While we saw no evidence of strict cellular compartmentalization along the proximal distal axis at the stages analyzed, we did observe a strong tendency for clonally related cells to segregate into either the dorsal or ventral regions of the mature limbs. Out of a total of 164 identified clonal cell populations from injections at stages HH16, HH18, and HH20, only one contained a single cell that crossed the dorsal-ventral boundary based on using the skeletal elements as the dorsoventral midline. Of the remaining clonal populations, 116 were completely restricted to either the dorsal or ventral regions of the limb (). However, at this stage of limb development, it is impossible to define a clear dorsal ventral boundary within the condensed cartilage and developing perichondrium. Because of this, clones that contained cartilage or perichondrium cells could not be classified as either dorsally or ventrally restricted. That is, in an otherwise dorsally restricted clone, cartilage cells belonging to that clone could be found at the ventral-most extent of the cartilage tissue and vice versa for otherwise ventrally-restricted clones. Despite the ambiguity in assignment of cells in the cartilage and perichondrium to either a dorsal or ventral identity, this observation that clonal descendants of single cells infected at stages 16–20 have a strong predilection toward dorsal or ventral restriction suggested to us that earlier in development there may exist a boundary in the limb bud mesenchyme that is restrictive to cellular mixing.
Figure 5 A dorsal/ventral boundary to cellular migration in the limb bud mesenchyme. (A) Summary of dorsal/ventral CHAPOL analysis of chicks harvested at HH35. Stages of embryo injections are listed on the left. Clones are classified as dorsally restricted (Dorsal), (more ...)
The LIM-homeobox gene Lmx1b
marks a dorsal domain in the mesenchyme of the early limb bud in both chick () and mouse and it has been demonstrated that Lmx1b
expression is both necessary (Dreyer et al., 1998
; Chen et al., 1998
) and sufficient (Riddle et al., 1995
; Vogel et al., 1995
) to direct dorsal pattern formation. Because lmx1b
is expressed in the dorsal mesenchyme from the earliest stage of limb development, it allows us to more accurately mark the dorsoventral location of cells within the early limb bud and to test whether an observed dorsoventral restriction obeys this molecular boundary.
We repeated the lineage analysis injecting at a stage HH11 which would generate two cell clones in the budding lateral plate mesoderm just as Lmx1b expression is first seen (early HH16). We harvested these samples at stage HH24 and analyzed the expansion of clonal populations relative to the Lmx1b expression boundary.
We identified a total of 18 clones in our analysis, 16 of which were restricted to either the dorsal Lmx1b positive compartment or to the ventral Lmx1b negative compartment (). One of the clones was classified as a border clone, all of its cells being right at the dorsal/ventral boundary. Finally, one clone was predominantly ventral, but contained a single cell that was unambiguously in the Lmx1b positive dorsal domain. Thus, as in the original analysis, we observe a strong predilection toward restriction along the D/V axis, in this case with the Lmx1b expression domain marking the boundary. These observations could arise from three possible scenarios. The first is that the limb bud mesenchymal cells are actually restricted in their movements along the dorsal ventral axis during limb outgrowth, second we may see an apparent restriction due to a lack of dorsal to ventral spread of clonally related cells, and third we may have misidentified unrestricted clones as being restricted because of a combination of attrition during processing of the picked cells and of small clone sizes. The second possibility is unlikely since among the 16 restricted clones 12 (7 dorsal and 5 ventral) were large clones that approached the dorsal/ventral boundary, but failed to cross and 4 of the clones (3 dorsal and 1 ventral), spanned the entire domain in which they were restricted, still failing to cross (). Finally, it is unlikely that the observed restriction is due to a sampling error resulting from attrition and small clone sizes since many of the clones in this analysis were quite large compared to the original lineage analysis at stage HH35 (averaging 10 and going as high as 20 cells per clone).
The observation that Lmx1b marks a dorsal/ventral boundary that is resistant to cellular mixing raises the possibility that Lmx1b plays a causal role in preventing dorsal/ventral mixing. We attempted to test this possibility, re-engineering the CHAPOL library to misexpress Lmx1b in each infected cell and retesting the restriction of clones along the dorsal/ventral axis of the limb relative to endogenous Lmx1b. If Lmx1b indeed acts as a dorsal selector gene establishing the dorsal-ventral compartment boundary, then ventral clones of cells in which Lmx1b is misexpressed should be capable of crossing that boundary. However, in our analysis we were unable to identify Lmx1b misexpressing clones crossing the endogenous Lmx1b expression boundary (data not shown).
Recent work in two other laboratories using the mouse limb bud as their model system has suggested the existence of a dorsoventral compartment border defined by Lmx1b
expression (Arques et al. 2007
, and Qui et al., 2007
) and one of those additionally obtained genetic evidence that Lmx1b
itself is responsible for establishing the compartment (Qui et al., 2007
). Thus, it is likely that technical limitations, such as levels of Lmx1b
expression from our retroviral system, have prevented us from identifying boundary crossing clones with the Lmx1b-CHAPOL vector.
Our data also indicate that, since the D/V restricted clones in our experiment are the progeny of cells labeled in the lateral plate mesoderm, the dorsal ventral boundary is established very early and even prior to definitive limb bud outgrowth. This would predict that the dorsoventral axis of the limb is established when the limb field of the lateral plate mesoderm is still a relatively flat sheet of tissue and thus that the ultimate dorsal/ventral axis of the limb bud originates as the medial/lateral axis of the lateral plate mesoderm. A prediction of this model would be that early midline signals are responsible for establishing the future D/V axis of the limb bud. It has, in fact, been beautifully demonstrated that a signal from the developing somites initiates a program that establishes the dorsal limb identity in the lateral plate mesoderm (Michaud et al., 1997
; Ohuchi et al. 1999
). We also see that implanting a barrier between the somites and the lateral plate mesoderm to block signals from the midline at HH15, just prior to lmx1b
induction, will prevent the primary induction of lmx1b
in the nascent limb bud (). Taken together, the preponderance of data generated from multiple labs on the establishment if the limb D/V axis leads us to the conclusion that this axis of the limb is established in two distinct phases. The early phase consists of a signal originating from the HH13–14 somites and effecting events that lead to the primary induction of lmx1b
in the medial lateral plate mesoderm (Michaud et al., 1997
; Ohuchi et al. 1999
; and our data). This signal would appear to be independent of Wnt7a in the ectoderm as ectodermal reversals prior to HH15 have no effect on D/V patterning and Wnt7a
mutants still show Lmx1b
induction in the proximal limb bud (Geduspan and MacCabe 1987
; Cygan et al. 1997
). The second phase, starting at HH15 (Geduspan and MacCabe 1987
; Michaud et al. 1997
; Altebef et al. 1997
), is regulated by Wnt7a secreted from the dorsal ectoderm and is responsible for D/V patterning in the most distal limb tissues (autopod). Ectodermal reversals and loss of ectodermal Wnt7a result in alterations of D/V patterning in distal (autopodial), tissues (Geduspan and MacCabe 1987
; Geduspan and MacCabe 1989
; Riddle et al. 1995
; Cygan et al. 1997
). Thus, the early midline lmx1b
induction seems to be responsible for D/V polarity of the proximal (stylopod, zeugopod), elements of the limb while the later ectodermally maintained signal is responsible for distal (autopodial) D/V patterning.
We have shown that undifferentiated progenitor cells in the early limb bud are not committed to individual cell fates nor do they appear to be restricted to any particular proximodistal segment along the length of the limb bud. In contrast, the limb progenitors are specified at a very early stage to either a dorsal or ventral fate, although they are not restricted from moving freely within the dorsal or ventral compartments. Elucidating the mode by which fates are determined in the limb provides a deeper understanding of how limb morphogenesis is organized and will help pave the way for future studies directed towards identifying the factors and cell interactions that direct these fate choices.