Professor Emeritus (Active), Department of Biology
Ph.D., John Hopkins
Research Interests: Morphogenesis and evolutionary developmental biology of the skull
Overview: Our lab focuses on the cellular and genetic mechanisms that control the shaping of skull cartilages and bones during development, and on how development changes during evolution to produce the marvelous diversity that we observe the skulls in different kinds of animals. We use teleost fish for these studies. We take advantage of zebrafish as a model system for development and genetics, of stickleback as a model for evolutionary biology and evo devo, and of teleosts more generally, the most species rich of all vertebrate groups, for their outstanding phenotypic variation.
Cellular and genetic basis of skeletal shaping.
A general hypothesis that guides much of our work is that the key cellular determinant of skeletal shaping is the patterning of the local spatial arrangements of skeleton forming cells. Our work on development of the zebrafish opercle, a bone that provides support for the gill cover, shows aspects of this control remarkably clearly (Fig. 1). The bone lineage arises in a specific preskeletal condensation, under control of upstream extracellular signals including Endothelin1, which we have studied for many years [1,2].
Fig. 1. The zebrafish opercle (Alizarin red) acquires a new form as it develops by reorganizing osteoblast (green, sp7:eGFP) recruitment . Live imaging from 3 to 5 days post fertilization. From our web-based developmental Atlas FishFace.
Structured phenotypic variation in evoluationary change.
We see outstanding diversity of the shape of this very same (homologous) bone among teleosts. For example in many neoteleosts the opercle takes on a protective function, developing bony spines in a variety of configurations (Fig. 2).
Fig. 2. Varying arrangements of protective opercle spines.
A. hake, B. rockfish, C. paradox fish, D. weak fish.
We hypothesize that developmental modularity, which we discovered to underlie patterning of the bone’s shape, facilitates shape evolution. In this scenario, the development can evolve to produce a seemingly inexhaustible array of bone shapes because each local region of a bone (that we consider to be a developmental module) can be modified nearly independently of the others. Subsequently, osteoblasts differentiate at precisely regulated positions within the condensation, first appearing in an elongating linear row to produce a bony strut (A). Then, at the growing end of this row, osteoblast addition abruptly switches to a new pattern to locally broaden the region of bone outgrowth at this region (B), resulting in a fan-shaped(C). Other similar reorganizations occur subsequently . These first two phases are under separate genetic control: Mutation of the transcription-factor encoding gene mef2ca specifically disrupts the first . Mutation of Indian hedgehog_a gene, encoding a local extracellular protein signal, disrupts the second . This temporal and spatial specificity in morphogenesis and its control reveals modularity, meaning that separate local regions of the bone develop largely autonomously from one another. Modularity is an important concept in our understanding of developmental patterning, and particularly, of how development evolves [3-7]. Such dissociability in phenotype is a hallmark of modularity. We can use stickleback to test this hypothesis. The opercle and associated facial bones all evolve new shapes as stickleback evolve from ocean-dwelling to freshwater forms [6,7]. Dorsally the bone expands and ventrally it contracts. We predicted that the dorsal and ventral regions, showing separate kinds of evolutionary modification, are separate developmental modules. In the example shown, our hypothetical boundary between the modules, shown by the thick line crossing the two bones examined (Fig. 3A), is supported by multivariate statistical analysis (Fig. 3B; see citations [7-9]). These results are important because they indicate that the way development structures phenotype into modules can impact morphological evolutionary change. The findings provide an answer, at least for this system, to long-standing central question in the field of evo devo: Can development bias evolution? Our evidence suggests that, indeed, it can.
Fig. 3. Evolutionary divergence in stickleback predicts a module boundary crossing the opercle & subopercle (A). Partial least squares analysis reveals that the covariance between these hypothetical modules is among the lowest of all possible subdivisions of the two bone configuration (B, arrow), supporting the hypothesis. This is because separate models, by definition, are semiautonomous, and hence show only low covariance between them .
'Unstructured' phenotypic variation in development and disease.
Phenotypic variation in evolving natural populations (as just described) clearly is highly structured by Darwinian selection, and likely by developmental modularity as well. In contrast, there are plenty of examples of rather ‘unstructured’ variation showing up nonadaptively in genetic mutants. This variation is thought to be due to loss of a process of buffering of stochastic noise associated with developmental processes, and understanding the buffering is important because its loss occurs in many kinds of human diseases. In ongoing collaborative studies with John Postlethwait’s lab we are studying mutations in two zebrafish genes that show ‘noisy’ variation in their phenotypes, to try to learn more about the mechanism of phenotypic buffering. A first step is to find out where in the developmental process the variation seems to arise. One of these zebrafish genes is fras1, encoding an extracellular matrix protein that is a critical component of a signaling/adhesion complex. Mutations in the human FRAS1 ortholog underlie Fraser’s syndrome, a rare but serious disorder that also shows a good deal of phenotypic noise. Our time-lapse studies in zebrafish reveal that the very early stages of cartilage morphogenesis, when the cartilage primordia outgrow in association with pharyngeal epithelia, appear variably disrupted . We hypothesize that the Fras1 protein complex is an essential component of morphogenesis, mediating mesenchymal-epithelial interaction, as we are testing with conditional mutant alleles. The second gene, already mentioned above, is mef2ca. The level of noisy phenotypic variation in mef2ca mutants is remarkable (Fig. 4) . We carried out a systematic study to see when we could first detect the variation, and pinpointed this time to the earliest stage of morphogenesis (i.e. corresponding to the stage shown in Fig. 1A, above). It may be more than coincidence that both fras1 and mef2ca appear to be required for buffering morphogenesis specifically (cartilage on one hand and bone on the other). Morphogenesis may be a particularly intricate process, especially prone to disturbance by stochastic noise.
Fig 4. Loss of developmental buffering in mef2ca mutants produces outstanding phenotypic variation, here shown in eight individual mutant larvae. The first example shows an essentially wild-type morphology of both the opercle (Op) and branchiostegal ray (BR). Live imaging, Alizarin red staining.
A rich diversity of opercle bone shape among teleost fishes.
PLoS One. 2017;12(12):e0188888
Authors: Kimmel CB, Small CM, Knope ML
The opercle is a prominent craniofacial bone supporting the gill cover in all bony fish and has been the subject of morphological, developmental, and genetic investigation. We surveyed the shapes of this bone among 110 families spanning the teleost tree and examined its pattern of occupancy in a principal component-based morphospace. Contrasting with expectations from the literature that suggest the local morphospace would be only sparsely occupied, we find primarily dense, broad filling of the morphological landscape, indicating rich diversity. Phylomorphospace plots suggest that dynamic evolution underlies the observed spatial patterning. Evolutionary transits through the morphospaces are sometimes long, and occur in a variety of directions. The trajectories seem to represent both evolutionary divergences and convergences, the latter supported by convevol analysis. We suggest that that this pattern of occupancy reflects the various adaptations of different groups of fishes, seemingly paralleling their diverse marine and freshwater ecologies and life histories. Opercle shape evolution within the acanthomorphs, spiny ray-finned fishes, appears to have been especially dynamic.
PMID: 29281662 [PubMed - indexed for MEDLINE]
Ligament versus bone cell identity in the zebrafish hyoid skeleton is regulated by mef2ca.
Development. 2016 12 01;143(23):4430-4440
Authors: Nichols JT, Blanco-Sánchez B, Brooks EP, Parthasarathy R, Dowd J, Subramanian A, Nachtrab G, Poss KD, Schilling TF, Kimmel CB
Heightened phenotypic variation among mutant animals is a well-known, but poorly understood phenomenon. One hypothetical mechanism accounting for mutant phenotypic variation is progenitor cells variably choosing between two alternative fates during development. Zebrafish mef2cab1086 mutants develop tremendously variable ectopic bone in their hyoid craniofacial skeleton. Here, we report evidence that a key component of this phenotype is variable fate switching from ligament to bone. We discover that a 'track' of tissue prone to become bone cells is a previously undescribed ligament. Fate-switch variability is heritable, and comparing mutant strains selectively bred to high and low penetrance revealed differential mef2ca mutant transcript expression between high and low penetrance strains. Consistent with this, experimental manipulation of mef2ca mutant transcripts modifies the penetrance of the fate switch. Furthermore, we discovered a transposable element that resides immediately upstream of the mef2ca locus and is differentially DNA methylated in the two strains, correlating with differential mef2ca expression. We propose that variable transposon epigenetic silencing underlies the variable mef2ca mutant bone phenotype, and could be a widespread mechanism of phenotypic variability in animals.
PMID: 27789622 [PubMed - indexed for MEDLINE]
Pharyngeal morphogenesis requires fras1-itga8-dependent epithelial-mesenchymal interaction.
Dev Biol. 2016 08 01;416(1):136-148
Authors: Talbot JC, Nichols JT, Yan YL, Leonard IF, BreMiller RA, Amacher SL, Postlethwait JH, Kimmel CB
Both Fras1 and Itga8 connect mesenchymal cells to epithelia by way of an extracellular 'Fraser protein complex' that functions in signaling and adhesion; these proteins are vital to the development of several vertebrate organs. We previously found that zebrafish fras1 mutants have craniofacial defects, specifically, shortened symplectic cartilages and cartilage fusions that spare joint elements. During a forward mutagenesis screen, we identified a new zebrafish mutation, b1161, that we show here disrupts itga8, as confirmed using CRISPR-generated itga8 alleles. fras1 and itga8 single mutants and double mutants have similar craniofacial phenotypes, a result expected if loss of either gene disrupts function of the Fraser protein complex. Unlike fras1 mutants or other Fraser-related mutants, itga8 mutants do not show blistered tail fins. Thus, the function of the Fraser complex differs in the craniofacial skeleton and the tail fin. Focusing on the face, we find that itga8 mutants consistently show defective outpocketing of a late-forming portion of the first pharyngeal pouch, and variably express skeletal defects, matching previously characterized fras1 mutant phenotypes. In itga8 and fras1 mutants, skeletal severity varies markedly between sides, indicating that both mutants have increased developmental instability. Whereas fras1 is expressed in epithelia, we show that itga8 is expressed complementarily in facial mesenchyme. Paired with the observed phenotypic similarity, this expression indicates that the genes function in epithelial-mesenchymal interactions. Similar interactions between Fras1 and Itga8 have previously been found in mouse kidney, where these genes both regulate Nephronectin (Npnt) protein abundance. We find that zebrafish facial tissues express both npnt and the Fraser gene fibrillin2b (fbn2b), but their transcript levels do not depend on fras1 or itga8 function. Using a revertible fras1 allele, we find that the critical window for fras1 function in the craniofacial skeleton is between 1.5 and 3 days post fertilization, which coincides with the onset of fras1-dependent and itga8-dependent morphogenesis. We propose a model wherein Fras1 and Itga8 interact during late pharyngeal pouch morphogenesis to sculpt pharyngeal arches through epithelial-mesenchymal interactions, thereby stabilizing the developing craniofacial skeleton.
PMID: 27265864 [PubMed - indexed for MEDLINE]
Epigenetic regulation of hematopoiesis by DNA methylation.
Elife. 2016 Jan 27;5:e11813
Authors: Gore AV, Athans B, Iben JR, Johnson K, Russanova V, Castranova D, Pham VN, Butler MG, Williams-Simons L, Nichols JT, Bresciani E, Feldman B, Kimmel CB, Liu PP, Weinstein BM
During embryonic development, cell type-specific transcription factors promote cell identities, while epigenetic modifications are thought to contribute to maintain these cell fates. Our understanding of how genetic and epigenetic modes of regulation work together to establish and maintain cellular identity is still limited, however. Here, we show that DNA methyltransferase 3bb.1 (dnmt3bb.1) is essential for maintenance of hematopoietic stem and progenitor cell (HSPC) fate as part of an early Notch-runx1-cmyb HSPC specification pathway in the zebrafish. Dnmt3bb.1 is expressed in HSPC downstream from Notch1 and runx1, and loss of Dnmt3bb.1 activity leads to reduced cmyb locus methylation, reduced cmyb expression, and gradual reduction in HSPCs. Ectopic overexpression of dnmt3bb.1 in non-hematopoietic cells is sufficient to methylate the cmyb locus, promote cmyb expression, and promote hematopoietic development. Our results reveal an epigenetic mechanism supporting the maintenance of hematopoietic cell fate via DNA methylation-mediated perdurance of a key transcription factor in HSPCs.
PMID: 26814702 [PubMed - indexed for MEDLINE]
Patterns of variation and covariation in the shapes of mandibular bones of juvenile salmonids in the genus Oncorhynchus.
Evol Dev. 2015 Sep-Oct;17(5):302-14
Authors: Kimmel CB, Watson S, Couture RB, McKibben NS, Nichols JT, Richardson SE, Noakes DL
What is the nature of evolutionary divergence of the jaw skeleton within the genus Oncorhynchus? How can two associated bones evolve new shapes and still maintain functional integration? Here, we introduce and test a "concordance" hypothesis, in which an extraordinary matching of the evolutionary shape changes of the dentary and angular articular serves to preserve their fitting together. To test this hypothesis, we examined morphologies of the dentary and angular articular at parr (juvenile) stage, and at three levels of biological organization—between salmon and trout, between sister species within both salmon and trout, and among three types differing in life histories within one species, Oncorhynchus mykiss. The comparisons show bone shape divergences among the groups at each level; morphological divergence between salmon and trout is marked even at this relatively early life history stage. We observed substantial matching between the two mandibular bones in both pattern and amount of shape variation, and in shape covariation across species. These findings strongly support the concordance hypothesis, and reflect functional and/or developmental constraint on morphological evolution. We present evidence for developmental modularity within both bones. The locations of module boundaries were predicted from the patterns of evolutionary divergences, and for the dentary, at least, would appear to facilitate its functional association with the angular articular. The modularity results suggest that development has biased the course of evolution.
PMID: 26372063 [PubMed - indexed for MEDLINE]
Building the backbone: the development and evolution of vertebral patterning.
Development. 2015 May 15;142(10):1733-44
Authors: Fleming A, Kishida MG, Kimmel CB, Keynes RJ
The segmented vertebral column comprises a repeat series of vertebrae, each consisting of two key components: the vertebral body (or centrum) and the vertebral arches. Despite being a defining feature of the vertebrates, much remains to be understood about vertebral development and evolution. Particular controversy surrounds whether vertebral component structures are homologous across vertebrates, how somite and vertebral patterning are connected, and the developmental origin of vertebral bone-mineralizing cells. Here, we assemble evidence from ichthyologists, palaeontologists and developmental biologists to consider these issues. Vertebral arch elements were present in early stem vertebrates, whereas centra arose later. We argue that centra are homologous among jawed vertebrates, and review evidence in teleosts that the notochord plays an instructive role in segmental patterning, alongside the somites, and contributes to mineralization. By clarifying the evolutionary relationship between centra and arches, and their varying modes of skeletal mineralization, we can better appreciate the detailed mechanisms that regulate and diversify vertebral patterning.
PMID: 25968309 [PubMed - indexed for MEDLINE]
Skull developmental modularity: a view from a single bone - or two.
J Appl Ichthyol. 2014 Aug 01;30(4):600-607
Authors: Kimmel CB
I review recent studies that connect development and evolution of skull bones in teleosts. Development uses genetic information to build a structured, modular phenotype, and since selection acts on the phenotype, developmental modularity may influence evolvability. Just how is a complex developing morphology spatially partitioned into modules? Here I briefly examine cellular, molecular genetic, and multivariate statistical approaches to the identification of developmental modules. Furthermore I review our evidence that developmental modularity provides evolutionarily labile regions within the skull and hence potentially biases evolutionary change in a positive manner. This view is rather different from early ones in the field of evolutionary developmental biology, in which developmental constraint due to patterns such as heterochronies were supposed to negatively impact evolution.
PMID: 25294950 [PubMed]
Role of mef2ca in developmental buffering of the zebrafish larval hyoid dermal skeleton.
Dev Biol. 2014 Jan 15;385(2):189-99
Authors: DeLaurier A, Huycke TR, Nichols JT, Swartz ME, Larsen A, Walker C, Dowd J, Pan L, Moens CB, Kimmel CB
Phenotypic robustness requires a process of developmental buffering that is largely not understood, but which can be disrupted by mutations. Here we show that in mef2ca(b1086) loss of function mutant embryos and early larvae, development of craniofacial hyoid bones, the opercle (Op) and branchiostegal ray (BR), becomes remarkably unstable; the large magnitude of the instability serves as a positive attribute to learn about features of this developmental buffering. The OpBR mutant phenotype variably includes bone expansion and fusion, Op duplication, and BR homeosis. Formation of a novel bone strut, or a bone bridge connecting the Op and BR together occurs frequently. We find no evidence that the phenotypic stability in the wild type is provided by redundancy between mef2ca and its co-ortholog mef2cb, or that it is related to the selector (homeotic) gene function of mef2ca. Changes in dorsal-ventral patterning of the hyoid arch also might not contribute to phenotypic instability in mutants. However, subsequent development of the bone lineage itself, including osteoblast differentiation and morphogenetic outgrowth, shows marked variation. Hence, steps along the developmental trajectory appear differentially sensitive to the loss of buffering, providing focus for the future study.
PMID: 24269905 [PubMed - indexed for MEDLINE]