Research Departments & Organizations
Limbs have played a crucial role in animal evolution. The adaptive evolution of vertebrates to aquatic, terrestrial, and aerial environments involved the acquisition and modification of their limbs. Over the past several decades, labs studying limb development in the mouse and chicken have identified a number of important signaling centers in the limb as well as several key molecules required for patterning the limb. Despite these advances, there are gaps in our understanding of how a limb is built. There is much to learn about the early stages of limb development and the identification of new genes required for limb growth and patterning is needed. We use mice to fill in some of these gaps by exploring limb formation, patterning and growth.
Specialized Terms: Limb development; Developmental genetics; Organogenesis; Mouse genetics; Signaling pathways; Embryogenesis
Extensive Research Description
Limbs have played a crucial role in animal evolution. The evolution of wings is recognized as the key innovation
behind the evolutionary success of the insects, while the adaptive evolution of
vertebrates to aquatic, terrestrial, and aerial environments involved the
acquisition and modification of their limbs. My interests in the establishment, patterning and
development of limbs have led me to work in both arthropods and chordates.
Graduate research: As a graduate student with Sean Carroll at the University of Wisconsin, Madison, I investigated the regulatory networks governed by the Homeotic (Hox) genes that control the differentiation of serially homologous structures within an individual species and the divergence of homologs between species. I utilized the insect wing (in the fruitfly Drosophila melanogaster and the butterfly Precis coenia) as a model system to gain a fundamental understanding of how the Hox class of transcription factors controlled the placement and morphology of insect flight appendages and how this regulation might have changed over the course of evolution. We initially set out to better define the role of Hox genes in regulating wing number. I discovered that no Hox genes were required to promote wing development, in Drosophila contrasting with previous notions, but instead that Hox genes in wingless segments repress wing development.
I further examined Hox gene expression in a primitive wingless insect and discovered that the domains of Ultrabithorax (Ubx) and Abdominal-A (Abd-A) were largely similar to those in Drosophila. As wing-like protrusions first appear on all segments in fossil insects, this suggests that Hox genes acquired their wing-repressive role over the course of insect wing evolution. I went on to show that in addition to its wing-repressive ability in the abdomen, Ubx regulates the identity of the D. melanogaster hindwing (haltere) by acting independently on selected subsets of genes downstream of the global wing organizing signals and at multiple levels of the wing developmental patterning hierarchy. Further, using a unique strain of P. coenia, which displayed homeotic transformations on their hindwings, I showed that Ubx regulates a different set of target genes in butterflies compared to fruit flies. Together, these studies have led to a deeper understanding of how the Hox genes control and diversify body plans and of their role in the evolution of body parts and patterns.
Postdoctoral research: As a postdoctoral fellow, I examined the genetic and molecular mechanisms that regulate patterning and morphogenesis of the mammalian limb. I worked with Drs. Lee Niswander and Kathryn Anderson at Memorial Sloan Kettering Cancer Center. Part of my work focused on a gene, Lrp4 that is required for limb patterning. I have studied how Lrp4 acts in the limb in conjunction with other developmental pathways to specify the AER and have explored how this gene ties into known developmental genetic pathways. In the course of these studies, I found that Lrp4 is required for a variety of different developmental processes, including formation of the neuromuscular junction. In parallel studies, I also studied how homologous limbs diverge in different mammalian lineages by comparing the mechanisms of limb patterning between the mouse and the bat. Lrp4 is a critical factor for limb patterning and development I identified Lrp4 as a gene mutated in 2 mouse lines derived from a forward genetics screen. Lrp4 is required at the early limb bud stage (E9.5), a time when the AER is first formed. In the absence of Lrp4 function, the AER cells fail to compact at the dorsal-ventral interface of the limb; the result is a broadened AER at the distal edge of the limb with cells that lack their characteristic columnar shape. As the AER is a crucial organizer of limb outgrowth, disruptions in its morphogenesis can explain most of the subsequent defects in limb development in Lrp4 mutants. Based on homology, Lrp4 is most closely related to Lrp5 and Lrp6, which are required for canonical Wnt signaling, however I tested the hypothesis that Lrp4 affects Wnt signaling and concluded that this is not the case in the early limb. Lrp4 is expressed in a complex pattern during development, including the teeth, kidneys, mammary buds and vibrissae, and that mutants show defects in each of the tissues where the gene is expressed.
Divergence of vertebrate limb morphology: how the bat got its wings I have continued my interest in the morphological diversification of homologous structures by studying the bat wing. The evolution of bat wing membranes and powered flight must have depended on mechanisms to retain and elaborate interdigit tissue. I identified a novel mechanism utilized in the bat wing to prevent apoptosis and interdigital regression involving a unique domain of Fgf8 expression in bat forelimb interdigits. In addition, high expression levels of the potent Bmp inhibitor Gremlin is found in bat forelimb interdigits but not in the un-webbed hindlimbs. By growing bat limbs in culture and experimentally manipulating these signals, I found that the combined action of increased Bmp and decreased Fgf signaling results in extensive forelimb interdigital apoptosis. Thus it appears that in the bat wing, inhibition of Bmp and activation of Fgf signaling cooperate to prevent interdigital cell death. These data indicate that although there is not a conserved mechanism for maintaining interdigit tissue across amniotes. In the bat, the expression in the bat forelimb interdigits of Gremlin and Fgf8 suggests that these key molecular changes underlie the evolution of the bat wing membrane.
Ongoing and future research: Characterization of additional genes affecting limb morphology From a subsequent round of mutagenesis screening in Dr. Anderson’s lab, I identified 3 additional mutant lines that display abnormal limb patterning and morphogenesis, as well as defects in other organs. We are continuing to work on these lines to identify the affected gene and determine the roles of these genes in limb and organ development. Lrp4 and Shd, two genes that control formation of the AER The AER plays a pivotal role in limb development through its signaling interactions with limb bud mesenchyme. Despite recent advances in defining some of the major genes necessary to form a limb, our understanding of how the AER is established and refined through the migration and compaction of Fgf8-expressing cells is not understood. I am currently working with two mutant lines, Lrp4 and a new mutant line, shorthand (shd), that show dorsal-ventral expansion of the AER during early limb development to examine the early steps of AER morphogenesis and AER maintenance. While the role of Lrp4 in limb development is unresolved, the early expansion of the AER suggests that Lrp4 is critical for determining the number of cells adopting an AER fate and also for setting the boundaries of the AER. My in vivo studies demonstrate expansion of both Fgf and Bmp signaling in the early Lrp4 mutant limbs. As both of these signaling pathways contribute to AER formation and Fgf8 expression in the early limb, the expansion of either could underlie the broadening of the AER in Lrp4 mutants.
Our preliminary experiments in Xenopus (in collaboration with Dr. Xi He’s laboratory) do not corroborate this. Dorsal injection of Lrp4 mRNA does not inhibit Wnt-mediated dorsal axis specification and co-injection of Lrp4 and Wnt8 did not inhibit activation of the Wnt-target Xnr3. Thus, further study is required to determine the signaling pathway(s) affected by Lrp4. The expanded AER phenotype in line shd occurs slightly later than Lrp4 mutants, suggesting that maintenance of the normal boundaries of the AER is disrupted in shd mutants. Exploration of this phenotype will shed light on the factors required for maintenance of a discreet border between AER and limb ectoderm. Line shd has been mapped to a gene-dense genomic interval and should be cloned shortly. How the early signaling pathways, including Bmps and Wnts converge on Fgf8 in the early limb and what factors regulate the morphology of AER are not understood. In my laboratory, I will use genetic interaction experiments between Lrp4, shd, Wnt and Bmp pathway components and their inhibitors to define the role of these genes in establishing normal number of cells in the AER, and setting its borders and directing AER morphogenesis. We are continuing to work with these mutants to better define how Lrp4 and shd regulate the AER.
In terms of human relevance, one of the most common types of birth defects is deformation of the limbs. However, little is known about the developmental, genetic and molecular mechanisms underlying these defects. Identification of the molecular mechanisms that pattern limbs will lead to a better understanding of the possible causes of human limb defects.
- To broaden our insight into the genetic mechanisms underlying limb formation and patterning we are studying novel mutants with limb defects that were identified in a forward genetics screen in the mouse. Two of these mutants, schlei and kerouac show preaxial polydactyly and affect anterior-posterior patterning. In contrast, shorthand displays brachydactyly, or shortening of the phalanges. The genomic regions linked to these mutants do not contain known limb patterning genes, indicating that we have identified novel genes required for limb development. The characterization and cloning of the genes affected in these mutants should provide a better understanding of anterior-posterior and proximal-distal patterning during limb development.
- The lab is also continuing to work on a pair of mutants that were identified in two independent screens. These lines both contain mutations in the low density lipoprotein related 4 gene (Lrp4). Lrp4 is essential for proper patterning along all three of the major axes during limb development. In addition, Lrp4 is required for neuromuscular junction formation and normal development of ectodermal organs such as mammary buds, teeth and vibrissae. We are interested in defining how Lrp4 regulates the development of these vastly different organs and in determining which signaling pathways Lrp4 interacts with.
- We feel that we’ve only seen the tip of the iceberg and that there are many more genes required for limb development that remain to be discovered. We plan to continue screening to identify additional novel genes that are required for normal limb development. Together, these studies will give depth to the current models of limb developmental regulatory hierarchies and provide a basis for understanding human birth defects, specifically those affecting the limb.
Forward genetics defines Xylt1 as a key, conserved regulator of early chondrocyte maturation and skeletal length.
Mis EK, Liem KF, Kong Y, Schwartz NB, Domowicz M, Weatherbee SD. Forward genetics defines Xylt1 as a key, conserved regulator of early chondrocyte maturation and skeletal length. Developmental Biology 2014, 385:67-82. 2014
Forward genetics identifies Kdf1/1810019J16Rik as an essential regulator of the proliferation-differentiation decision in epidermal progenitor cells.
Lee S, Kong Y, Weatherbee SD. Forward genetics identifies Kdf1/1810019J16Rik as an essential regulator of the proliferation-differentiation decision in epidermal progenitor cells. Developmental Biology 2013, 383:201-13. 2013
Forward genetics uncovers Transmembrane protein 107 as a novel factor required for ciliogenesis and Sonic hedgehog signaling.
Christopher KJ, Wang B, Kong Y, Weatherbee SD. Forward genetics uncovers Transmembrane protein 107 as a novel factor required for ciliogenesis and Sonic hedgehog signaling. Developmental Biology 2012, 368:382-92. 2012
Renal cystic disease proteins play critical roles in the organization of the olfactory epithelium.
Pluznick JL, Rodriguez-Gil DJ, Hull M, Mistry K, Gattone V, Johnson CA, Weatherbee S, Greer CA, Caplan MJ. Renal cystic disease proteins play critical roles in the organization of the olfactory epithelium. PloS One 2011, 6:e19694. 2011
Synergistic interaction between ciliary genes reflects the importance of mutational load in ciliopathies.
Lee S, Weatherbee SD. Synergistic interaction between ciliary genes reflects the importance of mutational load in ciliopathies. Journal Of The American Society Of Nephrology : JASN 2010, 21:724-6. 2010
A mouse model for Meckel syndrome reveals Mks1 is required for ciliogenesis and Hedgehog signaling.
Weatherbee SD, Niswander LA, Anderson KV. A mouse model for Meckel syndrome reveals Mks1 is required for ciliogenesis and Hedgehog signaling. Human Molecular Genetics 2009, 18:4565-75. 2009
Mammalian limbs take flight.
Weatherbee SD. Mammalian limbs take flight. Developmental Cell 2008, 14:149-50. 2008