Figures and figure supplements A minimally sufficient model for rib proximal-distal patterning based on genetic analysis and agent-based simulations

For decades, the mechanism of skeletal patterning along a proximal-distal axis has been an area of intense inquiry. Here, we examine the development of the ribs, simple structures that in most terrestrial vertebrates consist of two skeletal elements—a proximal bone and a distal cartilage portion. While the ribs have been shown to arise from the somites, little is known about how the two segments are specified. During our examination of genetically modified mice, we discovered a series of progressively worsening phenotypes that could not be easily explained. Here, we combine genetic analysis of rib development with agent-based simulations to conclude that proximal-distal patterning and outgrowth could occur based on simple rules. In our model, specification occurs during somite stages due to varying Hedgehog protein levels, while later expansion refines the pattern. This framework is broadly applicable for understanding the mechanisms of skeletal patterning along a proximal-distal axis.

Compare to F1. Brackets outline comparable somite regions, curved line indicates forelimb bud location.Phenotypes were consistently observed in at least 3/3 animals of each genotype.When null for skeletal muscle genes, embryos can also display skeletal abnormalities, including rib fusions, truncations and sternal defects (Pax3: [Henderson et al., 1999;Vivian et al., 2000]).In Shh KO embryos epaxial progenitors are reportedly absent, while the hypaxial progenitors are reduced (Borycki et al., 1999;Gustafsson et al., 2002) and our results looking at Myf5 and MyoD expression confirmed this observation (A2, B2).Perhaps, absence of the epaxial compartment explains the failure of the proximal rib to form in both situations while additional defects in the hypaxial compartment are responsible for lost distal segments in the Shh;Apaf1 DKO embryos.However, we found that Shh;Apaf1 DKO embryos displayed the same pattern as Shh KO embryos (A3, B3) suggesting that a failure to specify the hypaxial muscle compartment is not the cause of the Shh;Apaf1 DKO phenotype.Pax9 and Pax1 are expressed in similar patterns in the sclerotome and are required for rib and vertebrae development (Furumoto et al., 1999;Peters et al., 1999;Rodrigo et al., 2003).Previous studies have shown that Pax1 is initially expressed in the Shh KO embryo but then lost (Chiang et al., 1996;Borycki et al., 1998;Zhang et al., 2001).As previously reported, we found Pax1 expressed in a smaller domain in Shh KO embryos compared to normal or Apaf1 KO embryos at E9.0 (C2).However, in Shh;Apaf1 DKO embryos, Pax1 expression was similar suggesting that some sclerotome specification still occurs (C3).As development continues, Pax1 expression is eventually lost in Shh KO embryos as well as in the Shh;Apaf1 DKO embryos.Neither Shh KO nor Shh;Apaf1 DKO embryos exhibited Pax9 expression in somites at E9.5 (B2-B3).
We then determined if the expression of Foxc2, a winged helix/forkhead transcription factor required for axial skeletal development (Kume et al., 2001;Wilm et al., 2004) is altered.Foxc2, which is expressed in the early unsegmented paraxial mesoderm and later becomes restricted to the sclerotome, may be a more comprehensive sclerotome marker than Pax1/9 (C1, D1) (Furumoto et al., 1999).Like Pax1, Foxc2 was expressed in a reduced domain in both Shh KO and the Shh;Apaf1 DKO embryos compared to normal embryos prior to E9.0, (E2, E3).However, in contrast to Pax1, the smaller domain of Foxc2 expression continued to be present after E10.0 in both Shh KO embryos and somite-matched Shh;Apaf1 DKO embryos (F2, F3).In summary, the loss of Shh correlates with smaller Pax1 and Foxc2 expression domains.In addition, the Pax1 domain disappears while the smaller FoxC2 expression domain persists, suggesting that at least a portion of the sclerotome is maintained in both contexts.Compared to control embryos, Apaf1 KO embryos did not display disruptions in sclerotome patterning as assessed by RNA in situ marker expression analysis (G, H, I).DOI: https://doi.org/10.7554/eLife.29144.007Specification appears to occur early (during somitogenesis), while the population of cells resides within the range of the Hh gradient, and then expansion and differentiation occur during embryo growth with proximal and distal fates being maintained by some sort of community effect independent of the influence of Hh. (B) We suggest that during normal development, Phase 1 involves the biological processes of sclerotome induction, cell death, and proximal-distal patterning, while Phase 2 involves expansion of cell number dependent on: (1) the events of Phase 1, (2) the proliferation rate, and (3) the constraints of the space in which expansion happens (body size).In the absence of Apaf1, development is largely normal as a slighly smaller initial progenitor pool due to decreased cell proliferation is mostly compensated for by decreased cell death.In Shh KO embryos, Hh signaling is greatly reduced during Phase 1 and thus the number of cells induced to become sclerotome is reduced (much smaller initial size).In addition, low Hh signaling specifies predominantly distal sclerotome.Despite a high level of cell death, a sufficient number of cells proliferate and are able to differentiate into the most distal segment during Phase 2. When Hh signaling is not as dramatically reduced (as in Shh hypomorphs) distal and some proximal specification can occur.In Shh:Apaf1 DKO embryos, however, along with reduced sclerotome due to both loss of Shh (smaller initial size), cell proliferation in the somite is also further reduced.Thus, ultimately fewer skeletal progenitors are generated during Phase 2. These embryos Figure 7

Figure 1 .Figure 2 .
Figure 1.Rib skeletal development is compromised in Shh null animals.(A) Frontal 1/3 view of the thoracic cage depicting the orientation of the proximal and distal ribs.Mice have 13 pairs of ribs.(B) Schematic of a vertebra and rib, transverse view.Red represents bone including the proximal/ vertebral rib and blue represents the cartilaginous distal/sternal rib.(C) The somite (Som), neural tube (NT), and notochord diagramed in cross-section.The dermatome and myotome (dark and light green) gives rise to the dermis and muscles while the sclerotome (yellow) gives rise to the vertebrae and ribs.Markers for these compartments are indicated.The location of Shh-expressing cells is indicated in black.(D-G) Alcian blue and alizarin red skeletal staining of the rib cage: (D) Normal mouse skeletal development at E18.5 (lower arms removed).(E) Without Apaf1, the axial skeleton develops normally (n = 12/12, lower arms removed).(F) Without Shh, embryos develop without vertebrae and the proximal portion of the ribs.Distal ribs form but are not patterned correctly (n = 17/17).(G) Double knock out (DKO) of both Shh and Apaf1 results in a more severe phenotype.DKO neonates develop without vertebrae, proximal and distal ribs (n = 7/7).The sternum is still present and ossifies on schedule.(H-J) Schematics representing skeletal preparations of normal (H) and null neonates.(I) The loss of the proximal ribs is consistent amongst all Shh KO neonates, however, the Figure 1 continued on next page Figure 3:

Figure 3 .Figure 3 -Figure 3 Figure 3 -
Figure 3. Conditional removal of Shh shows an early role for Hh signaling in rib development.(A, B) Shh is expressed not only in the floor plate but also in the dorsal root ganglia, ventral neural tube, developing lungs (yellow arrowheads), as well as the developing ribs themselves (white arrowhead).(C-H) Alcian blue and alizarin red staining of neonates in which an inducible Foxa2-CRE-ERT2 was used to ablate Shh only in the notochord and floor plate.(C) An E18.5 Foxa2-CRE-ERT2;Shh -/fl neonate after a Tamoxifen injection at E7.0 exhibits a similar phenotype to Shh null animals.Neonates develop without vertebrae and proximal ribs (inset -dorsal view).Distal ribs develop with mild segmental defects.(D) The additional loss of a Casp3 in the Shh hypomorph worsened the phenotype.Note a reduction of the distal segments compared to the Shh hypomorph alone (inset -dorsal view shows lack of vertebrae and proximal ribs).(E) Normal embryo, ventral (top) and dorsal view (bottom).(F-H) Range of hypomorph phenotypes.Those with the least severe phenotype (F) have some disruption of the vertebrae and the most proximal ends of the proximal ribs, while the most severe phenotype is similar to Shh KO embryos with complete loss of vertebrae and proximal ribs (H).Phenotypes were observed in at least 3/3 animals of each genotype.Ptch1 expression after Tamoxifen injection at E7.0 and E8.0 was assessed at E8.0 and E.9.0, respectively, see Figure 3-figure supplement 1. Skeletal defects could be due to alterations in somite patterning.Experiments addressing this possibility can be found in Figure 3figure supplement 2. DOI: https://doi.org/10.7554/eLife.29144.005

FigureFigure 4 .
Figure 4: Cartilage differen tiation fails in double null embryos A. differentiation B-D.shows an external lateral view, second pan an internal lateral view, asterisk indicate Sox9 embryos express from the dorsal midline toward the sternu (S).expression domain (thinner and shorter) an located distally.have a further reduced the distal domain.E-J expressing condensed cartilage forming normal and DKO embryos have only a loose aggregate Sox9-expressing cells and very few loose distributed cells, if any, expressing K-M.band of cells from the vertebrae towards th sternum ( Col2a1 (arrowheads).observed in thoracic area.N-P. in controls ( embryos, distally (arrowheads).undetectable in the entire thoracic area Shh;Apaf1 Q-S.embryos is shown in black and white f greater contrast.normal embryos extend from the vertebra towards the sternum.embryos have distally-located rib cartilage (arrowheads).rib cartilages are observed in DKO embryos.Exp served in at least 3/3 animals of each gen type.

Figure 5 .
Figure 5. Alterations in somite size and proliferation are evident in the different genotypes.(A-D) Representative longitudinal views (top, limb bud indicated by grey oval) and transverse sections (bottom) from somite-matched E9.0 mouse embryos at equivalent locations (based on anatomical landmarks).Phosphorylated histone H3 (pHH3) immunofluorescence reveals cells in mitosis (green: pHH3; blue: DAPI).(E).(Top) Ratio of average somite size to average litter-matched controls size is plotted directly.(Bottom) The probability distribution of Bayesian estimates for size ratios is shown in box plot format.Bayesian estimates indicate that Shh KO and Shh;Apaf1 DKO embryos have smaller somites than either controls or Apaf1 KO embryos.Median estimates are Apaf1 KO = 0.92, Shh KO = 0.59 and Shh;Apaf1 DKO = 0.57, probability that the ratios are less than 1 are 0.67, 0.99, and 0.99, Figure 5 continued on next page