Here I review the two major lines of research in which my laboratory has been engaged for the past 35 years. The first of these research programs concerns the description and analysis of social learning processes influencing food choices of Norway rats ( Rattus norvegicus ). The second involves social interactions affecting mate choices of Japanese quail ( Coturnix japonica ). Study of these model systems has shown that at least two biologically important behaviors, food choice and mate choice, can be shaped by social interactions and that the social interactions that bias behavioral development are open to reductionist analysis in terms of the behavior of interacting individuals.

Publish in high visibility journals. Acknowledge connection with comparative psychology. Stress positive features of comparative psychology rather than weaknesses of alternative approaches.

Spinal motor neurons (MNs) are a diverse group of specialized neurons that innervate and control all muscles of the body, thereby allowing locomotion and respiration. Over the past decade, many groups have used directed differentiation of pluripotent stem cells into MNs to model human MN development and disease in vitro. Despite these efforts, it remains unknown whether stem cell-derived MNs fully recapitulate endogenous MN populations. To address this question, this thesis presents the characterization of human embryonic and stem cell-derived MNs on molecular and physiological levels. We find that the processes of MN development and organization are largely conserved between mice and humans. However, the majority of stem cell-derived MNs only express characteristics of MNs that normally innervate axial muscles in vivo. To expand the diversity of stem cell-derived MNs, we propose an alternative method of differentiation using overexpression of MN fate determinants. Specifically, we find that overexpression of the transcription factor Foxp1 during stem cell differentiation results in a large number of dorsal and ventral limb-innervating MNs. Finally, we demonstrate that stem cell-derived MNs are capable of forming functional synapses with muscle cells in vitro. Together, this work provides a detailed analysis of the strengths and weaknesses of current MN differentiation protocols, and presents new approaches for increasing the functional diversity of stem cell-derived MNs. In addition to valuable insights into human development, this body of work provides important information for ongoing efforts of modeling MN disease with stem cells.

One of the key challenges of neural development is achieving the precise balance between progenitor self-renewal and differentiation. The production of early born neurons must be controlled such that the necessary numbers are generated without depleting the pool of progenitors for subsequent glial differentiation. This work reports the identification of two transcription factors, PLZF and RP58, as pivotal factors in the preservation of neural progenitor cells in the developing spinal cord through the period of neurogenesis. PLZF has been previously shown to promote the maintenance of both hematopoietic and male germline stem cells. This work will demonstrate that PLZF is expressed within a central domain of the developing spinal cord of chick and mouse embryos, that it is sufficient to oppose neuronal differentiation, and that its loss results in compromised progenitor maintenance and excessive neuron formation. Furthermore, it will be shown that PLZF functions in part by promoting the expression of FGF receptor 3 and thereby enhancing neural progenitor receptivity for the pro-progenitor FGF signaling pathway. Unlike PLZF, previous research has indicated that RP58 acts to promote cellular differentiation in the context of neocortical, hippocampal, and cerebellar development. However, in the context of the developing spinal cord, RP58 acts as a potent anti-differentiation factor. RP58 misexpression results in a greatly expanded neural progenitor pool whereas its reduction through RNAi knockdown results in rapid neuronal differentiation. This work will propose that this divergent activity of RP58 in the two fundamental regions of the CNS can be explained as different manifestations of the same gene regulatory module achieving different effects depending on the relative order in which its component parts are activated. Both PLZF and RP58 are members of the BTB-ZnF family of transcription factors, a family previously with only a limited known role in the development of the CNS. This work will close with a consideration of the prospects of PLZF and RP58 not only being important factors in maintaining neural progenitor cells, but as potential forerunners of a previously unappreciated family of spinal developmental regulators.

In a series of studies undertaken to determine the conditions under which naive house mice (observers) develop preferences for foods eaten by recently-fed conspecifics (demonstrators), we found that observer mice exhibited enhanced preference for a food following interaction with either a healthy or an ill recently-fed demonstrator that had eaten that food. We also found that house mice developed an enhanced preference for a food after exposure to an anesthetized conspecific demonstrator powdered with that food, but not after exposure to a cotton-batting, conspecific-sized surrogate powdered with the same food. Results of other studies have indicated that, for both rats and mice, the presence in a food of carbon disulfide (a substance found on the breath of rats) increases preference for a carbon-disulfide-contaminated food. taken together, the parallels between Norway rats and house mice in social learning processes suggest homologous rather than analogous systems of communication about distant foods in these two murid rodents.

The faithful and sophisticated control of motor neurons (MNs) allows for our ability to walk, chew, breathe, and speak. This dissertation is focused on the characterization of MNs and the intricate circuitry that are involved in the control of these MNs that surround the airways. The relevance of this study goes beyond satisfying our curiosity of the physiology of breathing. This knowledge is critical in their application in respiratory medicine and speech rehabilitation – as the same set of MNs generate rudimentary vocal sounds in rodents and may provide us with a greater understanding about speech motor pathways in humans.

The dissertation begins with a literature review of principles of circuit assembly predominantly centered in the spinal cord. In Chapter 2, I define the molecular organization of the cranial MNs in the brainstem. Unique transcription factors and guidance cues including Foxp1, Pou3f1, Etv4, and Npn2 label specific cranial motor pools that match previously described motor populations identified by classic retrograde labeling. This molecular definition of cranial motor pools opens up new doors for us to understand their origins to harness the potential for rehabilitation when these MNs are diseased or injured.

In Chapter 3, I demonstrate that the respiratory drive can extend to brachial limb-innervating MNs, a feature normally suppressed by Foxp1. In the absence of Foxp1, the limb-innervating MNs exhibit molecular and anatomical characteristics that resemble that of the thoracic respiratory MNs. The findings of this study provide another compelling evidence that the respiratory motor circuit can integrate MNs out of their usual field of innervation to harbor ectopic respiratory MNs – similar to the respiratory drive crossing the midline in the crossed phrenic phenomenon.

In Chapter 4, through genetic manipulation I show that Pou3f1, a marker for phrenic and other respiratory MN populations, is essential for proper the phrenic nerve branch projection and intercostal cell fate specification. Finally in Chapter 5, I summarize the key findings of my dissertation and discuss the limitations as well as remaining questions. Together, the results presented in my dissertation have a significant impact on our understanding of the respiratory motor pathways, which could be applied for the regeneration of diseased cells or rehabilitation efforts to accelerate the recovery of motor circuitry involved in breathing, speech, and language.

The central nervous system (CNS) is a very complex and highly organized structure. In the beginning the CNS is a sheet of cells. Over time external cues encourage this sheet to become a tube and from this tube an abundance of cell types are generated. These cells then migrate out, establish contacts with other cells, and form functional circuits. Given the complexity of the CNS, one of the fundamental goals of developmental neurobiology is to understand how this wealth of cellular diversity is generated and organized given the initial guidance of just a few signaling cues. Through the work of many groups, the emerging solution to this unbalanced equation appears to be signaling pathway interactions. To address this we focused our study on the roles and interactions of two major developmental signaling pathways: Notch and Sonic hedgehog (Shh). In the developing CNS, the major role of Notch signaling is progenitor maintenance and the major role of Shh signaling is progenitor patterning. However, when studied together, we observed that Notch signaling was able to modulate a progenitor cell’s response to Shh and in doing so influenced cell fate choices made within the developing spinal cord. Interested in these initial findings, we wanted to study other functions of Notch signaling. In a second study we observed that interactions between Notch and retinoic acid (RA) signaling contributed to neuronal diversity within the caudal hindbrain and rostral spinal cord. Then, in a third study, we observed that Notch signaling maintained apical cell contacts within the developing brain. Collectively, this work illustrates that, within the context of neural development, Notch signaling has multiple functions and this functional diversity is largely facilitated through interactions with other signaling pathways. While the functions of Notch signaling were enriched by pathway interactions, we also took a closer look at the factors that influence Shh signaling activity. In a final study we show that Shh signaling activity can be also be modulated through the protein kinase A (PKA)-direct and PKA-indirect downstream phosphorylation of Gli proteins, bifunctional transcriptional effectors of Shh signaling activity.

Localized diffusible chemotropic signals are canonical sources of guidance information for axons extending towards their synaptic targets. Equally important, but less well understood, are the contact-dependent regional boundaries that provide either permissive or non-permissive substrates for axon growth. Classic work has demonstrated the importance of netrin1 as a floor plate chemoattractant for commissural axons in the developing spinal cord; subsequent studies in different systems have suggested that netrin1 also has short-range guidance activities. Here, I have further analyzed the role of netrin1 in the spinal cord and find that netrin1 mediates short-range growth boundaries that guide many additional classes of spinal axons. All spinal axons grow precisely around the ventricular zone (VZ), without innervating it, suggesting that the edge of the VZ represents a growth boundary. Multiple lines of evidence suggest that this boundary is mediated by netrin1: first, netrin1 is expressed by neural progenitors in the VZ and transported to the progenitor end feet at the pial surface; second, neurofilament (NF)+ axons initiate oriented growth coincident with the dorsal boundary of VZ-netrin1; third, several axons project aberrantly into the VZ in the absence of netrin1. This phenotype is observed only when netrin1 is ablated from the VZ, not the FP. Moreover, the selective ablation of netrin1 from the VZ is sufficient to locally reshape the trajectories of NF+ axons. Our studies have shown that netrin1 mediates the growth boundary acting primarily through the Dcc receptor and that netrin1 accumulates in axons in a Dcc dependent manner. Taken together, our data suggest that netrin1 establishes local “hederal” boundaries that provide an adhesive substrate while also preventing local innervation.