tc-eve, Tc-run, and Tc-odd, wired into a negative feedback loop. Note that this is a parsimonious wiring explaining observed gene expression dynamics in WT and knockdown phenotypes. Actual wiring might differ from the one shown, especially that pair-rule genes are known to act as repressors rather than activators. (D) Experimental evidence of segmentation clock wiring in Tribolium. (E) A sketch of the genetic wiring of the Tribolium gap gene cascade. Note that this is a parsimonious wiring explaining observed gene expression dynamics in WT and knockdown phenotypes. Actual wiring might differ from the one shown, especially that most gap genes are known to act as repressors rather than activators. (F) Experimental evidence that gap genes are wired into a genetic cascade in Tribolium: repressing a single gap gene results in the up-regulation of upstream genes in the cascade and down-regulation of downstream genes. (G) Experimental evidence that the Wnt/Cad gradient (shown in gray) acts as a speed regulator of the segmentation clock in Tribolium. In Tc-lgs RNAi embryos: Tc-cad gradient is reduced (i.e., its peak concentration is lower than in WT) and shifted toward posterior; concomitantly, Tc-eve oscillation frequency is reduced and Tc-eve waves are shifted toward posterior. In Tc-pan RNAi embryos: Tc-cad gradient is reduced, shifted toward anterior, and stretched; concomitantly, Tc-eve oscillation frequency is reduced and Tc-eve waves are shifted toward anterior and stretched. In Tc-zen RNAi embryos: Tc-cad gradient has the same peak level and slope as in WT, but just shifted toward anterior; concomitantly, Tc-eve waves are shifted toward anterior without any sign of spatial stretch or time dilation. (H) Further evidence that gap genes are wired into a genetic cascade in Tribolium. Upon reinducing the leading gap gene in the cascade (Tc-hb) using a transgenic line where Tc-hb minigene is placed downstream of a heat-shock promoter, the whole gap gene sequence is reinduced in the SAZ. (I) A possible model for how the speed of the pair-rule clock or the gap gene cascade is modulated by a Wnt/Cad gradient in Tribolium: Wnt/Cad activates the pair-rule clock and/or gap gene cascade, but represses a multistable gene regulatory network. The gradual switch between the 2 gene networks results in the gradual slowing down of pair-rule oscillations and/or gap gene sequential activation. Throughout the figure, for all embryo schematics: anterior to the left and posterior to the right. RNAi, RNA interference; WT, wild-type.

Fig 5. The RD model (see also Box 2).

In the RD model (top panel), a slow-diffusing molecule (molecule A) activates itself as well as a fast-diffusing molecule (molecule B), which is also an inhibitor of A. The RD model can pattern nonelongating (bottom left) as well as elongating tissues (bottom right). RD, reaction–diffusion.

Fig 6. The SR model (see also Box 3).

(A) Top panel: In nonperiodic SR model, a speed regulator (shown in gray) modulates the speed of cellular state transitions (each cellular state is shown in a different color) in a dose-dependent fashion (top panel, right). At a very low or zero concentration of the speed regulator, cellular state transitions are arrested (top panel, left). (A) Bottom panel: The SR model can operate in a gradient-based mode to pattern nonelongating tissues (left) or in a wavefront-based mode to pattern elongating tissues (right). (B) If the processes driving cellular state transitions is periodic (i.e., driven by a clock, which expression is shown in brown; top panel), the SR model can generate periodic patterns in both elongating and nonelongating tissues (via a wavefront-based and gradient-based modes of the model, respectively). SR, speed regulation.

Fig 7. Mechanisms of segmentation and regionalization in vertebrates.

(A) Delayed negative feedback loop giving rise to oscillations in Hes/Her expression. In the absence of transcriptional repression by autoinhibition (1), Hes/Her genes are activated and mRNA transcribed (2). This leads to HES/HER protein translation (3) and accumulation. After a time delay associated with gene expression steps, HES/HER proteins reach sufficient levels to bind the Hes/Her regulatory regions and inhibit transcription (4). Autoinhibition is relieved by HES/HER degradation (5) and the cycle begins again. (B) Mechanism of FGF gradient formation. Only progenitor cells (pink) actively express the fgf8 ligand. Once cells ingress into the PSM (purple), they cease to transcribe fgf8. Progressive degradation of fgf8 mRNA and protein leads to gradient formation as cells acquire more anterior positions within the paraxial mesoderm. (C) Genomic organization of the HoxA cluster. HoxA1-13 genes are arranged colinearly within the cluster in the 3′ to 5′ orientation. Chromatin opening and gene expression start at the 3′ end and proceed in the 3′ to 5′ direction. Genes are colored according to the vertebral identities they specify. The 2 TADs (3′ and 5′) are shown as gray triangles. Anterior Hox genes are activated by Wnt signaling (red), central Hox genes by Wnt/Cdx (green), and posterior Hox genes by Gdf11/TGFβ (blue). PSM, presomitic mesoderm; TAD, topologically associated domain.

Fig 8. A model toward reconciliation of simultaneous and sequential modes of AP patterning in insects.

(A) Right: In a loss of function mutant of a Drosophila gap gene (here shown only Dm-kni mutant), gap gene expression anterior to it is extended (here Dm-Kr), and gap gene expression posterior to it is missing (here shown for Dm-gt). This is reminiscent of the gap gene phenotypes in Tribolium (compare with Fig 4E and 4F) and suggests that gap genes in Drosophila are wired into a genetic cascade as well (see the sketch of a gene network to the left; note that this is a parsimonious wiring explaining observed gene expression dynamics in WT and mutant phenotypes, where wiring might differ from the one shown, especially that most gap genes are known to act as repressors rather than activators). (B) A model for the evolution of gap gene regulation in insects from a sequential mode of patterning (like in Tribolium) to a simultaneous mode (like in Drosophila). Left: Typical speed regulation model for producing nonperiodic patterns (Box 3), in which the leading gene in the gene sequence (blue) is initially uniformly expressed. Right: Expressing the leading gene in the gene sequence (blue) in a graded fashion result in speedy and seemingly simultaneous patterning [9]. AP, anterior–posterior.

Fig 9. Comparison of segmentation and regionalization in short-germ insects versus vertebrates.

PSM, presomitic mesoderm; SAZ, segment addition zone.