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Pax group III genes and the evolution of insect pair-rule patterning

Gregory K. Davis^1,*, Carlos A. Jaramillo^1,* and Nipam H. Patel^1,2,3,

¹ Committee on Developmental Biology, University of Chicago, Chicago, IL 60637, USA
² Department of Organismal Biology and Anatomy, University of Chicago, Chicago, IL 60637, USA
³ Howard Hughes Medical Institute, University of Chicago, Chicago, IL 60637, USA
^* These authors contributed equally to this work

View larger version (96K): [in a new window]   Fig. 1. Anti-Pby crossreacts with Prd, Gsb and Gsbn. Drosophila embryos are stained with anti-Pby (A-J) and gene-specific monoclonal antibodies (mAbs) against Prd (K-T), Gsb (U-AA) and Gsbn (BB-EE). (A-J) As anti-Pby reacts to Prd, Gsb and Gsbn protein, the Pby pattern is a fusion of the Prd, Gsb and Gsbn patterns, as well as an additional glial pattern appearing at stage 13. (K-T) At stage 4 Prd is expressed in an anterior broad stripe (K), which itself resolves into stripes 1 and 2 as it is joined by stripes 3-7 (L). During stage 5, the primary stripes resolve into a segmental pattern of 14 secondary stripes of alternating intensity as an anterior dorsal domain appears (M). By stage 10, Prd stripes are mostly absent, but the protein persists in the gnathal protuberances (Q-T). (U-AA) At stage 7, Prd and Gsb are expressed in a coincident pattern (N,U), although Gsb stripes persist through stage 10, at which time they restrict to the neuroectoderm (X-Y). (BB-EE) Gsbn is expressed in a defined neuroectodermal pattern at stage 10, and persists after the neural stripes of Gsb have diminished. The Prd-specific MAb DP201 has not been previously described. Staining in the gnathal protuberances (Q-T) is also present in G,I but is not apparent because focal planes are sagittal. Anterior is towards the left and all views are lateral, except F,H,J,P,R,T,W,Y,AA,CC,EE, which are ventral views.

View larger version (69K): [in a new window]   Fig. 2. The Pairberry pattern in Tribolium mimics Drosophila. Tribolium embryos are stained with anti-Pby (in black; A,C,D,F,G,I,J-S) with Dapi counterstains (B,E,H) that correspond to (A,D,G), respectively. In addition, embryos are immunostained with second labels (in brown) of Eve (C,F,I,J,L) or En (M-S). (Q,R) Higher magnification images of A4 and T2 segments of P. Embryos are blastoderm stage (A-C) and increasingly older from D to S. (A-C) At the blastoderm stage, anti-Pby reveals protein in a broad anterior domain, while Eve is expressed in a posterior domain (C). (D-H) The mandibular Pby stripe (Mn arrow) appears anterior to the first of two nascent primary Eve stripes (F). No additional stripes appear before the amniotic fold advances (asterisk, H), concurrent with the onset of gastrulation. (I-K) A weak broad Pby stripe appears between the two primary Eve stripes (arrowhead, I,J) and splits, giving rise to the maxillary and labial stripes (Mx and La arrows, K). (L-N) A second broad stripe appears between two primary Eve stripes (arrowhead, L) and splits (M), giving rise to the first and second thoracic stripes (T1 and T2 arrows, N). Stripes of En appear just posterior to individuated stripes (M,N). (O-S) The third thoracic and first abdominal stripes have appeared, as well as a de novo broad stripe (O). The broad stripe corresponding to the eighth and ninth abdominal stripes splits (P, arrowhead), while more anterior segmental stripes overlap with stripes of En by one cell row (P,Q). Still more anterior stripes are restricted to the neuroectoderm (P,R). Later in development, all stripes are present and restricted to the neuroectoderm, with additional Pby staining in preantennal domains, the mandibles, maxillae and labial appendages, as well as the tips of the gnathal palps and limb primordia (S). Anterior is towards the left and all views are ventral except F, which is slightly oblique. J-S have been dissected off of the yolk and L-S have had the amnion removed. Scale bar: 250 µm for A-P,S; 19 µm for Q; and 28 µm for R.

View larger version (49K): [in a new window]   Fig. 3. Schistocerca possesses at least two Pax group III genes. (A) Amino acid sequence alignment of Pby1 and Pby2 (Schistocerca americana) with Gsb, Gsbn and Prd (Drosophila melanogaster) reveals conservation in the paired domain, homeodomain and octapeptide (black underline). The extended region of the homeodomain is also shown (gray underline). The addition and deletion of amino acids that result from the alternative splice forms of pby1 and pby2, respectively, are shown above the alignment. Available cDNA sequence for both pby1 and pby2 have been deposited in GenBank (Accession Numbers, AY040535 and AY040536, respectively). (B) Schematic of Schistocerca, Drosophila and vertebrate PgIII gene products. The relative lengths and conserved regions of each gene product are shown. PD, paired domain; HD, extended homeodomain; O, octapeptide. Hatching indicates presumed regions not yet sequenced. Pax3 and Pax7 are from mouse. (C) Phylogenetic analysis of Pby1 and Pby2 amino acid sequences using maximum parsimony. Based on the alignments shown in A, Tree I (partial paired domain alone) is the most parsimonious tree and Tree II (partial paired domain + homeodomain proper) is a strict consensus of the three most parsimonious trees. Both trees support the placement of Pby1 and Pby2 within PgIII. Pax genes that lack full homeodomains were not included in Tree II. Pax groups I, II, III and IV are those originally defined (Balczarek et al., 1997). Pax 1-9 are from mouse; Gsb, Gsbn, Prd, Eyeless, Sparkling and Pox Meso are from Drosophila; and Pax B and Pax D are from the cnidarian Acropora millepora (Miller et al., 2000). Numbers shown reflect the percentage of 1000 bootstrap replicates supporting the indicated node (only values >50% for PgIII nodes are shown, other PgIII nodes are shown in gray).

View larger version (96K): [in a new window]   Fig. 4. The Pairberry pattern comprises both pby1 and pby2 in Schistocerca. Grasshopper embryos stained for pby1 mRNA (A,F,K) and pby2 mRNA (B,G,L), En protein (C,H,M), Pby protein (D,I,N), or both Pby (black) and En (brown) protein (E,J,O). Approximate stages are as indicated. Open arrowheads (<) mark T1 in Pby embryos. (A-E) 18%: pby1 mRNA is found in seven stripes that correspond to the antennal, gnathal and thoracic segments, as well as a broad posterior domain and pre-antennal eyespots (brown arrows, A,D,E). (B,C) pby2, by contrast, is not expressed at this stage (B) while only the antennal stripes of En (C) have appeared. Anti-Pby staining reveals a pattern similar to pby1 mRNA as well as several, more anterior, pre-antennal domains of the head lobes (brown arrowheads, D,E). (F-J) 23%: additional pby1 stripes have appeared in the intercalary segment and A1-A6, along with a broad posterior domain (A7/A8). Note that the gnathal, thoracic, and A1 pby1 stripes are now restricted to the neuroectoderm (F). pby2 mRNA is found in stripes, coincident with pby1, that are entirely restricted to the neuroectoderm and are, in this embryo, only just appearing in A4 (G). pby2 mRNA appears about the same time as En, which in this embryo is found only as far posterior as A3 (H). The pby1 eyespots have refined to two lateral domains, which are still not present in the pby2 pattern (compare F with G). The two Pby embryos are slightly older, showing laterally restricted stripes in A3 and A4 as well individuated A7 and A8 stripes (I,J). The Pby/En embryo reveals that En stripes appear before the restriction of Pby1 protein (J). (K-O) 35%: ventrally restricted stripes of pby1, as well as pby2, are found in the antennal, intercalary as well as all gnathal, thoracic and abdominal segments (K,L). pby1 and pby2 stripes are also found in A11 (black arrows, K,L), which does not express En (the A11 stripe is present, but not visible in O). At this stage, pby1 is also expressed in at least one ring in the limb primordia, of which three are visible in the Pby embryos (red arrows, K,N,O). For the first time, pby2 is also expressed in pre-antennal domains coincident with pby1, as well as the gnathal appendages (red arrowheads, L,N,O). Anti-Pby staining recapitulates the combined pby1 and pby2 patterns, while also revealing non-PgIII domains found in the pre-antennal regions of the head lobes, the mandibles (in which staining appears before the appearance of pby2 mRNA in the mandible) (asterisk, I,J,N,O), as well as tips of gnathal palps, limbs, abdominal buds and the telson (where En is also expressed) (black arrowheads, M-O). Scale bar: 1.0 mm.

View larger version (73K): [in a new window]   Fig. 5. Early Pairberry pattern in Shistocerca. (A-D) Anti-Pby immunostaining of early grasshopper embryos. (A) Dapi stain of embryo, 9% of development. (B) Same embryo, showing weak posterior Pby domain also observed with pby1 mRNA (not shown). (C) 11% embryo showing gnathal arc (black arrowhead). The amniotic fold has been left intact (asterisk). (D) 15% embryo showing gnathal arc (black arrowhead) and T2/T3 broad domain with higher levels at the anterior edge where the T2 Pby stripe will form (open arrowhead). (E-G) Anti-Pby (black) and anti-Hb (brown) immunostaining of 16-17% embryos. (E) At 16% the Mn, La and T2 Pby stripes have formed, while T1 is just beginning to appear. The strong subdomain of Hb protein extends from the Mn Pby stripe to just posterior of the La Pby stripe, where the weak subdomain continues through the T1 Pby stripe. Low levels of Hb are also found throughout the more anterior head lobes (asterisk). (F) Slightly later, the Mx Pby stripe has formed while the Hb domain remains static. (G) At 18%, extension results in the concomitant separation of Pby stripes and increased length of the Hb domain, particularly in T1. (H) 17% embryo after the split of the gnathal arc and intercalation of the Mx and T1 Pby stripes, plus addition of antennal (red arrowhead) and T3 Pby stripes. Open arrowhead indicates position of T2 Pby stripe. Embryo also shows several pre-antennal domains, which include the eyespot present in the pby1 mRNA pattern (red arrow) and domains not seen in either the pby1 or pby2 pattern (black arrows). Scale bar: 300 µm for A-D,H; and 170 µm for E-G.

View larger version (49K): [in a new window]   Fig. 6. Striped patterns of Pby1, Pby2, En and Hunchback protein during Schistocerca embryogenesis. (A-C) The horizontal axis represents time, as measured in percentage development, while the vertical axis represents the length of the embryo, as measured from the tip of the head lobes to the end of the abdomen. 9-26%: extension occurs throughout the embryo but is concentrated in the abdomen. 26-28%: overall length decreases, owing to morphological segmentation, which contracts the embryo in accordian-like fashion, despite the persistence of extension in the abdomen. >28%: overall length increases as the result of nondifferential growth throughout embryo. (A) The schematic shows the timing of appearance and position along the anteroposterior axis of En protein (red) and Pby2 protein (green) during 9-31% of development. Estimates of the timing of appearance of Pby2 protein are based on pby2 mRNA. (B) Same schematic as A, but showing Pby1 protein (purple), coincident Pby1 and Pby2 protein (purple and green hatched), and En protein (red) stripes. Broken purple lines indicate relatively weak expression as detected with anti-Pby. Vertical black bars indicate when Pby1 becomes restricted to the neuroectoderm (see text). Note that stripes of En join Pby1 stripes prior to their restriction. Also note that T2, T3 and the abdominal Pby1 stripes originate from within broader stripes of a two-segment periodicity (see text and Fig. 6). The A11 Pby1 and Pby2 stripe, which does not appear until 35%, is not shown. Non-stripe domains of Pby1 and Pby2 have also been omitted. A,B are based on 23 embryos stained with anti-Pby and anti-En, which were used to measure embryo length and the position of stripes; tracings of four representative embryos are shown (the intercalary stripe, Ic, is shown through the tissue of the antennae in the 31% embryo). The distribution of Pby1 and Pby2 protein was estimated by comparing the Pby and En pattern with the distribution of pby1 and pby2 mRNA (Fig. 4). (C) Same schematic as B, with the estimated position of three ectodermal Hb expression domains (Patel et al., 2001). The early anterior band occupies parasegments 1-3 and thus extends from the Mn to T1 segments, while concurrent weaker expression extends throughout the head (brown, strong subdomain; yellow, weak subdomain). It is not known when the domain extending from A4 to A5 disappears, as the mesodermal expression obscures the domain at 22%. The posterior extent of the domain extending from A7 to A9 should also be regarded as provisional. Staging estimates are ±1%. Oc, ocular; T, telson.

View larger version (70K): [in a new window]   Fig. 7. Early pby1 stripes are of a two-segment periodicity. (A) The posterior portion of 18-20% grasshopper embryos stained for pby1 mRNA are shown. A1 and A2 pby1 stripes arise from within an initial A1/A2 broad domain, comprising the future pby1 stripes of A1 and A2. Note residual expression connecting A1 and A2 stripes during the individuation process (red arrow), as well as the low level of expression posterior to the A1 stripe through to the posterior tip. The individuation of stripes A1 and A2 is accompanied by the appearance of the A3/A4 broad domain, which will later refine into stripes A3 and A4. (B) The extending abdomens of 21-24% embryos immunostained for Pby (black) and En (brown) are shown. A5 and A6 Pby stripes arise from within the broad ‘A5/A6’ domain and A7 and A8 Pby stripes from within the broad ‘A7/A8’ domain. As in A, note the low level of expression throughout the posterior beginning from the most recently individuated anterior stripe. Also note that En stripes appear before the restriction of Pby stripes to the ventral neuroectoderm: the third embryo from the left shows both a restricted stripe (arrowhead, A2) with En and an unrestricted stripe (arrow, A3) posterior to which an En stripe is just beginning to appear. Although some broad domains appear after the previous (more anterior) broad domain has split (A), other broad domains appear before the anterior nascent stripes have fully individuated (B), though this may reflect a difference in the turnover rates of transcript versus protein. Scale bar: 100 µm. Embryos in A have swelled somewhat as a result of the in situ protocol.